Rabbit hemorrhagic disease virus vaccine and uses thereof

Abstract

The present invention relates to an immunogenic or vaccine composition which provides protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus of variant strain 1 (RHDV-1) and of variant strain 2 (RHDV-2) and uses thereof.

Description

[0001] RABBIT HEMORRHAGIC DISEASE VIRUS VACCINE AND USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of medicine, more particularly to a recombinant immunogenic or vaccine composition for the protection of rabbits from rabbit hemorrhagic disease (RHD) caused by infection of rabbit hemorrhagic disease virus (RHDV) and variant strains.

[0004] BACKGROUND OF THE INVENTION

[0005] Rabbit hemorrhagic disease (RHD) is caused by caliciviruses of the species Lagovirus europaeus, which mainly affect European rabbits (Oryctolagus cuniculus).

[0006] Rabbit hemorrhagic disease (RHD) is a rapidly fatal, highly infectious disease of European feral and domestic rabbits characterized by hemorrhagic lesions affecting, in particular, the liver and lungs, with around 90% mortality in adult rabbits occurring mostly 24- 48 h. after infection. RHD is caused by strains of RHDV (virus of the family Caliciviridae) a non-enveloped virus with a diameter around 35-40 nm, icosahedral symmetry with a single capsid subunit formed by 90 dimers of the VP60 (also known as VP1 ) protein, and a linear single-stranded positive-sense RNA genome of 6.4-8.5 kb.

[0007] RHDV (also referred to as RHDV1 , RHDV-1, or as classic RHDV) only affects adult European rabbits (Oryctolagus cuniculus). This virus was first reported in China in 1984, from which it spread worldwide, mainly in Asia, Europe, Australia, and New Zealand. Recent outbreaks have also been reported in USA, Canada and Mexico.

[0008] Until recently, and following the new nomenclature proposed by Le Pendu et al. (2017), all RHD-viruses belonged to the GL1 genotype (also known as classic RHDV, with the genogroups G1-G5, all isolated from 1984). In 1996, the antigenic variant RHDVa (G6) was detected. In 2010, a new virus causing RHD outbreaks in France was identified. This new lagovirus, referred first to as the RHDVb variant or RHDV-2, differs phylogenetically, antigenically and pathogenically from other lagoviruses, being included in the new genotype, GI.2. This new virus, GI.2 also known as RHDV-2, RHDVb or simply RHDV2, causes lower average mortality than G 1.1 -genotype viruses, although induced mortality in very young rabbits is higher than that caused by GI.1.

[0009] Importantly, RHDV-2 killed rabbits previously vaccinated with RHDV vaccines, and affected young European rabbits, as well as hares (Lepus spp.). All these features strongly suggest that the virus was not derived from RHDVa, but from some other unknown source. RHDV-2 has since rapidly spread to most European countries, as well as to Australia, Canada, and the United States, replacing the previously circulating viruses, particularly those belonging to the GI.1b / RHDV variants. This rapid widespread of RHDV-2 has generated a new threat to rabbit farming and negative impacts on feral rabbit populations.

[0010] The first commercial vaccines against rabbit hemorrhagic disease (RHD) were developed in the early 1990s and they were based on inactivated classic rabbit hemorrhagic disease virus (RHDV) prepared from the liver of RHDV-infected rabbits. After the introduction of such inactivated liver-derived vaccines, several vaccines containing the capsid protein VP60 were developed. Currently, existing vaccines based on RHDV-1 are largely ineffective against RHDV-2 viruses, and vice versa, because they cannot confer homologous protection and heterologous cross-protection against both RHDV-1 and RHDV-2, increasing the number of vaccinations required to protect rabbits from both variant strains of rabbit hemorrhagic disease virus, and consequently, increasing associated economic costs. Therefore, there is a need for vaccines which provide effective protection for both variants’ strains of the rabbit hemorrhagic disease virus in a single dose.

[0011] SUMMARY OF THE INVENTION

[0012] The inventors of the present invention have encountered that an immunogenic or vaccine composition comprising a recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain, provides effective protection to rabbits from infection with both rabbit hemorrhagic disease virus 2 (RHDV-2) and rabbit hemorrhagic disease virus 1 (RHDV- 1) variant strains. Surprisingly, the inventors further observed that said protection is obtained with the administration of a single dose of the immunogenic or vaccine composition to a subject. Furthermore, the inventors also observed that said protection does not appear to be affected by the presence of circulating anti-RHDV antibodies, in particular, of maternal origin also known as maternally derived antibodies (MDA).

[0013] Therefore, a first aspect of the present invention relates to an immunogenic or vaccine composition comprising a recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain, wherein the composition is capable of a) inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2) and b) inducing heterologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 1 (RHDV-1 ), wherein the immunogenic or vaccine composition is devoid of immunogens derived from RHDV-1 variant strains and wherein said protection is induced after the administration of a single dose of the composition.

[0014] Another aspect of the present invention relates to the immunogenic or vaccine composition according to the invention for use as a medicament. A further aspect of the present invention relates to the immunogenic or vaccine composition according to the invention for use in the treatment and / or prophylaxis of rabbit hemorrhagic disease (RHD) and associated diseases in a subject.

[0015] A further aspect of the present invention relates to the immunogenic or vaccine composition according to the invention for use in a method for inducing an immune response against rabbit hemorrhagic disease virus in a subject.

[0016] Yet another aspect of the present invention relates to a pre-filled vaccine delivery device comprising an immunogenic or vaccine composition according to the invention.

[0017] A further aspect of the present invention relates to a method for the production of the immunogenic or vaccine composition according to the invention, comprising: a) culturing a yeast host cell comprising a nucleotide sequence encoding a VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain to obtain the recombinant VP60 protein; b) separating the recombinant VP60 protein of a RHDV-2 variant strain from the yeast host cell non-soluble components; and c) obtaining the composition comprising the recombinant VP60 protein of a RHDV- 2 variant strain and the yeast host cell soluble components.

[0018] DESCRIPTION OF THE FIGURES

[0019] Figure 1. Mean serological response against rabbit hemorrhagic disease virus 2 (RHDV-2) (as Iog2 HI / 50pl antibodies against RHDV-2) (ordinate) per group and different days (abscissa).

[0020] Figure 2. Mean serological response against rabbit hemorrhagic disease virus 2 (RHDV-2) (as Iog2 HI / 50pl antibodies against RHDV-2) (ordinate) per group and per day postvaccination (abscissa).

[0021] Figure 3. Mean serological response against rabbit hemorrhagic disease virus 2 (RHDV-2) (as Iog2 HI / 50yl antibodies against RHDV-2) (ordinate) per group and per day postvaccination (abscissa).

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] As previously mentioned, the present invention relates to an immunogenic or vaccine composition comprising a recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain that provides homologous protection to subjects against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2) variant strains and heterologous protection, also referred as cross-protection, against RHD caused by rabbit hemorrhagic disease virus 1 (RHDV-1) variant strains, wherein, surprisingly, said protection is obtained after the administration of a single dose of the composition. Further, the protection is not affected by the presence of circulating anti-RHDV antibodies, particularly of maternally derived antibodies (MDA).

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs at the time of filing. However, in the event of any latent ambiguity, definitions provided herein take precedent over any other definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular forms as well.

[0025] Immunogenic or vaccine compositions

[0026] Hence, as first aspect of the present invention relates to an immunogenic or vaccine composition comprising a recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain, from here onwards the composition of the invention, wherein the composition is capable of a) inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2) and b) inducing heterologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 1 (RHDV-1), wherein the composition is devoid of immunogens derived from RHDV-1 variant strains and wherein said protection is induced after the administration of a single dose of the composition.

[0027] Another aspect of the present invention relates to a single dose immunogenic or vaccine composition comprising a recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain, wherein the composition is capable of a) inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2) and b) inducing heterologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 1 (RHDV-1), wherein the composition is devoid of immunogens derived from RHDV-1 variant strains.

[0028] The expression “composition of the invention” is to be understood as meaning both the immunogenic composition of the invention as well as the vaccine composition of the invention. When “composition of the invention” is used in reference to a particular embodiment and / or definition, said particular embodiment is to be understood to be applicable to both the immunogenic composition as well as the vaccine composition, i.e., the composition of the invention refers to the immunogenic composition and / or the vaccine composition of the invention. In cases where a particular embodiment or a definition does not apply to both the immunogenic composition and the vaccine composition, the individual expression “immunogenic composition of the invention” or “vaccine composition of the invention” is used instead. The term “immunogenic composition” refers to a composition that is capable of eliciting, establishing, inducing or improving an immune response in a subject of a cellular or antibody- mediated immune response type to the composition upon administration to the subject. An “immunogenic composition” comprises molecules with antigenic properties, such as killed or attenuated bacteria or virus, and also immunogenic polypeptides. An immunogenic polypeptide is generally referred to as antigenic. A molecule is “antigenic” when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor. It is to be understood, that in the present invention, the immunogenic composition of the invention comprises as the immunogenic polypeptide, a recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain and said agent elicits an immune response against rabbit hemorrhagic disease (RHD) in a subject.

[0029] The term “vaccine” or “vaccine composition”, as used herein, refers to an immunogenic composition that is able to elicit, establish, induce or improve an immune response in a subject to a particular disease of cellular or antibody-mediated type upon the administration to the subject that is protective. A vaccine or vaccine composition typically contains an agent that resembles a disease-causing microorganism or a part thereof (e.g. a polypeptide). Vaccines or vaccine compositions can be prophylactic and / or therapeutic. The term “vaccine” or “vaccine composition”, as also used herein, refers to an immunogenic composition of the invention complemented by pharmaceutically acceptable excipients and / or carriers, that when administered to a subject, elicits, or is able to elicit directly or indirectly, an immune response in the subject against rabbit hemorrhagic disease-causing microorganism. It is to be understood, that in the present invention, in the composition of the invention, the agent which is able to elicit an immune response to a particular disease is a recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain and said agent elicits an immune response towards the disease-causing microorganism RHDV whose infection causes rabbit hemorrhagic disease (RHD).

[0030] The composition of the invention described herein may lead to the generation of an immune response in the subject. In the context of the present invention, the term “immune response” or “immunological response” refers to the development of a cellular and / or antibody-mediated immune response by the composition of the invention. Usually, an immune or immunological response includes, one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition of the invention. Preferably, the subject displays a therapeutic and / or a prophylactic immunological (memory) response, such that the clinical severity of the disease is reduced and / or resistance to new infection is enhanced. In a particular embodiment, the immunological response is protective. Such protection can be demonstrated by a reduction in number of symptoms, a reduction in the severity of symptoms, or the lack of one or more of the symptoms associated with the infection of the RHDV-1 and / or RHDV-2, a delay in the onset of viremia, a reduction in viral persistence, a reduction in the overall viral load and / or a reduction of viral excretion, and a reduction and / or prevention of mortality.

[0031] The term “recombinant”, as used herein, refers to a biological molecule or biological structure which is artificially produced (e.g., by laboratory methods), synthetic, and / or has a different structure and / or function than the molecule or structure from which it was obtained or than its wild type counterpart. For the sake of clarity, a recombinant molecule or recombinant structure that is synthetic may nonetheless function comparably to its wild type counterpart. A “recombinant protein / polypeptide” thereby encompasses a protein / polypeptide produced by expression of a recombinant polynucleotide.

[0032] An “isolated protein”, as used herein, refers to a protein altered by the hand of man from its natural state. This means a protein that, if occurring in nature, has been removed from its natural and original environment. Accordingly, a polypeptide naturally present in a living organism is not considered an "isolated protein". The term “purified protein” refers to a protein that is present in a different environment than in the crude protein (or extract, or culture) from which it is obtained. In a particular embodiment, the purified protein is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% pure; more preferably, at least 97% pure, and more preferably still at least 99% pure. In a more particular embodiment, the purified protein is substantially free of contaminants, which means that the purified protein is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% pure; more preferably, at least 97% pure, and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art. In a particular embodiment, purified means that the level of contaminants is below a level acceptable for administration to a human or non-human subject. In other particular embodiment, the term purified protein means a protein suitable for pharmaceutical use. Therefore, in the context of the present invention the terms “recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2)” variant strain or “recombinant VP60 protein of a RHDV-2” variant strain refer to an artificially or synthetically produced VP60 protein of a RHDV-2 variant strain, which may be isolated. In a particular embodiment of the composition of the invention, the recombinant VP60 protein of a RHDV-2 variant strain is isolated. In a particular embodiment of the composition of the invention, the recombinant VP60 protein of a RHDV-2 variant strain is isolated but not purified. In another particular embodiment of the present invention, the recombinant VP60 protein of RHDV-2 is isolated and purified material that is substantially free of contaminants. Both, the recombinant VP60 protein of a RHDV-2 variant strain that is isolated but not purified, and the recombinant VP60 protein of a RHDV-2 variant strain that is isolated but purified material substantially free of contaminants are suitable for pharmaceutical use.

[0033] In the context of the present invention the term “VP60 protein” refers to the major structural capsid protein of rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain, which can self-assemble to form virus-like particles (VLPs) of approximately 33-40 nm. In a particular embodiment of the composition of the invention, the composition further comprises virus-like particles assembled of the recombinant VP60 protein of a RHDV-2 variant strain.

[0034] The term "VLP or virus-like particle", as used herein, relates to a self-assembling macromolecular structure formed by VP60 protein dimers which are devoid of the virus genetic material. The term "self-assembled", also called “auto-assembled”, or simply “assembled”, as used herein refers to a macromolecular structured of proteins formed spontaneously through protein-protein interactions in between the VP60 proteins themselves. As the skilled person will be well aware, the fact that the recombinant VP60 protein is capable of self-assemble into VLPs does not mean that all of the VP60 protein is assembled into VLPs. The expression “the composition of the invention further comprises VLPs” as used herein refers to the fact that the composition of the invention may comprise recombinant VP60 protein of a RHDV-2 strain in all stages ofassembly of a VLP, i.e., monomers, dimers, oligomers (partially assembled VLPs) and VLPs. In a particular embodiment of the composition of the invention, the composition comprises monomers and / or dimers and / or oligomers and / or VLPs of the recombinant VP60 protein of a RHDV-2 variant strain. In a particular embodiment of the composition of the invention, at least 10%, at least 30%, at least 40%, at least 50% of the VP60 protein of a RHDV-2 variant strain is assembled in the form of a VLP. In a more particular embodiment of the composition of the invention, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% of the VP60 protein of a RHDV-2 variant strain is assembled in the form of a VLP. In an even more particular embodiment of the composition of the invention, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% of the VP60 protein of a RHDV-2 variant strain is assembled in the form of a VLP.

[0035] In the present invention the VP60 protein is capable of eliciting an immune response in the subject to which is administered. In a particular embodiment of the composition of the invention, the VP60 protein is of a RHDV-2 variant strain. In a particular embodiment of the composition of the invention, the VP60 protein of a RHDV-2 variant strain comprises the sequence according to SEQ ID NO: 1 or a functionally equivalent variant. In another particular embodiment of the composition of the invention, the VP60 protein of a RHDV-2 variant strain consists of the sequence according to SEQ ID NO: 1 or a functionally equivalent variant.

[0036] The terms “functional variant” and “functionally equivalent variant” are interchangeable and are herein understood as all those peptides derived from the VP60 protein of a rabbit hemorrhagic disease virus (RHDV-2) variant strain by means of modification, insertion and / or deletion of one or more amino acids, provided that the function of eliciting an immune response described above, in particular an homologous and / or heterologous immune response, in the subject to which is administered is substantially maintained. As used herein, functional variant is also meant to include the full-length sequence of any VP60 protein of a RHDV-2 variant strain, analogs thereof, or immunogenic fragments thereof. The term "immunogenic fragment" refers to a fragment of a protein which includes one or more epitopes and thus elicits the immunological response described above. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,705,871 ; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Synthetic VP60 protein of RHDV-2 variant strains is also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens. See, e.g., Bergmann etal. (1993) Eur. J. Immunol. 23:2777-2781 ; Bergmann et al. (1996) J. Immunol. 157:3242-3249; Suhrbier, A. (1997) Immunol, and Cell Biol. 75:402- 408.

[0037] In order to determine if a functional variant or functionally equivalent variant of a VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain is still capable of eliciting an immune response as described above, in particular an homologous and / or heterologous immune response, in the subject to which is administered, traditional experiments well known by an expert in the field may be performed, such as vaccination of subjects with the composition of the invention comprising the functional or functionally equivalent VP60 protein of a RHDV-2 variant strain and challenging the subjects with an infectious virus strain, as described in the Examples section of the present invention. In a particular embodiment, the functionally equivalent variants of the VP60 protein of a RHDV-2 variant strain maintains at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, and at least 100% or more of the activity of the VP60 protein of a RHDV-2 variant strain, in particular, of the ability of the VP60 protein of a RHDV- 2 variant strain to induce homologous protection against RHD caused by rabbit hemorrhagic disease virus 2 (RHDV-2) and heterologous protection against RHD caused by rabbit hemorrhagic disease virus 1 (RHDV-1) variant strain when administered in a single dose. In a particular embodiment of the composition of the invention, the functionally equivalent variants of the VP60 protein of a RHDV-2 variant strain are those that have a degree of identity with respect to SEQ ID NO: 1 greater than at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 99% sequence identity.

[0038] The terms “identity”, “identical” or “percent identity” in the context of two or more amino acid or nucleotide sequences, refer to two or more sequences or fragments of said sequences that are the same or have a specified percentage of nucleotide or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Publicly available software programs can be used to align sequences. Appropriate parameters for maximal alignment by particular alignment software can be determined by one skilled in the art. In certain embodiments, the default parameters of the alignment software are used. In certain embodiments, the percentage identity “X” of a first nucleotide sequence to a second nucleotide sequence is calculated as 100 x (Y / Z), where Y is the number of nucleotide residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the second sequence is longer than the first sequence, then the global alignment taken the entirety of both sequences into consideration is used, therefore all letters and gaps in each sequence must be aligned. In this case, the same formula as above can be used but using as Z value the length of the region wherein the first and second sequence overlaps, said region having a length, which is substantially the same as the length of the first sequence.

[0039] For instance, 95% identical to a reference sequence according to the present invention, the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide or polypeptide sequence and that gaps in homology of up to 5% of the total number of nucleotides or amino acid residues in the reference sequence are allowed.

[0040] In a particular embodiment of the composition of the invention, the composition comprises at least about 28hemagglutination units (HU) per dose, at least about 210HU per dose, at least about 211HU per dose, at least about 212HU per dose, at least about 213HU per dose, at least about 214HU per dose, at least about 215HU per dose, at least about 216HU per dose of recombinant VP60 protein of a RHDV-2 variant strain, at least about 218HU per dose. In another particular embodiment of the composition of the invention, the composition comprises between about 28hemagglutination units (HU) to about 218HU per dose, preferably between about 29HU to about 218HU per dose, preferably between about 210HU to about 218HU per dose, more preferably between about 211HU to about 218HU per dose, even more preferably between about 212HU to about 218HU per dose of recombinant VP60 protein of a RHDV-2 variant strain.

[0041] The term “hemagglutination units”, or its acronym “HU”, as used herein, refers to an operational unit defined as the amount of recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain which is required to agglutinate an equal volume of a standardized red blood cell suspension. This agglutination of red blood cells is due to a phenomenon whereby cells infected with the recombinant VP60 protein of a RHDV-2 variant strain adsorb erythrocytes (red blood cells) on their surface.

[0042] The term “hemagglutinin inhibition", or its acronym “HI”, as used herein, refers to an operational unit that is closely related to the hemagglutination assay but includes anti-viral antibodies as “inhibitors” to interfere with the interaction of a recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain.

[0043] The hemagglutination assay (HA) and hemagglutination inhibition assay (HIA) are techniques well known in the art developed in 1941-42, described in, e.g., Hirst, G K. (1942, The Journal of experimental medicine 75, 1 :49-64) and carried out in the examples, e.g. Example 2.

[0044] The terms “about” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 10- or 5-fold, and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.

[0045] The recombinant VP60 protein of the composition of the invention is of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain. The recombinant VP60 protein of a RHDV-2 variant strain used in the composition according to the present invention can be derived in any fashion including isolation and purification from the RHDV-2 variant strain nucleotide sequence coding for the VP60 capsid protein, standard protein synthesis and recombinant techniques. The recombinant DNA techniques and methods to obtain recombinant proteins are well-known by the skilled in the art and described in detail in the manuals by Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rdEd. Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York 5 (2001) and by Ausubel et al., Current Protocols In Molecular Biology, John Wiley and Sons, Inc. (1998). In a particular embodiment the recombinant VP60 protein of a RHDV-2 variant strain of the composition of the invention is a VP60 protein having a sequence identity of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO: 1 . In a particular embodiment the recombinant VP60 protein of a RHDV-2 variant strain of the composition comprises the sequence according to SEQ ID NO: 1. In a particular embodiment the recombinant VP60 protein of a RHDV-2 variant strain of the composition consists of the sequence according to SEQ ID NO: 1.

[0046] The composition of the invention is capable of inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2) and heterologous protection, also referred to as cross-protection, against RHD caused by rabbit hemorrhagic disease virus 1 (RHDV-1 ) after administration of a single dose of the composition of the invention. The term “homologous protection” in the context of the present invention refers to protection conferred by or obtained due to the composition of the invention against a strain of the same genotype of the recombinant VP60 protein of a RHDV-2 variant strain of the composition of the invention, i.e. , a RHDV-2 variant strain. On the other hand, the term “heterologous protection”, or “cross-protection”, as used herein, refers to the protection conferred by, or obtained due, to the composition of the invention against a strain of a different genotype of the recombinant VP60 protein of a RHDV-2 variant strain of the composition of the invention, i.e., a non RHDV-2 variant strain, specifically but not limited to a RHDV-1 variant strain. In a particular embodiment, the “heterologous protection” (“cross-protection”) is against a RHDV-1 variant strain.

[0047] The term “strain” or “variant strain”, as used herein, refers to a genetic variant, subtype or genotype of a microorganism, in particular to a genetic variant, subtype or genotype of a rabbit hemorrhagic disease virus. According to the proposed classification and nomenclature of Caliciviridae family by Le Pendu et al. [(2017) Journal of General Virology; 98:1658-1666], the RHD causing virus can be classified into two genotypes: GI.1 and GI.2. GL1 genotype (classic RHDV, comprises the genogroups G1-G5, all isolated from 1984, and the antigenic variant RHDVa / G6 identified in 1996). In addition to this, GL1 comprises 4 variants: Gl.1a, GI.1 b, Gl.1c and Gl.1d. In the context of the present invention, the terms “RHDV-1 strain”, “RHDV-1 variant strain”, “RHDV strain”, “RHDV1 strain”, “classic RHDV” or “RHDV variant strain”, as used herein, are interchangeable between them and with the term “GI.1 genotype” and are meant to include all its variants, i.e., GL1a, GI.1 b, Gl.1c and Gl.1d. Likewise, the terms “RHDV-2 strain”, “RHDV2” or “RHDV-2 variant strain”, as used herein, are interchangeable between them and with the term “GL2 genotype”.

[0048] The expressions “inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2)” or “inducing homologous protection against RHDV-2”, as used herein, refer to the immune response provided by the composition of the invention which leads to a therapeutic and / or a prophylactic immunological (memory) response, such that the clinical severity of the RHD disease by RHDV-2 variant strain is reduced and / or resistance to new infection by RHDV-2 variant strain is enhanced, as previously defined. In a particular embodiment, the immunological response is protective. In particular, the homologous protection against RHDV-2, as used herein, refers to the immune response provided or conferred by the composition of the invention that leads to a reduction of mortality and / or prevention of mortality caused by RHDV-2 variants strains in subjects, in particular in rabbits. Likewise, the expressions “inducing heterologous protection against RHD caused by rabbit hemorrhagic disease virus 1 (RHDV-1 )” or “inducing heterologous protection against RHDV-1” refers to the immune response provided by the composition of the invention which leads to a therapeutic and / or a prophylactic immunological (memory) response, such that the clinical severity of the RHD disease by RHDV-1 variant strain is reduced and / or resistance to new infection by RHDV-1 variant strain is enhanced, as previously defined. In a particular embodiment, the immunological response is protective. In particular, the heterologous protection against RHDV-1 , as used herein, refers to the immune response provided or conferred by the composition of the invention that leads to a reduction of mortality and / or prevention of mortality caused by RHDV-1 variant strains in subjects, in particular in rabbits.

[0049] The protection conferred by the composition of the invention to RHD can be determined by measuring the severity of the clinical symptoms of RHD in animals infected by rabbit hemorrhagic disease virus 1 (RHDV-1 ) and / or rabbit hemorrhagic disease virus 2 (RHDV-2), in particular the survival rate of vaccinated infected subjects in comparison with survival rate of non-vaccinated infected subjects. In a particular embodiment of the composition of the invention, the protection against RHD is measured as survival rate of infected subjects. In a particular embodiment of the composition of the invention, the protection against RHD is measured as mortality rate of infected subjects

[0050] The term “survival rate”, as used herein, refers to the percentage of subjects alive after a certain time post-infection by rabbit hemorrhagic disease virus 1 (RHDV-1 ) and / or rabbit hemorrhagic disease virus 2 (RHDV-2) (natural or experimental infection or challenge) or post diagnosis of RHD, or until the infection has been dealt with by the animals which survive. The survival rate can be performed in a control environment, wherein different groups of vaccinated subjects and non-vaccinated subjects (control group) are challenged with a virulent strain of RHDV-1 and / or RHDV-2, and the number of survivor subjects is counted at different times after challenge, as described in the Examples 2 to 6 of the present description. The “survival rate” can be determined directly from the “mortality rate”, since the mortality rate plus the survival rate must equal 1 (when in fractions) or 100% (when in percentage). Therefore, a mortality rate of 80% corresponds to a survival rate of 20%. The terms “mortality” and “mortality rate” are used interchangeably with the terms “lethality” and “lethality rate”, respectively.

[0051] In a particular embodiment of the composition of the invention, the protection against rabbit hemorrhagic disease (RHD) is determined by the survival rate of subjects vaccinated with the composition of the invention and infected with rabbit hemorrhagic disease virus 1 (RHDV-1 ) and / or rabbit hemorrhagic disease virus 2 (RHDV-2). In another particular embodiment, the survival rate of subjects vaccinated with the composition of the invention and infected with RHDV-1 is of at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100% of naturally or experimentally infected subjects. In another particular embodiment of the composition of the invention the survival rate of subjects vaccinated with the composition of the invention and infected with RHDV-2 is of at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100% of naturally or experimentally infected subjects. In another particular embodiment of the composition of the invention, the survival rate of the subjects vaccinated with the composition of the invention and infected with RHDV-2 is of at least 90%, preferably 100%. Thus, in a particular embodiment of the composition of the invention, the survival rate is 100%, i.e., prevention, against RHDV-2 variant strains. In another particular embodiment, the survival rate of the subjects vaccinated with the composition of the invention and infected with RHDV- 1 is of at least 70%, preferably 100%. Thus, in a particular embodiment of the composition of the invention, the survival rate is 100%, i.e., prevention, against RHDV-1 variant strains. In another particular embodiment of the composition of the invention, the survival rate of subjects vaccinated with the composition of the invention and infected with RHDV-1 and RHDV-2 is of at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100% of naturally or experimentally infected subjects.

[0052] In a particular embodiment of the composition of the invention, the protection against rabbit hemorrhagic disease (RHD) is determined by the mortality rate of subjects vaccinated with the vaccine composition of the invention and infected with rabbit hemorrhagic disease virus 1 (RHDV-1 ) and / or rabbit hemorrhagic disease virus 2 (RHDV-2). In another particular embodiment, the mortality rate of subjects vaccinated with the composition of the invention and infected with RHDV-1 is of less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, about 0% of naturally or experimentally infected subjects. In another particular embodiment of the composition of the invention, the mortality rate of subjects vaccinated with the composition of the invention and infected with RHDV-2 is of less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, about 0% of naturally or experimentally infected subjects. In another particular embodiment of the composition of the invention, the mortality rate of the subjects vaccinated with the composition of the invention and infected with RHDV- 2 is of less than 10%, preferably 0%. Thus, in a particular embodiment of the composition of the invention, the mortality rate is 0%, i.e., prevention, against RHDV-2 variant strains. In another particular embodiment, the mortality rate of the subjects vaccinated with the composition of the invention and infected with RHDV-1 is of less than 30%, preferably 0%. Thus, in a particular embodiment of the composition of the invention, the mortality rate is 0%, i.e., prevention, against RHDV-1 variant strains. In another particular embodiment of composition of the invention, the mortality rate of subjects vaccinated with the composition of the invention and infected with RHDV-1 and RHDV-2 is of less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 3%, less than about 2%, less than about 1%, about 0% of naturally or experimentally infected subjects. Thus, in a particular embodiment of the composition of the invention, the mortality rate of the subjects vaccinated with the composition of the invention is 0%, i.e., prevention, against rabbit hemorrhagic disease virus 1 (RHDV-1 ) and rabbit hemorrhagic disease virus 2 (RHDV-2) variant strains.

[0053] The term “rabbit hemorrhagic disease” or its acronym “RHD”, as used herein, refers to a disease also known as viral hemorrhagic disease (VHD), which is extremely contagious and highly infectious and lethal to domesticated and wild rabbits belonging to the Oryctolagus cuniculus species. Severe losses are common in unvaccinated animals, and in intensive farms a variable proportion of rabbits may die according to the circulating rabbit hemorrhagic disease virus. RHD is characterized by affecting primarily the liver of the infected animals, triggering the mass death of hepatocytes which can in turn lead to disseminated intravascular coagulation, hepatic encephalopathy, and nephrosis. The disease caused by the classic RHDV-1 may cause death of most or all rabbits (80-90% lethality) whereas a mortality rate caused by RHDV-2 variant strains is up to 70%. This disease, independently from the causative RHD virus strain, also causes dramatic losses in wild or feral rabbit populations, particularly when it is first introduced. Rabbit hemorrhagic disease virus spreads very readily. While the clinical evolution of the disease can be peracute, acute, subacute or chronic, clinical manifestations have been described mainly in the acute infection, as there are usually no clinical signs of disease in the peracute form, and the subacute form is characterized by similar but milder signs. The incubation period varies between 1 and 5 days depending on the type of causative agent, and death may occur 12-36 hours after the onset of fever (>40°C). During this phase, various signs can be observed, such as anorexia, apathy, dullness, prostration, nervous signs (convulsion, ataxia, paralysis, opisthotonos, paddling), groans and cries, respiratory sign (dyspnoea, frothy and bloody nasal discharge), and cyanosis of mucous membranes. During an outbreak, a certain number of rabbits (5-10% in the case of RHDV / RHDVa and significantly more if the infection is caused by RHDV-2) may show a chronic or subclinical evolution of the disease, which is characterised by severe and generalised jaundice, loss of weight and lethargy. These animals often die 1-2 weeks later, probably due to liver dysfunction. Due to the rapid course of this disease, the animals are usually found in good condition after death. Gross pathological lesions are variable and may be subtle and include circulatory and degenerative disorders. Liver necrosis and splenomegaly are the primary lesions. The liver appears yellowish-brown in color, brittle and degenerated, with a marked lobular pattern. The tracheal mucosa is hyperaemic, containing abundant frothy fluid, and the lungs are oedematous and congested. The spleen is engorged, with rounded edges and enlarged (splenomegaly). The presence of clotted blood in blood vessels is due to disseminated intravascular coagulation (DIC). Such massive coagulopathy is usually the cause of hemorrhages in a variety of organs and sudden death. In subacute and chronic disease, an icteric discoloration of the ears, conjunctiva and subcutis is clearly evident.

[0054] Increased virulence of RHDV-2 variant strains has been reported since their first appearance in the field among others by Capucci, L., et al., “Increased pathogenicity in rabbit hemorrhagic disease virus type 2 (RHDV2)” Vet. Rec. 2017, 180, 426, resulting in highly pathogenic and highly virulent RHDV-2 variant strains.

[0055] In a particular embodiment, the immunogenic or vaccine composition of the invention is capable of inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2) and heterologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 1 (RHDV-1 ), wherein the RHDV-2 is a highly virulent RHDV-2 variant strain. In a particular embodiment, the immunogenic or vaccine composition of the invention is capable of inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2) and heterologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 1 (RHDV-1), wherein the RHDV-2 is a highly virulent RHDV-2 variant strain which causes a mortality of at least 70%.

[0056] The terms “highly pathogenic” or “highly virulent” RHDV-2 variant strain as used herein, refer to a RHDV-2 variant strain that causes a mortality percentage of at least 70%, preferably, at least 80%, preferably, at least 90%, preferably, at least 95%, preferably, at least 99%, and more preferably at least 100%. Furthermore, as used herein the terms “highly pathogenic” or “highly virulent” RHDV-2 variant strain refer to a RHDV-2 variant strain that also causes acute infection forms of the disease wherein the infected subjects typically exhibit at least one of the following clinical signs: pyrexia, lethargy, weight loss, or terminal seizures. Pyrexia is the most consistently observed clinical sign, which usually develops first in infected subjects. The onset of pyrexia typically correlates with an exponential increase in viraemia, which frequently coincides with the onset of lethargy and weight loss. Lethargy affects the regular cyclical activity patterns of a subject over the infection resulting in a reduction of the activity levels in said subject, which generally concurs with the onset of pyrexia. Weight loss is also typically observed in infected subjects, likely due to decreased feed intake and / or dehydration. Terminal seizures are observed only in the terminal phase of the disease, and they are characterized as intermittent episodes of generalized tonic-clonic seizure activity which usually commence prior to death. In a particular embodiment, the immunogenic or vaccine composition of the invention is capable of inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2) and heterologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 1 (RHDV-1 ), wherein the RHDV-2 is a highly virulent RHDV-2 variant strain which causes at least one clinical sign selected from the group consisting of a mortality of at least 70%, pyrexia, lethargy, weight loss, terminal seizures, and any combination thereof.

[0057] The “highly pathogenic” or “highly virulent” RHDV-2 variant strains do not cause subacute forms of the disease infection.

[0058] The term "associated disease", as used herein, refers to any disease, condition or disorder other than rabbit hemorrhagic disease (RHD) that may occur concomitantly with RHD caused by the infection of an additional pathogen selected from a group of microorganisms consisting of myxoma virus, shope fibroma virus (SFV), Pasteurella multocida, Staphylococcus aureus, E. coll, Salmonella enterica, Salmonella typhimurium, Clostridium spiroforme, Clostridium perfringens, Clostridium cuniculi, Eimeria sp., Encephalitozoon cuniculi and Trichophyton mentagrophytes. Preferably, the one additional pathogen is selected from a group of microorganisms consisting of myxoma virus, shope fibroma virus (SFV), Pasteurella multocida, Staphylococcus aureus, E. coli, and Trichophyton mentagrophytes. More preferably, the additional pathogen is selected from a group of microorganisms consisting of myxoma virus and shope fibroma virus (SFV).

[0059] The term “immunogen” or “antigen”, as used herein refers to a component against which a subject can initiate an immune response, e.g. humoral and / or cellular immune response. As used herein, the term “immunogen” or “antigen” refers to a protein or peptide sequence capable of inducing an immune response in a subject. In the context of the composition of the invention the immunogen capable of inducing an immune response is a VP60 polypeptide or protein from a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain, in particular the recombinant VP60 capsid protein. Despite this, the composition of the invention may comprise other immunogens as long as said immunogens are not derived from rabbit hemorrhagic disease virus 1 (RHDV-1 ) variant strains. In fact, the composition of the invention is devoid of immunogens derived from RHDV-1 variant strains. The term “devoid”, as used herein, refers to the complete absence of immunogens derived from RHDV-1 variant strains, or the presence at such low levels which are undetectable and do not elicit an immune response in a subject. In a particular embodiment the “immunogen” or “antigen” is a VP60 polypeptide from a RHDV- 2 variant strain, preferably is the VP60 capsid protein of a RHDV-2 variant strain, and still more preferably, is the recombinant VP60 capsid protein of a RHDV-2 variant strain.

[0060] The terms “derived” or "derived from", as used herein, refer to the origin or source, and may include naturally occurring, isolated, recombinant, unpurified or purified molecules.

[0061] In a particular embodiment of the composition of the invention, the composition is devoid of further immunogens derived from rabbit hemorrhagic disease virus 2 (RHDV-2) variant strains, besides the recombinant VP60 protein. In a particular embodiment of the composition of the invention, the antigen consists of the recombinant VP60 protein of a RHDV-2 variant strain.

[0062] The composition of the invention is capable of inducing heterologous protection, also herein referred as cross-protection, after the administration of a single dose. In order to administer the composition of the invention, said composition must be formulated in accordance with the type of administration to be carried out. In a particular embodiment of the composition of the invention, the composition is formulated to be administered by parental, oral, mucosal or intranasal route. In a particular embodiment the composition of the invention is formulated to be administered by parental route, more particularly by subcutaneous, intradermal, or intramuscular route. In a more particular embodiment, the composition of the invention is formulated to be administered subcutaneously. The skilled person in the art is well aware of the best practices and methodology to formulate the composition of the invention to be in accordance with the requirements of the administration route.

[0063] In a particular embodiment, the immunogenic or vaccine composition is formulated to be administered in a volume of about 0.10 ml per dose, about 0.20 ml per dose, about 0.30 ml per dose, about 0.35 ml per dose, about 0.40 ml per dose, about 0.45 ml per dose, about 0.50 ml per dose, about 0.55 ml per dose, about 0.60 ml per dose, about 0.65 ml per dose, about 0.70 ml per dose, about 0.75 ml per dose, about 0.80 ml per dose, about 0.85 ml per dose, about 0.90 ml per dose, about 0.95 ml per dose. In a more particular embodiment, the composition of the invention is formulated to be administered in a volume ranging between about 0.10 ml to about 10 ml per dose. In a particular embodiment, the composition of the invention is formulated to be administered in a volume ranging between about 0.20 ml to about 5.0 ml per dose. Preferably, said composition is formulated to be administered in a volume ranging between about 0.20 ml to about 1 .0 ml per dose. More preferably, the said composition is formulated to be administered in a volume of about 0.50 ml per dose.

[0064] In an embodiment, the composition of the invention may further comprise one or more veterinary acceptable excipient.

[0065] The term "veterinary acceptable” excipient means a component that can be administered to a subject along with the immunogenic or vaccine composition of the invention without causing any undesirable biological effect or interacting in a deleterious manner with any of the other components of the composition. It is usually approved by a regulatory agency of a state or federal government or is included in the Eur. Ph. or the U.S. Pharmacopoeia or other generally recognized pharmacopoeia, including those that apply for use in animals, and more particularly in rabbits.

[0066] The term "excipient" refers to a vehicle, or diluent that is administered with the active ingredient and includes solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption-delaying agents, and the like. Such pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and similars. Water or saline aqueous solutions and aqueous dextrose and glycerol solutions, particularly for injectable solutions, are preferably used as vehicles. Suitable pharmaceutical vehicles are described in Remington: The Science and Practice Of Pharmacy, 21stEd. Philadelphia, PA. Lippincott Williams & Wilkins (2005); or by Rowe etal., Handbook of Pharmaceutical Excipients, Pharmaceutical Press, 6thEd. (2009).

[0067] Suitable veterinary acceptable vehicles include, for example, water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, monoglycerides and diglycerides of fatty acids and any combinations thereof.

[0068] The vaccine composition of the invention further comprises one or more acceptable pharmaceutical adjuvant.

[0069] The immunogenic composition of the invention may further comprise one or more adjuvants as to aid in the several aspects of maintaining and applying the said composition.

[0070] The term “adjuvant” or “acceptable pharmaceutical adjuvant”, as used herein, refers to a substance which, when added to the immunogen, non-specifically enhances or potentiates an immune response to said immunogen in a recipient host upon exposure to the mixture. As such, in another particular embodiment, the composition of the invention further comprises an acceptable pharmaceutical adjuvant. Illustrative non-limitative examples of acceptable pharmaceutical adjuvants that can be included in the immunogenic or vaccine composition of the invention include, mineral oil, ginseng, chitosan, squalane, squalene, dimethylaminoethyl (DEAE), vitamin E, zymosan, glucans, non-ionic block copolymers, monophosphoryl lipid A, aluminum hydroxide, aluminum phosphate, phosphate buffer, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), vegetable oils, water-in-oil emulsion, oil-in-water emulsion, and water-in-oil- in-water emulsion, and any combination thereof. In a particular embodiment of the immunogenic or vaccine composition of the invention the acceptable pharmaceutical adjuvant is selected from a group consisting of: mineral oil, ginseng, chitosan, dimethylaminoethyl (DEAE), aluminum hydroxide, phosphate buffer and any combination thereof. In a further particular embodiment of the composition of the invention, the acceptable pharmaceutical adjuvant is mineral oil. In an embodiment of the composition of the invention, the acceptable pharmaceutical adjuvant is light mineral oil, preferably is liquid paraffin.

[0071] The adjuvant of the composition of the invention may be present at different concentrations. Therefore, in another particular embodiment of the composition of the invention, the acceptable pharmaceutical adjuvant is at a concentration of between about 3% weight per volume (w / v) to about 30% w / v. In another particular embodiment, the concentration of the acceptable pharmaceutical adjuvant is at a concentration of about 4% w / v, about 5% w / v, about 6% w / v, about 7% w / v, about 8% w / v, about 9% w / v, about 10% w / v, about 11% w / v, about 12% w / v, about 13% w / v, about 14% w / v, about 15% w / v, about 16% w / v, about 17% w / v, about 18% w / v, about 19% w / v, about 20% w / v, about 21% w / v, about 22% w / v, about 23% w / v, about 24% w / v, about 25% w / v, about 26% w / v, about 27% w / v, about 28% w / v, about 29% w / v, about 30% w / v. In a more particular embodiment of the composition of the invention, the acceptable pharmaceutical adjuvant is at a concentration of about 20% w / v. In another particular embodiment of the composition of the invention, the acceptable pharmaceutical adjuvant is at a concentration of between about 50 g / L to about 500 g / L, between about 100 g / L to about 400 g / L, between about 150 g / L to about 300 g / L, or between about 200 g / L to about 300 g / L.

[0072] In a particular embodiment, the composition of the invention further comprises at least one additional antigen of other pathogens causing disease in rabbits. In a particular embodiment of the composition of the invention the at least one additional antigen is selected from a group of microorganisms consisting of myxoma virus, shope fibroma virus (SFV), Pasteurella multocida, Staphylococcus aureus, E. coli, Salmonella enterica, Salmonella typhimurium, Clostridium spiroforme, Clostridium perfringens, Clostridium cuniculi, Eimeria sp., Encephalitozoon cuniculi and Trichophyton mentagrophytes. Preferably, the at least one additional antigen is selected from a group of microorganisms consisting of myxoma virus, shope fibroma virus (SFV), Pasteurella multocida, Staphylococcus aureus, E. coli, and Trichophyton mentagrophytes. More preferably, the at least one additional antigen is selected from a group of microorganisms consisting of myxoma virus and shope fibroma virus (SFV).

[0073] The composition of the invention may be obtained by any method including isolation and purification from a rabbit hemorrhagic disease virus 2 (RHDV-2) nucleotide sequence coding for the VP60 capsid protein, either from wild-type RHDV-2 variant strains or by chemically protein synthesis using standard recombinant techniques. The recombinant techniques and methods to obtain recombinant proteins are well-known by the skilled in the art and described in detail in the manuals by Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rdEd. Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York 5 (2001 ) and by Ausubel et al., Current Protocols In Molecular Biology, John Wiley & Sons, Inc., Media, PA, (1988).

[0074] In a particular embodiment, the composition of the invention is obtained by a method based on recombinant techniques using expression vectors such as insect cells, mammalian cells, yeast cells or E. coli as host cells. In a particular embodiment, the immunogenic or vaccine composition of the invention is obtained by a method using yeast host cells as expression vector.

[0075] In a preferred embodiment, the immunogenic or vaccine composition of the invention is obtained by a method comprising: a) culturing a yeast host cell comprising a nucleotide sequence encoding a VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain to obtain the recombinant VP60 protein; b) separating the recombinant VP60 protein of a RHDV-2 variant strain from the yeast host cell non-soluble components; and c) obtaining the composition comprising the recombinant VP60 protein of a RHDV-2 variant strain and the yeast host cell soluble components and discarding the yeast host cell non-soluble components.

[0076] Step a)

[0077] The term “yeast host cell”, as used herein, refers to a yeast cell which contains the nucleotide sequence which when transcribed and / or translated leads to the production of the VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain. Several known yeast host cells are known and suitable for the method described above, namely and without limitation, Saccharomyces cerevisiae, Scizosacchromyces pombe, Komagataella phaffii (formerly known as Pichia pastoris), Hansanuela polymorpha and Yarrowia lipolytica. Said host cell can be obtained by transformation with lithium, electroporation, biolistic and glass bead methods of cells by conventional methods known by persons skilled in the art (Sambrook and Russell, Molecular cloning: A Laboratory Manual”, 3rdEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor NewYork (2001).

[0078] In a particular embodiment of the composition of the invention, the yeast host cell belongs to the species Komagataella phaffii.

[0079] The term "nucleotide sequence", as used herein, refers to an oligonucleotide sequence or polynucleotide sequence, and its variants. The nucleotide sequence may be DNA or RNA of genomic or synthetic or recombinant origin which may be double-stranded or singlestranded whether representing the sense or antisense strand. In a particular embodiment of the composition of the invention, the nucleotide sequence in step a) of the method for obtaining the composition of the invention is DNA. In a more particular embodiment of the composition of the invention, the nucleotide sequence in step a) of the method for obtaining the composition of the invention is DNA prepared by use of recombinant DNA techniques (i.e. recombinant DNA). In another particular embodiment of the composition of the invention, the nucleotide sequence is a sequence which encodes for the recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) according to SEQ ID NO: 1 or a functionally equivalent thereof. In a particular embodiment of the composition of the invention, the functionally equivalent variants of the recombinant VP60 protein of a RHDV-2 have a degree of sequence identity with SEQ ID NO: 1 of at least of 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%.

[0080] Step a) of the method for obtaining the composition of the invention relates to the process of culturing a yeast host cell. Said culturing is done in a suitable medium at suitable conditions for the growth of the yeast host cells and the expression of the VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain as a recombinant protein. It is well within the scope of knowledge of the expert in the field to be aware or known how to determine the best parameters for said culturing process. Suitable mediums for yeast growth and protein expression include both rich, minimal and synthetic mediums, which have different variations of their carbon sources, nitrogen sources, and different supplementations of essential amino acids and vitamins and minerals. Examples are, without limitation, yeast extract peptone dextrose (YEPD), yeast extract peptone (YEP), synthetic dextrose (SD) medium, buffered dextrose-complex medium (BMDY), synthetic complete (SC) medium and yeast nitrogen base (YNP). In a particular embodiment of the composition of the invention, step a) of the method for obtaining the composition of the invention is carried out in a buffered dextrose-complex medium (BMDY) comprising 1 % yeast extract, 2% peptone, 1 .34% yeast nitrogen base (YNB), 0.002% biotin and 1% dextrose. In another particular embodiment of the composition of the invention, in the method for obtaining the composition of the invention the BMDY medium is further supplemented with saline medium, preferably sodium phosphate, and more preferably 100 mM sodium phosphate. In addition to the medium the yeast host cells are growth at suitable conditions for the production of the recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV- 2) variant strain. In a particular embodiment of the composition of the invention, in step a) of the method for obtaining the composition of the invention the yeast host cells containing the nucleotide sequence encoding the VP60 protein of a RHDV-2 variant strain, are grown for between about 12 hours to about 36 hours, preferably 24 hours. In a particular embodiment of the composition of the invention, in step a) of the method for obtaining the composition of the invention the yeast host cells containing the nucleotide sequence encoding the VP60 protein of a RHDV-2 variant strain, are grown, at a pH of between about 5.0 to about 7.0, preferably a pH of about 6.0. In a particular embodiment of the composition of the invention, in step a) of the method for obtaining the composition of the invention the yeast host cells containing the nucleotide sequence encoding the VP60 protein of a RHDV-2 variant strain are grown at a temperature of between about 25°C to about 35°C, preferably about 30°C. In a particular embodiment of the composition of the invention, in step a) of the method for obtaining the composition of the invention the yeast host cells containing the nucleotide sequence encoding the VP60 protein of a RHDV-2 variant strain are grown for between about 12 hours to about 36 hours, preferably about 24 hours, at a pH of between about 5.0 to about 7.0, preferably a pH of about 6.0 and at a temperature of between about 25°C to about 35°C, preferably about 30°C.

[0081] Step b)

[0082] Once the recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) has been obtained in step a), the next step of the method for obtaining the composition of the invention is the process of separating the recombinant VP60 protein of a RHDV-2 variant strain from the yeast host cell non-soluble components.

[0083] The term “yeast host cell non-soluble components” as used herein refers to components associated with the yeast host cell wall, such as mannoproteins and glycans, and with the cell membranes, mainly lipids. In order to separate the recombinant VP60 protein of a RHDV-2 variant strain from the yeast host cell non-soluble components several techniques are available and form part of the general knowledge of the expert in the field. In a particular embodiment of the composition of the invention, step b) of the method for obtaining the composition of the invention is carried out by cell disruption followed by centrifugation. In a more particular embodiment, cell disruption is carried out by bead mill, high pressure homogenization and / or ultrasonication. Preferably cell disruption is carried out by high pressure homogenization, more preferably by high pressure homogenization at about 1000 bar, about 1500 bar, about 2000 bar. In another particular embodiment, the centrifugation is carried out at about 8000g to about 12000g, preferably at 10000g for between about 15 minutes to about 45 minutes, preferably 30 minutes. In another particular embodiment, the centrifugation is carried out at a temperature of between about 0°C to about 8°C, preferably 4°C. In a particular embodiment of the composition of the invention, after the centrifugation step, the supernatant comprises the recombinant VP60 protein of the RHDV-2 variant strain and yeast host cell soluble components, whereas the pellet comprises yeast host cell nonsoluble components and debris, which is discarded.

[0084] In a preferred embodiment, the composition of the invention is obtained by a method comprising: a) culturing a yeast host cell comprising a nucleotide sequence encoding a VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain to obtain the recombinant VP60 protein; b) separating the recombinant VP60 protein of a RHDV-2 variant strain from the yeast host cell non-soluble components by centrifugation; and c) obtaining the composition comprising the recombinant VP60 protein of a RHDV-2 variant strain and the yeast host cell soluble components and discarding the yeast host cell non-soluble components.

[0085] Step c)

[0086] The end result of step c) is the composition comprising the recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain and the yeast host cell soluble components. The terms “yeast host cell soluble components” or “soluble components of the yeast host cell” are used herein as all the components which are left in solution once the yeast host cells are disrupted and the resulting solution centrifuged. Therefore, the soluble components of the yeast host cell are those components that are not associated with cell debris and non-soluble components. The recombinant VP60 protein of a RHDV-2 variant strain of the composition of the invention can be further purified form the yeast host cell soluble components. However, in a particular embodiment of the composition of the invention, the method for obtaining the composition of the invention does not comprise a step of purifying the recombinant VP60 protein of a RHDV-2 variant strain from the composition obtained in step b).

[0087] In another particular embodiment of the composition of the invention, step c) may further comprise a step of sterilization of the composition comprising the recombinant VP60 protein of a RHDV-2 variant strain and the yeast host cell soluble components, preferably wherein said sterilization is carried out by filtration through a 0.22 pm filter.

[0088] In a particular embodiment of the composition of the invention, the method for obtaining the composition of the invention further comprises a step of mixing the composition obtained in step c) with an acceptable pharmaceutical adjuvant.

[0089] Thus, in a particular embodiment, the composition of the invention is obtained by a method comprising: a) culturing a yeast host cell comprising a nucleotide sequence encoding a VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain to obtain the recombinant VP60 protein; b) separating the recombinant VP60 protein of a RHDV-2 variant strain from the yeast host cell non-soluble components by centrifugation; c) obtaining the composition comprising the recombinant VP60 protein of a RHDV-2 variant strain and the yeast host cell soluble components and discarding the yeast host cell non-soluble components; and d) mixing the composition obtained in step c) with an acceptable pharmaceutical adjuvant.

[0090] Thus, in another particular embodiment, the vaccine composition of the invention is obtained by a method comprising: a) culturing a yeast host cell comprising a nucleotide sequence encoding a VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain to obtain the recombinant VP60 protein; b) separating the recombinant VP60 protein of a RHDV-2 variant strain from the yeast host cell non-soluble components by centrifugation; c) obtaining an immunogenic composition comprising the recombinant VP60 protein of a RHDV-2 variant strain and the yeast host cell soluble components and discarding the yeast host cell non-soluble components; and d) mixing the immunogenic composition obtained in step c) with an acceptable pharmaceutical adjuvant.

[0091] Medical uses of the invention

[0092] All of the definitions and embodiments described in relation to previous aspects of the present invention are equally valid and applicable for the following aspects.

[0093] In a particular embodiment of the medical use of the invention the composition comprises virus-like particles assembled of the recombinant VP60 protein of a RHDV-2 variant strain.

[0094] The composition of the invention finds uses in the treatment and / or prophylaxis of rabbit hemorrhagic disease (RHD) and associated diseases in a subject.

[0095] Another aspect of the present invention relates to the composition of the invention for use as a medicament. Alternatively, the present invention relates to the use of the composition of the invention for the manufacture of a medicament.

[0096] As used herein, the term “medicament” is meant to include vaccines and any other composition or formulation of the composition of the invention for the treatment and / or prophylaxis of an infection caused by rabbit hemorrhagic disease virus, either by rabbit hemorrhagic disease virus 1 (RHDV-1 ) and / or rabbit hemorrhagic disease virus 2 (RHDV-2) or variant strains thereof in a subject.

[0097] A further aspect of the present invention relates to the composition of the invention for use in a method for inducing an immune response against rabbit hemorrhagic disease virus in a subject, from here onwards the medical use of the invention. Alternatively, the invention relates to the use of the composition of the invention for the manufacture of a medicament for inducing an immune response against rabbit hemorrhagic disease virus in a subject. Alternatively, the invention relates to a method for inducing an immune response against rabbit hemorrhagic disease virus in a subject, comprising administering to said subject a therapeutically effective amount of the composition of the invention.

[0098] In a particular embodiment of the medical use of the invention, the method is for inducing a homologous immune response against rabbit hemorrhagic disease virus 2 (RHDV-2).

[0099] In another particular embodiment of the medical use of the invention, the method is for inducing a heterologous immune response against rabbit hemorrhagic disease virus 1 (RHDV-1 ), also referred as inducing a cross-protection.

[0100] In a particular embodiment of the medical use of the invention, the method is for inducing a homologous immune response against rabbit hemorrhagic disease virus 2 (RHDV-2) and a heterologous immune response against rabbit hemorrhagic disease virus 1 (RHDV-1 ).

[0101] In another particular embodiment of the medical use of the invention the immune response confers protection against rabbit hemorrhagic disease (RHD) in a subject. In a preferred embodiment of the medical use of the invention, the method is for inducing a homologous protection against rabbit hemorrhagic disease (RHD) caused by RHDV-2. In a preferred embodiment of the medical use of the invention, the method is for inducing a heterologous protection against RHD caused by RHDV-1. In another preferred embodiment of the medical use of the invention, the method is for inducing a homologous protection against rabbit hemorrhagic disease (RHD) caused by RHDV-2 and a heterologous protection against rabbit hemorrhagic disease (RHD) caused by RHDV-1. In another preferred embodiment of the medical use of the invention, the method is for inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by a highly virulent RHDV-2 variant. In another preferred embodiment of the medical use of the invention, the method is for inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by a RHDV-2 variant strain which causes at least 70% mortality.

[0102] In another preferred embodiment of the medical use of the invention, the method is for inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2) and heterologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 1 (RHDV-1 ), wherein the RHDV-2 is a highly virulent RHDV-2 variant strain. In another preferred embodiment of the medical use of the invention, the method is for inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2) and heterologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 1 (RHDV-1 ), wherein the RHDV-2 is a highly virulent RHDV-2 variant strain which causes a mortality of at least 70%.

[0103] A further aspect of the present invention relates to the immunogenic or vaccine composition of the invention for use in the treatment and / or prophylaxis of rabbit hemorrhagic disease (RHD) and associated diseases in a subject. Alternatively, the invention relates to the use of the composition of the invention for the manufacture of a medicament for the treatment and / or prophylaxis of rabbit hemorrhagic disease and associated diseases in a subject. Alternatively, the invention relates to a method for treating and / or preventing rabbit hemorrhagic disease and associated diseases in a subject, comprising administering to said subject a therapeutically effective amount of the composition of the invention.

[0104] The terms “prevention” or “prophylaxis”, as used herein, relate to the capacity to prevent, minimize, or delay the onset or development of a disease or condition before its onset. In the present context said disease is rabbit hemorrhagic disease (RHD). The term “prevention”, as used herein, also relates to resistance to new infection, where thanks to the administration of the composition of the invention the resistance to a new infection is enhanced and / or the clinical severity of the disease reduced. Such prevention also results in a reduction in number of symptoms or clinical signs, severity of symptoms or clinical signs, or the lack of one or more of the symptoms or clinical signs associated with the infection of the RHDV-1 and / or RHDV-2, a delay in the onset of viremia, reduced viral persistence, a reduction in the overall viral load and / or a reduction of viral excretion, and reduction or prevention of mortality.

[0105] As used herein, the terms "treat" or "treatment" refer to therapeutic treatment, the purpose of which is to reverse, reduce, suppress, delay or stop the progression or severity of the condition associated with the disease or disorder. The term "treatment" includes reducing or alleviating at least one adverse effect or condition of an ailment, a disease or disorder, such as an infection. Treatment is usually "effective" when one or more symptoms or clinical signs are reduced. Alternatively, treatment is "effective" if disease progression is delayed or halted. That is, "treatment" includes not only the improvement of symptoms or clinical signs, but also the interruption of at least a condition that indicates the progression or worsening of symptoms or clinical signs that would be expected in the absence of treatment. The beneficial or desirable clinical outcome, whether detectable or not, is a reduction in one or more symptoms or clinical signs, a reduction in the extent of the disease, a stable (not aggravated) condition of the disease. These include, but are not limited to, delayed or slowed progression, amelioration or alleviation of the disease state, and remission (partial or total). The term "treatment" of a disease also includes providing relief from symptoms, clinical signs or side effects of the disease (including symptomatic treatment).

[0106] As previously mentioned, the term “confers” is also meant to include terms such as provides, elicits, induces, furnishes, and other such terms. The expression “confers protection against rabbit hemorrhagic (RHD) in a subject” in the present context refers to the protective immunological (memory) response, such that resistance to new infection by RHDV strains is enhanced and / or the clinical severity of the RHD disease reduced or prevented, as previously defined. In a particular embodiment of the medical use of the invention, the RHD is caused by infection with a rabbit hemorrhagic disease virus 1 (RHDV-1) variant strain and / or a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain.

[0107] The term “subject”, as used herein, refers to the target individuals in need thereof to whom the composition of the invention is administered, among others, mammals, livestock, or any other animal species susceptible to be vaccinated with the composition of the invention. Preferably, the mammal is selected from the Lagomorpha order, preferably from the Leporidae family, and particularly are rabbits and hares, more preferably a rabbit or hare from the genus Lepus, Sylvilagus or Oryctolagus, and even more preferably Oryctolagus cuniculus species. As used herein, the term “rabbit” is intended for Oryctolagus cuniculus species including, wild or feral rabbits, farming or domesticated rabbits and pet rabbits of any age or in any phase of their production cycle. In a particular embodiment of the medical use of the invention, the composition of the invention is for use in rabbits from 30 days of age onwards to reduce mortality of rabbit hemorrhagic disease (RHD) caused by classic RHD virus (RHDV-1 ) and RHDV-2 variant strains (RHDV-2), including highly virulent strains. In a particular embodiment, the composition of the invention is for use in subjects of at least 1 days of age, at least 5 days of age, at least 10 days of age, at least 15 days of age, at least 20 days of age, at least 25 days of age, or at least 30 days of age, preferably for use in subjects of at least 20 days of age, at least 25 days of age or at least 30 days of age, more preferably for use in subjects of at least 30 days of age, and also preferably for use in subjects of 30 days of age of more.

[0108] The protection against RHD is conferred to a subject. In a particular embodiment of the medical use of the invention, the subject is a mammal, preferably a rabbit or a hare, more preferably a rabbit or hare belonging to the family Leporidae, more preferably a rabbit or hare from the genus Lepus or Sylvilagus or the species Oryctolagus cuniculus. In another particular embodiment of the medical use of the invention, the rabbit or hare belongs to the genus Sylvilagus and to a species selected from the group consisting of: Sylvilagus floridanus, Sylvilgus andinus, Sylvilagus aquaticus, Sylvilagus brasiliensis, Sylvilagus dicei, Sylvilagus insonus, Sylvilagus palustris, Sylvilagus varynaensis, Sylvilagus audubonii, Sylvilagus cognatus, Sylvilagus cunicularius, Sylvilagus floridanus, Sylvilagus graysoni, Sylvilagus nuttallii, Sylvilagus obscurus, Sylvilagus robustus, Sylvilagus transitionalis, Sylvilagus bachmani and Sylvilagus mansuetus.

[0109] In another particular embodiment of the medical use of the invention, the rabbit or hare belongs to the genus Lepus. In another particular embodiment of the medical use of the invention, the rabbit or hare is a species selected from the group consisting of: Lepus alien!, Lepus americanus, Lepus arcticus, Lepus othus, Lepus timidus, Lepus insularis, Lepus tibetanus, Lepus total, Lepus castroviejoi, Lepus comus, Lepus coreanus, Lepus europaeus, Lepus mandschuricus, Lepus starcki, Lepus fagani, Lepus microtis, Lepus hainanus, Lepus nigricollis, Lepus peguensis, Lepus sinensis, Lepus yarkandensis, Lepus brachyurus, Lepus californicus, Lepus callotis, Lepus capensis, Lepus corsicanus, Lepus flavigularis, Lepus granatensis, Lepus habessinicus, Lepus oiostolus, Lepus saxatilis and Lepus townsendii.

[0110] In a particular embodiment of the medical use of the invention, the protection against rabbit hemorrhagic disease (RHD) is determined by the survival rate of subjects vaccinated with the composition of the invention and infected with rabbit hemorrhagic disease virus 1 (RHDV-1) and / or rabbit hemorrhagic disease virus 2 (RHDV-2). In a particular embodiment of the medical use of the invention, the survival rate of the subjects vaccinated with the composition of the invention and infected with rabbit hemorrhagic disease virus 1 (RHDV-1 ) is of at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100% of naturally or experimentally infected subjects. In another particular embodiment of the medical use of the invention the survival rate of subjects vaccinated with the composition of the invention and infected with rabbit hemorrhagic disease virus 2 (RHDV-2) is of at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100% of naturally or experimentally infected subjects. In another particular embodiment, the survival rate of the subjects vaccinated with the composition for the medical use of the invention and infected with rabbit hemorrhagic disease virus 2 (RHDV-2) is of at least 90%, preferably 100%. Thus, in a particular embodiment of the medical use of the invention, the survival rate is 100%, i.e., prevention, against RHDV-2 variant strains. In another particular embodiment of the medical use of the invention, the survival rate of the subjects vaccinated with the composition for the medical use of the invention and infected with rabbit hemorrhagic disease virus 1 (RHDV-1 ) is at least 70%, preferably 100%. Thus, in a particular embodiment of the medical use of the invention, the survival rate of the subjects vaccinated with the composition of the invention is 100%, i.e., prevention, against RHDV-1 variant strains. In a particular embodiment of the medical use of the invention, the survival rate of the subjects vaccinated with the composition of the invention and infected with rabbit hemorrhagic disease virus 1 (RHDV-1 ) and rabbit hemorrhagic disease virus 2 (RHDV-2) is of at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100% of naturally or experimentally infected subjects.

[0111] In a particular embodiment of the medical use of the composition of the invention, the protection against rabbit hemorrhagic disease (RHD) is determined by the mortality rate of subjects vaccinated with the composition of the invention and infected with rabbit hemorrhagic disease virus 1 (RHDV-1 ) and / or rabbit hemorrhagic disease virus 2 (RHDV-2). In another particular embodiment of the medical use of the invention, the mortality rate of subjects vaccinated with the composition of the invention and infected with rabbit hemorrhagic disease virus 1 (RHDV-1 ) is of less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, about 0% of naturally or experimentally infected subjects. In another particular embodiment of the medical use of the composition of the invention, the mortality rate of subjects vaccinated with the composition of the invention and infected with rabbit hemorrhagic disease virus 2 (RHDV-2) is of less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, about 20%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, about 0% of naturally or experimentally infected subjects. In another particular embodiment, the mortality rate of the subjects vaccinated with the composition for the medical use of the invention and infected with rabbit hemorrhagic disease virus 2 (RHDV-2) is of less than 10%, preferably 0%. Thus, in a particular embodiment, the composition for the medical use of the invention, the mortality rate is 0%, i.e., prevention, against RHDV-2 variant strains. In another particular embodiment, the mortality rate of the subjects vaccinated with the composition for the medical use of the invention and infected with rabbit hemorrhagic disease virus 1 (RHDV-1 ) is of less than 30%, preferably 0%. Thus, in a particular embodiment of the medical use of the invention, the mortality rate of the subjects vaccinated with the composition of the invention is 0%, i.e., prevention, against RHDV-1 variant strains. In another particular embodiment of the medical use of the composition of the invention, the mortality rate of subjects vaccinated with the composition of the invention and infected with rabbit hemorrhagic disease virus 1 (RHDV-1 ) and rabbit hemorrhagic disease virus 2 (RHDV-2) is of less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, about 20%, less than about 10%, less than about 5%, less than about 96%, less than about 3%, less than about 2%, less than about 1 %, about 0% of infected subjects Thus, in a particular embodiment of the medical use of the invention, the mortality rate of the subjects vaccinated with the composition of the invention is 0%, i.e., prevention, against rabbit hemorrhagic disease virus 1 (RHDV-1 ) and rabbit hemorrhagic disease virus 2 (RHDV-2) variant strains. In order for the composition of the invention to confer protection against RHD in a subject, the composition has to be delivered to the subject in order to elicit an immune response. In a particular embodiment of the medical use of the invention, the composition is administered by parenteral, oral, mucosal or intranasal route. In a particular embodiment of the medical use of the invention, the composition is formulated to be administered by parenteral route, more particularly by subcutaneous, intradermal or intramuscular route. In a more particular embodiment of the medical use of the invention, the immunogenic or vaccine composition is administered subcutaneously.

[0112] In a particular embodiment of the medical use of the invention, the composition is administered in a volume ranging between about 0.10 ml to about 10 ml per dose. In a particular embodiment of the medical use of the invention, the composition is administered in a volume ranging between about 0.20 ml to about 5.0 ml per dose. Preferably, said composition is administered in a volume ranging between about 0.20 ml to about 1 .0 ml per dose. More preferably, said immunogenic or vaccine composition is administered in a volume of about 0.50 ml per dose.

[0113] In a particular embodiment the medical use of the invention, the composition can be administered in a single dose or in several doses, depending on the required treatment plan or the required plan to elicit an immune response. In a particular embodiment of the medical use of the invention, the immunogenic or vaccine composition of the invention is administered in two or more doses.

[0114] As previously mentioned, one of the advantages of the composition of the invention is that the protection against RHD is obtained after a single dose. Therefore, in another particular embodiment of the medical use of the invention, the immunogenic or vaccine composition is administered in a single dose.

[0115] The term “single dose", as used herein, in a broad sense as referring to any contained composition present in a predetermined or metered amount to be substantially administered for a single use purpose. The single dose may be administered in a single dosage form, such as a single injection or single shot. The single dose is effective to achieve a therapeutic and / or prophylactic effect against rabbit hemorrhagic disease (RHD) caused by all known RHD virus variant strains as previously defined. This means that a single dose is effective for treating, preventing, reducing, controlling and / or ameliorating clinical signs associated to rabbit hemorrhagic disease caused by all known RHD virus variant strains as previously defined. The single dose is also capable of inducing homologous protection against rabbit hemorrhagic disease caused by rabbit hemorrhagic disease virus 2 (RHDV-2) and inducing heterologous protection against rabbit hemorrhagic disease caused by rabbit hemorrhagic disease virus 1 (RHDV-1 ). After the administration of a single dose there is no need to administer a second or further doses (three, four, etc.) to maintain said therapeutic and / or prophylactic effect. The single dose, as used herein, is capable of maintaining said therapeutic or prophylactic effect at least 360 days, preferably at least 365 days, preferably at least 12 months, preferably at least 1 year. This means that the single dose, as used herein, has a duration of immunity (also referred as DOI) of at least 360 days, preferably at least 365 days, preferably at least 12 months, preferably at least 1 year. After the duration of immunity has elapsed, a further single dose herein referred as annual booster can be administered. Thus, in a particular embodiment of the immunogenic or vaccine composition of the invention, the single dose is administered once a year, preferably as an annual booster. Thus, in a particular embodiment of the medical use of the immunogenic or vaccine composition of the invention, the single dose is administered once a year as an annual booster. The single dose as annual booster is also capable of inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2) and inducing heterologous protection against rabbit hemorrhagic disease caused by rabbit hemorrhagic disease virus 1 (RHDV-1). In this way, the subject is protected over the years against rabbit hemorrhagic disease outbreaks.

[0116] The term “booster”, as used herein, refers to an extra administration of the immunogenic or vaccine composition of the invention which is given after an earlier dose known as primary vaccination or primer dose. After initial immunization, a booster dose provides a re-exposure to the immunizing antigen. It is intended to increase immunity against that antigen back to protective levels after memory against that antigen has declined through time.

[0117] Booster doses are administered when protection provided by the primary dose has begun to decrease over the time. In a particular embodiment, the immunogenic or vaccine composition of the invention is given once a year as an annual booster.

[0118] The meaning of a booster dose is different from the meaning of an additional dose. Additional dose of a vaccine may be needed as part of an extended primary vaccination for target populations where the immune response rate following the standard primary series is deemed insufficient to achieve an immune response and clinical protection.

[0119] The objective of a booster dose is to restore vaccine effectiveness from that deemed no longer sufficient.

[0120] The need for a booster dose following a primary vaccination is evaluated in several ways. One way is to measure the level of antibodies specific against a disease over the time after the primary dose is given. Anamnestic response, the rapid production of antibodies after a stimulus of an antigen, is a typical way to measure the need for a booster dose of an immunogenic or vaccine composition. If the anamnestic response is high after receiving a primary vaccine over the time, there is most likely little to no need for a booster dose. The skilled person can also measure the active B and T cell activity against the antigen after a certain amount of time that the primary vaccine was administered or determine the prevalence of the disease in vaccinated populations. Not only does the composition of the invention provide protection against rabbit hemorrhagic disease (RHD) from a single dose but said protection is effective early on after vaccination, in particular 7 days post administration.

[0121] The protection against rabbit hemorrhagic disease (RHD) conferred by the composition of the invention is a long-term protection, since the duration of the immunity is of at least 360 days, preferably 365 days, this means a duration of the immunity (also referred as DOI) of 1 year. The term “duration of immunity”, as used herein, refers to the time point at which vaccine- induced immunity begins to decline and provides less protection against a disease or a disease agent. The duration of immunity can be determined equally by HIA, as exemplified in the Examples 5 and 6 of the present description. In the present context, the disease agent is the rabbit hemorrhagic disease virus. In a particular embodiment of the medical use of the invention the protection against RHD is conferred for at least one year after administration of a single dose. In another particular embodiment of the medical use of the invention the protection against RHD is conferred for at least one year after administration of a single dose, wherein RHD is caused by infection with a rabbit hemorrhagic disease virus 1 (RHDV-1 ) variant strain and / or a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain. After the duration of immunity has elapsed, a further single dose herein referred as annual booster can be administered. Thus, in a particular embodiment of the immunogenic or vaccine composition of the invention, the single dose is administered once a year, preferably as an annual booster. In another particular embodiment of the medical use of the immunogenic or vaccine composition of the invention, the single dose is administered once a year as an annual booster.

[0122] In a particular embodiment of the medical use of the invention, the composition is administrated at a dose of at least about 210HU per dose, at least about 211HU per dose, at least about 212HU per dose, at least about 213HU per dose, at least about 214HU per dose, at least about 215HU per dose, at least about 216HU per dose of recombinant VP60 protein of RHDV-2 variant strain, at least about 218HU per dose. In another particular embodiment of the medical use of the invention, the composition is administrated at a dose of between about 28HU to about 218hemagglutination units (HU) per dose, preferably between about 29HU to about 218HU per dose, preferably between about 210HU to about 218HU per dose, more preferably between about 211HU to about 218HU per dose, even more preferably between about 212HU to about 218HU per dose of recombinant VP60 protein of a RHDV-2 variant strain.

[0123] A surprising advantage of the composition of the invention is the fact that its efficacy in providing protection against RHD in a subject was not affected by the presence of circulating antibodies against rabbit hemorrhagic disease virus 2 (RHDV-2) and / or against rabbit hemorrhagic disease virus 1 (RHDV-1) in said subject. In a particular embodiment of the medical use of the invention, the subject has circulating anti-RHDV-1 and / or anti RHDV-2 antibodies. In a particular embodiment of the medical use of the invention, the subject has circulating anti-RHDV-1 and / or anti RHDV-2 antibodies at the time of administration of the immunogenic or vaccine composition of the invention.

[0124] The term “circulating antibodies”, as used herein, refers to antibodies previously present in the subject as a response to having suffered rabbit hemorrhagic disease (RHD) by having developed either their own active immune response or by acquired (passive) immune response of maternal origin, i.e., maternally derived antibodies also known as MDAs. The term “maternally derived antibodies”, or its acronym “MDAs”, as used herein, refers to antibodies which are inherited by the litter of a mother rabbit and derived from exposure of said mother to natural infections or to previous inoculation with immunogenic or vaccine compositions. The MDAs derived protection is known to usually last up to at least 30 days of life. Despite conferring immediate protection, MDAs are also known for causing interference, by unclear mechanisms, with the development of active immunity against the specific infection or disease agent of which the MDAs provide protection. In the present case the specific disease agent is rabbit hemorrhagic disease virus 1 (RHDV-1 ) and / or rabbit hemorrhagic disease virus 2 (RHDV-2). In a particular embodiment of the medical use of the invention, the circulating anti- RHDV-1 and / or anti RHDV-2 antibodies are maternally derived antibodies (MDAs).

[0125] In a particular embodiment of the medical use of the invention, the composition further comprises an acceptable pharmaceutical adjuvant, more particularly, an acceptable pharmaceutical adjuvant selected from a group consisting of: mineral oil, ginseng, chitosan, dimethylaminoethyl (DEAE), aluminum hydroxide, phosphate buffer and any combination thereof, even more particularly mineral oil. In a particular embodiment of the medical use of the invention, the composition comprises an acceptable pharmaceutical adjuvant which is light mineral oil, preferably is liquid paraffin. In a particular embodiment of the medical use of the invention, the pharmaceutical adjuvant is at a concentration of between about 3% w / v to about 30% w / v, In another particular embodiment of the medical use of the invention the concentration of the acceptable pharmaceutical adjuvant is at a concentration of about 4% w / v, about 5% w / v, about 6% w / v, about 7% w / v, about 8% w / v, about 9% w / v, about 10% w / v, about 11% w / v, about 12% w / v, about 13% w / v, about 14% w / v, about 15% w / v, about 16% w / v, about 17% w / v, about 18% w / v, about 19% w / v, about 20% w / v, about 21% w / v, about 22% w / v, about 23% w / v, about 24% w / v, about 25% w / v, about 26% w / v, about 27% w / v, about 28% w / v, about 29% w / v, about 30% w / v. In a more particular embodiment of the medical use of the invention, the acceptable pharmaceutical adjuvant is at a concentration of about 20% w / v. In another particular embodiment of the medical use of the invention, the composition comprises the acceptable pharmaceutical adjuvant at a concentration of between about 50 g / L to about 500 g / L, between about 100 g / L to about 400 g / L, between about 150 g / L to about 300 g / L, between about 200 g / L to about 300 g / L.

[0126] In a particular embodiment of the medical use of the invention, the composition is obtained by a method comprising: a) culturing a yeast host cell comprising a nucleotide sequence encoding a VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain to obtain the recombinant VP60 protein; b) separating the recombinant VP60 protein of a RHDV-2 variant strain from the yeast host cell non-soluble components; and c) obtaining the composition comprising the recombinant VP60 protein of RHDV-2 variant strain and the yeast host cell soluble components, wherein the method preferably does not comprise a step of isolating the VP60 protein of a RHDV-2 variant strain from the composition obtained in step c).

[0127] Devices and methods of the invention

[0128] All of the definitions and embodiments described in relation to previous aspects of the present invention are equally valid and applicable for the following aspects.

[0129] A further aspect of the present invention relates to pre-filled vaccine delivery device comprising the composition of the invention.

[0130] The term “vaccine delivery device”, as used herein, refers to any instrument which is pharmaceutically suitable for containing the composition of the invention and administering it to a subject. Examples of such devices, without limitation, are sprays, drop containers and syringes, either manual or automatic syringes. In a particular embodiment of the pre-filled vaccine delivery device, the device is a syringe. In another particular embodiment of the pre-filled vaccine delivery device, the device is configured to administer the composition of the invention by parenteral, oral, mucosal or intranasal route. In a particular embodiment of the pre-filled vaccine delivery device, the device is configured to administer the composition of the invention by parenteral route, more particularly by subcutaneous, intradermal or intramuscular route. In another particular embodiment of the pre-filled vaccine delivery device, the device is a syringe and is configured to administer the vaccine composition of the invention subcutaneously.

[0131] In a particular embodiment of the pre-filled vaccine delivery device, the device contains one or more doses of the composition of the invention, wherein each dose comprises at least about 210HU, at least about 211HU, at least about 212HU, at least about 213HU, at least about 214HU, at least about 215HU, at least about 216HU of recombinant VP60 protein of RHDV-2 variant strain. In another particular embodiment of the pre-filled vaccine delivery device, the device contains a single dose of the composition of the invention, wherein the dose comprises at least 212HU of recombinant VP60 protein of RHDV-2. In another particular embodiment of the pre-filled vaccine delivery device, the device contains one or more doses of the composition of the invention, wherein each dose comprises between about 28HU to about 218HU, preferably between about 29HU to about 218HU, even more preferably between about 210HU to about 218HU, more preferably between about 211HU to about 218HU, even more preferably between about 212HU to about 218HU of recombinant VP60 protein of RHDV-2 variant strain.

[0132] In a particular embodiment of the pre-filled vaccine delivery device, the device contains a volume of the composition of the invention, ranging between about 0.10 ml to about 10 ml. Preferably, said device contains a volume of the composition of the invention ranging between 0.20 ml to about 5.0 ml. More preferably, the said device contains a volume of the composition of the invention ranging between about 0.20 ml to about 1.0 ml. Even more preferably, the said device contains a volume of the composition of the invention of about 0.50 ml.

[0133] Another aspect of the present invention relates to a method for the production of the immunogenic or vaccine composition of the invention, from here onwards the method of the invention, comprising: a) culturing a yeast host cell comprising a nucleotide sequence encoding a VP60 protein of a rabbit hemorrhagic disease virus (RHDV-2) variant strain to obtain the recombinant VP60 protein; b) separating the recombinant VP60 protein of a RHDV-2 variant strain from the yeast host cell non-soluble components; and c) obtaining the composition comprising the recombinant VP60 protein of a RHDV- 2 variant strain and the yeast host cell soluble components and discarding the yeast host cell non-soluble components.

[0134] In a particular embodiment of the method of the invention, the method does not comprise a step of isolating the recombinant VP60 protein of a RHDV-2 variant strain from the composition obtained in step c).

[0135] In a particular embodiment of the method of the invention, the method further comprises a centrifugation step, where the supernatant comprises the recombinant VP60 protein of a RHDV-2 variant strain and yeast host cell soluble components, whereas the pellet comprises yeast host cell non-soluble components and debris, which is discarded.

[0136] In a particular embodiment of the method of the invention the method comprises: a) culturing a yeast host cell comprising a nucleotide sequence encoding a VP60 protein of a rabbit hemorrhagic disease virus (RHDV-2) variant strain to obtain the recombinant VP60 protein; b) separating the recombinant VP60 protein of a RHDV-2 variant strain from the yeast host cell non-soluble components; and c) obtaining the composition comprising the recombinant VP60 protein of a RHDV- 2 variant strain and the yeast host cell soluble components and discarding the yeast host cell non-soluble components, wherein the composition obtained in step c) comprises virus-like particles assembled of the recombinant VP60 protein of a RHDV-2 variant strain.

[0137] In a preferred embodiment, the method of the invention comprises: a) culturing a yeast host cell comprising a nucleotide sequence encoding a VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain to obtain the recombinant VP60 protein; b) separating the recombinant VP60 protein of a RHDV-2 variant strain from the yeast host cell non-soluble components by centrifugation; and c) obtaining the composition comprising the recombinant VP60 protein of a RHDV-2 variant strain and the yeast host cell soluble components and discarding the yeast host cell non-soluble components.

[0138] In another particular embodiment of the method of the invention, the method further comprises the step of mixing the composition obtained in step c) with an acceptable pharmaceutical adjuvant.

[0139] In another particular embodiment, the vaccine composition of the invention is obtained by a method comprising: a) culturing a yeast host cell comprising a nucleotide sequence encoding a VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain to obtain the recombinant VP60 protein; b) separating the recombinant VP60 protein of a RHDV-2 variant strain from the yeast host cell non-soluble components; and c) obtaining an immunogenic composition comprising the recombinant VP60 protein of a RHDV-2 variant strain and the yeast host cell soluble components and discarding the yeast host cell non-soluble components.

[0140] In another particular embodiment of the method of the invention, the method further comprises the step of mixing the immunogenic composition obtained in step c) with an acceptable pharmaceutical adjuvant.

[0141] Thus, in a preferred embodiment, the method of the invention comprises: a) culturing a yeast host cell comprising a nucleotide sequence encoding a VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain to obtain the recombinant VP60 protein; b) separating the recombinant VP60 protein of a RHDV-2 variant strain from the yeast host cell non-soluble components by centrifugation; c) obtaining an immunogenic composition comprising the recombinant VP60 protein of a RHDV-2 variant strain and the yeast host cell soluble components and discarding the yeast host cell non-soluble components; and d) mixing the immunogenic composition obtained in step c) with an acceptable pharmaceutical adjuvant.

[0142] In yet another particular embodiment of the method of the invention, the acceptable pharmaceutical adjuvant is selected from a group consisting of: mineral oil, ginseng, chitosan, dimethylaminoethyl (DEAE), aluminum hydroxide, phosphate buffer and any combination thereof. In an embodiment of the method of the invention, the acceptable pharmaceutical adjuvant is light mineral oil, preferably is liquid paraffin.

[0143] In a more particular embodiment of the method of the invention, the acceptable pharmaceutical adjuvant is at a concentration of between about 3% w / v to about 30% w / v, preferably 20% w / v. In another particular embodiment of the method of the invention the concentration of the acceptable pharmaceutical adjuvant is at a concentration of about 4% w / v, about 5% w / v, about 6% w / v, about 7% w / v, about 8% w / v, about 9% w / v, about 10% w / v, about 11% w / v, about 12% w / v, about 13% w / v, about 14% w / v, about 15% w / v, about 16% w / v, about 17% w / v, about 18% w / v, about 19% w / v, about 20% w / v, about 21% w / v, about 22% w / v, about 23% w / v, about 24% w / v, about 25% w / v, about 26% w / v, about 27% w / v, about 28% w / v, about 29% w / v, about 30% w / v. In a more particular embodiment of the method of the invention, the acceptable pharmaceutical adjuvant is at a concentration of about 20% w / v. In another particular embodiment of the method of the invention, the acceptable pharmaceutical adjuvant is at a concentration of between about 50 g / L to about 500 g / L, between about 100 g / L to about 400 g / L, between about 150 g / L to about 300 g / L, between about 200 g / L to about 300 g / L.

[0144] In a further particular embodiment of the method of the invention, the acceptable pharmaceutical adjuvant is mineral oil, preferably liquid paraffin.

[0145] In one more particular embodiment of the method of the invention, the yeast host cell belongs to the species Komagataella phaffi (formerly Pichia pastoris).

[0146] In another particular embodiment of the method of the invention, the recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) comprises the sequence according to SEQ ID NO: 1 or a functionally equivalent variant.

[0147] Additional aspects of the invention

[0148] The present invention is further characterized by the following aspects:

[0149] 1 . An immunogenic or vaccine composition comprising a recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain, wherein the composition is capable of a) inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2) and b) inducing heterologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 1 (RHDV-1), wherein the immunogenic or vaccine composition is devoid of immunogens derived from RHDV-1 variant strains and wherein said protection is induced after the administration of a single dose of the composition. A single dose immunogenic or vaccine composition comprising a recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain, wherein the composition is capable of a) inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2) and b) inducing heterologous protection against RHD caused by rabbit hemorrhagic disease virus 1 (RHDV-1), wherein the composition is devoid of immunogens derived from RHDV-1 variant strains. The immunogenic or vaccine composition according to aspect 1 or 2, wherein the recombinant VP60 protein comprises the sequence according to SEQ ID NO: 1 or a functionally equivalent variant thereof. The immunogenic or vaccine composition according to aspect 3, wherein the functionally equivalent variant of the recombinant VP60 protein has a degree of sequence identity with respect to SEQ ID NO: 1 greater than at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 99% sequence identity. The immunogenic or vaccine composition according to any one of aspects 1 to 4, wherein the immunogenic or vaccine composition comprises between about 210hemagglutination units (HU) to about 218HU per dose of recombinant VP60 protein of RHDV-2 variant strain. The immunogenic or vaccine composition according to aspect 5 wherein the immunogenic or vaccine composition comprises at least about 211HU per dose, at least about 212HU per dose, at least about 213HU per dose, at least about 214HU per dose, at least about 215HU per dose, at least about 216HU per dose of recombinant VP60 protein of RHDV-2 variant strain. The immunogenic or vaccine composition according to aspect 5 wherein the immunogenic or vaccine composition comprises between about 211HU to about 218HU per dose, even more preferably between about 212HU to about 218HU per dose of recombinant VP60 protein of RHDV-2 variant strain. The immunogenic or vaccine composition according to any one of the aspects 1 to 7 further comprising at least one additional antigen of other pathogens causing disease in rabbits. The immunogenic or vaccine composition according to aspect 8, wherein the at least one additional antigen is selected from a group of microorganisms consisting of: myxoma virus, shope fibroma virus (SFV), Pasteurella multocida, Staphylococcus aureus, E. coll, Salmonella enterica, Salmonella typhimurium, Clostridium spiroforme, Clostridium perfringens, Clostridium cuniculi, Eimeria sp., Encephalitozoon cuniculi and Trichophyton mentagrophytes. The immunogenic or vaccine composition according to any one of aspects 1 to 9, wherein the composition is formulated to be administered by subcutaneous, intradermal, or intramuscular route, preferably by subcutaneous route. The immunogenic or vaccine composition according to any one of aspects 1 to 10, further comprising virus-like particles assembled of the recombinant VP60 protein of a RHDV-2 variant strain. The immunogenic or vaccine composition according to any one of aspects 1 to 11 , wherein the composition is capable of inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2) and heterologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 1 (RHDV-1 ), wherein the RHDV-2 is a highly virulent RHDV- 2 variant strain. The immunogenic or vaccine composition according to any one of aspects 1 to 12, wherein the composition is capable of inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2), wherein the RHDV-2 is a highly virulent RHDV-2 variant strain which causes a mortality of at least 70%. The immunogenic or vaccine composition according to any one of aspects 1 to 13, further comprising an acceptable pharmaceutical adjuvant. The immunogenic or vaccine composition according to aspect 14, wherein the acceptable pharmaceutical adjuvant is selected from a group consisting of: mineral oil, ginseng, chitosan, dimethylaminoethyl (DEAE), aluminum hydroxide, phosphate buffer and any combination thereof. The immunogenic or vaccine composition according to aspect 15, wherein the acceptable pharmaceutical adjuvant is light mineral oil, preferably is liquid paraffin. The immunogenic or vaccine composition according to any one of aspects 14 to 16, wherein the acceptable pharmaceutical adjuvant is at a concentration of between about 3% w / v to about 30% w / v, preferably 20% w / v. The immunogenic or vaccine composition according to any one of aspects 14 to 17, wherein the acceptable pharmaceutical adjuvant is at a concentration of between about 50 g / L to about 500 g / L, preferably between about 100 g / L to about 400 g / L, more preferably between about 150 g / L to about 300 g / L, even more preferably between about 200 g / L to about 300 g / L. The immunogenic or vaccine composition according to any one of aspects 1 to 18, wherein the immunogenic or vaccine composition is formulated to be administrated in a volume of about 0.10 ml per dose, about 0.20 ml per dose, about 0.30 ml per dose, about 0.35 ml per dose, about 0.40 ml per dose, about 0.45 ml per dose, about 0.50 ml per dose, about 0.55 ml per dose, about 0.60 ml per dose, about 0.65 ml per dose, about 0.70 ml per dose, about 0.75 ml per dose, about 0.80 ml per dose, about 0.85 ml per dose, about 0.90 ml per dose, about 0.95 ml per dose. The immunogenic or vaccine composition according to any one of aspects 1 to 19, wherein the composition is obtained by a method comprising: a) culturing a yeast host cell comprising a nucleotide sequence encoding a VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain to obtain the recombinant VP60 protein; b) separating the recombinant VP60 protein of a RHDV-2 variant strain from the yeast host cell non-soluble components, and c) obtaining the composition comprising the recombinant VP60 protein of a RHDV-2 variant strain and the yeast host cell soluble components and discarding the yeast host cell non-soluble components.

[0150] 21 . The immunogenic or vaccine composition according to aspect 20, wherein the yeast host cell belongs to the species Komagataella phaffii.

[0151] 22. The immunogenic or vaccine composition according to aspect 20 or aspect 21 , wherein step b) is carried out by centrifugation.

[0152] 23. The immunogenic or vaccine composition according to aspect 22, wherein after the centrifugation, the supernatant comprises the recombinant VP60 protein of a RHDV-2 variant strain and yeast host cell soluble components, whereas the pellet comprises yeast host cell non-soluble components and debris, which is discarded.

[0153] 24. The immunogenic or vaccine composition according to any one of aspects 20 to 23, wherein the method does not comprise a step of isolating the recombinant VP60 protein of a RHDV-2 variant strain from the immunogenic composition obtained in step c).

[0154] 25. The immunogenic or vaccine composition according to any one of aspects 20 to 24, wherein step c) further comprises a step of sterilization of the composition comprising the recombinant VP60 protein of a RHDV-2 variant strain and the yeast host cell soluble components, preferably wherein said sterilization is carried out by filtration through a 0.22 pm filter.

[0155] 26. The immunogenic or vaccine composition as defined in any one of aspects 1 to 25 for use as a medicament.

[0156] 27. The immunogenic or vaccine composition as defined in any one of aspects 1 to 25 for use in the treatment and / or prophylaxis of rabbit hemorrhagic disease (RHD) and associated diseases in a subject.

[0157] 28. The immunogenic or vaccine composition as defined in any one of aspects 1 to 25 for use in a method for inducing an immune response against rabbit hemorrhagic disease virus in a subject. 29. The immunogenic or vaccine composition for use according to aspect 28, in a method for inducing a homologous immune response against rabbit hemorrhagic disease virus 2 (RHDV-2).

[0158] 30. The immunogenic or vaccine composition for use according to aspect 28 or 29, in a method for inducing a heterologous immune response against rabbit hemorrhagic disease virus 1 (RHDV-1).

[0159] 31 . The immunogenic or vaccine composition for use according to any one of aspects 28 to 30 in a method for inducing a homologous immune response against RHDV-2 and a heterologous immune response against RHDV-1.

[0160] 32. The immunogenic or vaccine composition for use according to any one of aspects 28 to 31 , wherein the immune response confers protection against RHD in the subject.

[0161] 33. The immunogenic or vaccine composition for use according to aspect 32, wherein the RHD is caused by infection with a RHDV-1 variant strain and / or a RHDV-2 variant strain.

[0162] 34. The immunogenic or vaccine composition for use according to claim 33 wherein the RHDV-2 variant strain is a highly virulent RHDV-2 variant.

[0163] 35. The immunogenic or vaccine composition for use according to any one of aspects 32 to 34, wherein the protection against RHD is measured as survival rate or mortality rate of naturally or experimentally infected subjects.

[0164] 36. The immunogenic or vaccine composition for use according to aspect 35, wherein the survival rate of subjects infected with RHDV-1 is of at least about 60%, at least about 70%, at least about 80%, at least about 90%, about 100% of naturally or experimentally infected subjects.

[0165] 37. The immunogenic or vaccine composition for use according to aspect 35 or 36, wherein the survival rate of subjects infected with RHDV-2 is of at least about 60%, at least about 70%, at least about 80%, at least about 90%, about 100% of naturally or experimentally infected subjects.

[0166] 38. The immunogenic or vaccine composition for use according to any one of aspects 35 to 37, wherein the survival rate of subjects infected with RHDV-1 and RHDV-2 is of at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100% of naturally or experimentally infected subjects.

[0167] 39. The immunogenic or vaccine composition for use according to aspect 35, wherein the mortality rate of subjects infected with RHDV-1 is of less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1 %, about 0% of naturally or experimentally infected subjects.

[0168] 40. The immunogenic or vaccine composition for use according to aspect 35 or 39, wherein the mortality rate of subjects infected with RHDV-2 is of less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1 %, about 0% of naturally or experimentally infected subjects.

[0169] 41 . The immunogenic or vaccine composition for use according to any one of aspects 35, 39 or 40, wherein the mortality rate of subjects infected with RHDV-1 and RHDV-2 is of less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 96%, less than about 3%, less than about 2%, less than about 1%, about 0% of naturally or experimentally infected subjects.

[0170] 42. The immunogenic or vaccine composition for use according to any one of aspects 27 to

[0171] 41 , wherein the immunogenic or vaccine composition is administered by subcutaneous, intradermal, or intramuscular route, preferably by subcutaneous route.

[0172] 43. The immunogenic or vaccine composition for use according to any one of aspects 27 to

[0173] 42, wherein the immunogenic or vaccine composition is administered in a single dose.

[0174] 44. The immunogenic or vaccine composition or the vaccine for use according to any one of aspects 27 to 43 wherein the immunogenic or vaccine composition is administrated at a dose of between about 210hemagglutination units (HU) to about 218HU per dose of recombinant VP60 protein of RHDV-2 variant strain.

[0175] 45. The immunogenic or vaccine composition for use according to aspect 44 wherein the immunogenic or vaccine composition is administrated at a dose of at least about 211HU per dose, at least about 212HU per dose, at least about 213HU per dose, at least about 214HU per dose, at least about 215HU per dose, at least about 216HU per dose of recombinant VP60 protein of RHDV-2 variant strain.

[0176] 46. The immunogenic or vaccine composition for use according to aspect 44 or 45 wherein the immunogenic or vaccine composition is administrated at a dose of between about 211HU to about 218HU per dose, even more preferably between about 212HU to about 218HU per dose of recombinant VP60 protein of RHDV-2 variant strain.

[0177] 47. The immunogenic or vaccine composition for use according to any one of aspects 27 to 46 wherein the immunogenic or vaccine composition is administrated in a volume ranging between about 0.20 ml to about 1 .0 ml per dose.

[0178] 48. The immunogenic or vaccine composition for use according to aspect 47 wherein the immunogenic or vaccine composition is administrated in a volume of about 0.30 ml per dose, about 0.35 ml per dose, about 0.40 ml per dose, about 0.45 ml per dose, about 0.50 ml per dose, about 0.55 ml per dose, about 0.60 ml per dose, about 0.65 ml per dose, about 0.70 ml per dose, about 0.75 ml per dose, about 0.80 ml per dose, about 0.85 ml per dose, about 0.90 ml per dose, about 0.95 ml per dose.

[0179] 49. The immunogenic or vaccine composition for use according to aspect 47 or 48 wherein the immunogenic or vaccine composition is administrated in a volume of about 0.50 ml per dose.

[0180] 50. The immunogenic or vaccine composition for use according to any one of aspects 27 to 49, wherein the subject is a mammal from the Lagomorpha order, preferably from the Leporidae family.

[0181] 51. The immunogenic or vaccine composition for use according to aspect 50, wherein the subject is a rabbit or hare from the genus Lepus, Sylvilagus or Oryctolagus, preferably from the Oryctolagus cuniculus species.

[0182] 52. The immunogenic or vaccine composition for use according to any one of aspects 27 to 51 , wherein the subject is at least 1 days of age, at least 5 days of age, at least 10 days of age, at least 15 days of age, at least 20 days of age, at least 25 days of age or at least 30 days of age, preferably the subjects is of at least 30 days of age, preferably the subject is of 30 days of age of more. 53. The immunogenic or vaccine composition for use according to any one of aspects 27 to 52, wherein the subject has circulating anti-RHDV-1 and / or anti-RHDV-2 antibodies.

[0183] 54. The immunogenic or vaccine composition for use according to aspect 53, wherein the circulating anti-RHDV-1 and / or anti-RHDV-2 antibodies are maternally-derived antibodies (MDAs).

[0184] 55. The immunogenic or vaccine composition for use according to any one of aspects 27 to 54, wherein the treatment and / or prophylaxis of RHD or the homologous immune response against RHDV-2 and the heterologous immune response against RHDV-1 or the protection against RHD is conferred for at least one year after administration of a single dose.

[0185] 56. The immunogenic or vaccine composition for use according to aspect 55, wherein the RHDV-2 variant strain is a highly virulent RHDV-2 variant strain.

[0186] 57. The immunogenic or vaccine composition for use according to any one of aspects 26 to

[0187] 56, wherein a single dose is administered once a year as annual booster.

[0188] 58. The immunogenic or vaccine composition for use according to any one of aspects 26 to

[0189] 57, wherein the treatment and / or prophylaxis of RHD or the homologous immune response against RHDV-2 and the heterologous immune response against RHDV-1 or the protection against RHD is conferred by virus-like particles assembled of the recombinant VP60 protein of a RHDV-2 variant strain.

[0190] 59. The immunogenic or vaccine composition for use according to aspect 58, wherein the RHDV-2 variant strain is a highly virulent RHDV-2 variant strain.

[0191] 60. A pre-filled vaccine delivery device comprising an immunogenic or vaccine composition as defined in any one of aspects 1 to 25.

[0192] 61. The pre-filled vaccine delivery device according to aspect 60, wherein the device contains one or more doses of the immunogenic or vaccine composition, wherein each dose comprises at least about 210HU, at least about 211HU, at least about 212HU, at least about 213HU, at least about 214HU, at least about 215HU, at least about 216HU of recombinant VP60 protein of RHDV-2 variant strain.

[0193] 62. The pre-filled vaccine delivery device according to aspect 60 or 61 , wherein the device contains one or more doses of the immunogenic or vaccine composition, wherein each dose comprises between 28HU to about 218HU, between about 29HU to about 218HU, between about 210HU to about 218HU, between about 211HU to about 218HU, or between about 212HU to about 218HU of recombinant VP60 protein of RHDV-2 variant strain.

[0194] 63. The pre-filled vaccine delivery device according to any one of aspects 60 to 62, wherein the device is a syringe and is configured to administer the immunogenic or vaccine composition subcutaneously.

[0195] 64. A method for the production of the immunogenic or vaccine composition as defined in any one of aspects 1 to 25, comprising: a) culturing a yeast host cell comprising a nucleotide sequence encoding a VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain to obtain the recombinant VP60 protein; b) separating the recombinant VP60 protein of a RHDV-2 variant strain from the yeast host yeast cell non-soluble components; and c) obtaining the composition comprising the recombinant VP60 protein of a RHDV-2 variant strain and the yeast host cell soluble components and discarding the yeast host cell non-soluble components.

[0196] 65. The method according to aspect 64, wherein the method does not comprise a step of isolating the recombinant VP60 protein of a RHDV-2 variant strain from the composition obtained in step c).

[0197] 66. The method according to aspect 64 or 65, further comprising the step of mixing the composition obtained in step c) with an acceptable pharmaceutical adjuvant.

[0198] 67. The method according to aspect 66, wherein the acceptable pharmaceutical adjuvant is selected from a group consisting of: mineral oil, ginseng, chitosan, dimethylaminoethyl (DEAE), aluminum hydroxide, phosphate buffer and any combination thereof.

[0199] 68. The method according to aspect 67, wherein the acceptable pharmaceutical adjuvant is light mineral oil, preferably is liquid paraffin.

[0200] 69. The method according to any one of aspects 66 to 68, wherein the acceptable pharmaceutical adjuvant is at a concentration of between about 3% w / v to about 30% w / v, preferably 20% w / v. 70. The method according to any one of aspects 64 to 69, wherein the yeast host cell belongs to the species Komagataella phaffi.

[0201] 71. The method according to any one of aspects 64 to 70, wherein the recombinant VP60 protein of a RHDV-2 variant strain comprises the sequence according to SEQ ID NO: 1 or a functionally equivalent variant.

[0202] 72. The method according to any one of aspect 64 to 71 , wherein step b) is carried out by centrifugation.

[0203] 73. The method according to aspect 72, wherein after the centrifugation, the supernatant comprises the recombinant VP60 protein and yeast host cell soluble components, whereas the pellet comprises yeast host cell non-soluble components and debris, which is discarded.

[0204] 74. The method according to any one of aspects 64 to 73, wherein step c) further comprises a step of sterilization of the composition comprising the recombinant VP60 protein of a RHDV-2 variant strain and the yeast host cell soluble components, preferably wherein said sterilization is carried out by filtration through a 0.22 pm filter.

[0205] 75. The method according to any one of aspects 64 to 74, wherein the composition of step c) comprises virus-like particles assembled of the recombinant VP60 protein of a RHDV- 2 variant strain.

[0206] ***

[0207] The invention is described by way of the following examples which are to be considered as merely illustrative and not limitative of the scope of the invention.

[0208] EXAMPLES

[0209] EXAMPLE 1 : Expression and recombinant production of a heterologous protein of a viral capsid protein (VP60) from rabbit hemorrhagic disease virus 2 (RHDV-2) in Komagataella phaffi.

[0210] The VP60 capsid protein of RHDV-2 used in the following examples was produced by recombinant DNA technology as follows:

[0211] The polypeptide sequence corresponding to the viral capsid protein (VP60) of SEQ ID NO: 1 from rabbit hemorrhagic disease virus 2 (RHDV-2) was used as a template. First, a codon optimized nucleotide sequence for expression of said VP60 capsid protein in Komagataella phaffi (K. phaffi, formerly known as Pichia pastoris) was generated using the OptimumGene™ algorithm (GenScript Biotech Corp., USA). The codon optimization analysis was done by well-known bioinformatic tools such as GenSmart™ from GenScript available at genscript.com / tools / gensmart-codon-optimization GenScript, which allowed to adapt the heterologous gene coding for the VP60 capsid protein to the host organism K. phaffi codon usage. The resulting codon-optimized nucleotide sequence corresponds to SEQ ID NO: 2. The said sequence was then cloned into an intracellular expression plasmid (pPHICEiB, 4040 base pairs, provided by GenScript) containing, hence, the synthetized nucleotide sequence (SEQ ID N0:2) coding for the VP60 capsid protein of the variant of rabbit hemorrhagic disease virus 2 (RHDV-2) comprising 579 amino acid residues (SEQ ID NO: 1 ) under the control of the constitutive promoter of GAP (glyceraldehyde-3-phosphate dehydrogenase) (pGAP). The insertion of the synthetic VP60 capsid protein encoding gene into the plasmid pPHICEiB was performed by standard restriction enzymes using Basl restriction sites added at the 5’ and 3’ extremity of the synthetic VP60 capsid protein encoding gene and used for the subcloning of the coding gene. A0X1 terminator was included into the plasmid as transcription terminator signal. A pUC Ori was used as origin of replication of the plasmid. A Zeocin resistance cassette was included in the plasmid in order to select further the transformed cells. After cloning, the presence and orientation of the whole insert in the expression vector were confirmed by single strand sequencing and restriction mapping according to standard techniques.

[0212] The recombinant VP60 protein was produced in K. phaffi strain BG10 (commercially available from ATUM). In order to obtain the transformed K. phaffi BG10 strain, electrocompetent cells were transformed with the cloned expression plasmid following a standard yeast electroporation protocol, namely, by first mixing the competent K. phaffi cells with 5 pg of the linearized plasmid in purified water and incubated for 5 minutes at 4°C. Then, a single electric pulse was performed at 2000V in an electroporation 0.2 cm cuvette. Once the electric pulse was emitted, the cells were recovered adding ice-cold 1M sorbitol. Transformed cells incorporating the linearized plasmid were selected in plates containing 100 pg / ml of Zeocin and incubated at 30°C for 72 hours. As the mutant K. phaffi was resistant to Zeocin, the transformed cells incorporating the linearized plasmid grew in this selective media.

[0213] K. phaffi transformants successfully incorporated the expression plasmid in their genome and were able to express the nucleotide sequence coding for the recombinant VP60 capsid protein of RHDV-2 in amounts that could be detected by standard Western blot and hemagglutination assay (HA).

[0214] Finally, K. phaffi transformants were cultivated in BMDY medium (Buffered Dextrose- complex Medium) containing 1% yeast extract, 2% peptone, 1.34% Yeast Nitrogen Base (YNB), 0.002% biotin, 1% dextrose, and 100 mM sodium phosphate at 30°C and pH 6 for 24 hours under agitation (240 rpm) to obtain the VP60 capsid protein expressed as intracellular recombinant soluble protein (of about 60.2 kDa). The final culture was washed twice with PBS and centrifuged to obtain the harvested transformed cells and discard the PBS (supernatant). Then, the harvested cells were adjusted by percentage of biomass with PBS prior to the final disruption of K. phaffi transformed cells. In order to release the intracellular content of the transformed cell culture, the adjusted-biomass was disrupted by high-pressure homogenization at 1500 bar by a homogenizer. Then, the disrupted culture was clarified by centrifugation at 10000 g during 30 minutes at 4°C. The supernatant containing the recombinant VP60 protein and yeast cell soluble components were selected while the sediment containing yeast cell non-soluble components was discarded. Finally, the supernatant was sterilized by filtration through a 0.22 pm sterilizing filter (Millex-GP) and stored at -20°C for further use.

[0215] The recombinant VP60 viral capsid protein sequence from RHDV-2 obtained following Example 1 corresponds to SEQ ID NO: 1.

[0216] EXAMPLE 2: Efficacy study of vaccine compositions comprising recombinant VP60 protein of RHDV-2 against rabbit hemorrhagic disease virus 2 (RHDV-2) (homologous protection).

[0217] The primary aim of this study was to assess whether administration of vaccine compositions comprising the VP60 capsid protein as obtained in Example 1 induced a serological immune response against rabbit hemorrhagic disease virus 2 (RHDV-2) and whether this immune response was protective against a homologous challenge after a single dose administration.

[0218] A total of 80 New Zealand white rabbits (Oryctolagus cuniculus) of 4 weeks of age were included in the study. All rabbits were free (seronegative) for rabbit hemorrhagic disease virus antibodies. Rabbits were randomly allocated into three different treatment groups which received a different vaccine composition comprising recombinant VP60 protein of RHDV-2 as obtained in Example 1 and one strict control group as detailed below.

[0219] On Day 0 of the study (DO), animals were vaccinated with a single dose of 0.5 ml of one of the following vaccine compositions formulated to be administered by subcutaneous (SC) route (Table 1).

[0220] Table 1. Summary of vaccination and challenge study

[0221] - Group 1 (G1): animals were vaccinated with 0.5 ml of a vaccine composition comprising 215-86HU (hemagglutination units) of recombinant VP60 protein of RHDV-2 formulated with 20% (w / v) mineral oil (liquid paraffin).

[0222] - Group 2 (G2): animals were vaccinated with 0.5 ml of a vaccine composition comprising 215-86HU (hemagglutination units) of recombinant VP60 protein of RHDV-2 formulated with 10% (w / v) mineral oil (liquid paraffin).

[0223] - Group 3 (G3): animals were vaccinated with 0.5 ml of a vaccine composition comprising 215-86HU (hemagglutination units) of recombinant VP60 protein of RHDV-2 formulated with 3.7% (w / v) mineral oil (liquid paraffin).

[0224] - Group 4 (G4 or Control Group): animals were vaccinated with 0.5 ml of a mock-vaccine comprising PBS (phosphate buffered saline) solution and served as a strict control.

[0225] The recombinant VP60 protein of RHDV-2 was produced as described in Example 1.

[0226] The different vaccine compositions were formulated by mixing the aqueous phase comprising the recombinant VP60 protein of RHDV-2 with the oily phase in a ratio of 66%:33% under agitation and performing a final homogenization under high-pressure to obtain an oil-in- water emulsion.

[0227] Blood samples were collected from the rabbits periodically throughout the vaccination period and assayed for evidence of seroconversion. Assessment of clinical signs were checked every day after vaccination and throughout the end of study. Local and general systemic clinical signs such as depression, body condition, dyspnea, nasal discharge, ocular discharge and mortality, were carefully monitored daily (i.e., two times a day). Any other abnormal clinical sign such as diarrhoea, respiratory signs, among other were also monitored and recorded.

[0228] Seven days after vaccination (Day 7) animals were experimentally infected (challenged) by intramuscular (IM) route to confirm the correlation between serological response and protection. Animals were challenged with 1 ml of a virulent RHDV-2 strain sufficient to cause signs of RHD in rabbits, particularly with a 1 ml solution of virulent RHDV-2 strain V-1037 (highly virulent field strain isolated from clinically infected animals in 2013) at a titer of 214-5HU (hemagglutination units).

[0229] To assess the serological immune response and protection induced by the different vaccine compositions tested against RHDV-2 infection, clinical signs as described above, and mortality caused by the RHDV-2 challenge were monitored on a daily basis from Day 7 to Day 12 post-challenge. To assess the serological response against RHDV-2, blood samples from all animals were extracted on Day -3 (3 days before the start of the study vaccination), Day 7 and Day 12 of the study. The serological response against RHDV-2 was measured by Hemagglutination Inhibition Assay (HIA) according to standard protocols, expressed as Iog2 HI / 50pl antibodies against RHDV-2.

[0230] Efficacy of the vaccine compositions were evaluated following these criteria:

[0231] Serology: The main evaluated parameter was serology response. Seroconversion against RHDV-2 was quantified in serum samples by HIA as described above. Serum samples were considered seropositive for antibodies against RHDV-2 when the HI value was > 3 Iog2 HI / 50jjl.

[0232] Mortality: The main evaluated parameter was mortality caused by RHDV-2 virus challenge. The vaccine composition was considered effective when a percentage equal or higher than 85% of vaccinated animals showed no signs of rabbit hemorrhagic disease (RHD) mortality.

[0233] Clinical signs: The secondary parameter to assess efficacy of the vaccine compositions tested was the presence or absence of typical clinic symptomatology of RHD caused by RHDV-2. The disease is well characterized for causing a rapid mortality so that clinical signs are usually less evident.

[0234] Results from the serological response against RHDV-2:

[0235] Prior to challenge, there was no detection of antibodies against RHDV-2 indicating a valid study. In contrast, a clear seroconversion was observed in all vaccinated groups, i.e. Group 1 to Group 3, seven days after the vaccination (Day 7) (Figure 1 ) with an increase in antibodies against RHDV-2 post-challenge. On Day 7 (D7) all vaccinated groups seroconverted and the serological response was significant different from the non-vaccinated group (Control Group) (Table 2). Moreover, there were no significant differences between the three vaccinated groups. On the contrary, no seroconversion was observed in animals of the Control Group (Group 4). Thus, the vaccine compositions tested against RHDV-2 comprising recombinant VP60 protein of RHDV-2 were able to elicit a potent antibody response against RHDV-2.

[0236] Table 2. Mean antibody levels against RHDV-2 (mean Iog2 HI / 50pl)

[0237] 1vaccination

[0238] 2challenge

[0239] Results from mortality and prevention after a challenge with a virulent RHDV-2 strain:

[0240] The results from all vaccinated groups (groups 1 to 3) showed a complete prevention of mortality caused by a RHDV-2 virulent challenge. All animals for each of the three treated groups survived to the challenge achieving a zero (0%) mortality at the end of the study (Table 3).

[0241] Thus, the presence of antibody titers in vaccinated rabbits lead to clinical protection.

[0242] In contrast, 75% of mortality was observed in the non-vaccinated group, Control Group 4 (Table 3).

[0243] Table 3. Overall mortality rate of the different groups

[0244] Group Mortality (%)

[0245] The above results clearly indicate that the three tested vaccine compositions (Group 1 to Group 3) comprising the recombinant VP60 capsid protein of RHDV-2 were effective in preventing mortality caused by an RHDV-2 infection. Furthermore, all the vaccine compositions tested conferred homologous protection against an experimental RHDV-2 infection regardless of the percentage of mineral oil included in the final formulation.

[0246] EXAMPLE 3: Efficacy study of vaccine compositions comprising recombinant VP60 protein of RHDV-2 against the classic rabbit hemorrhagic disease virus (also known as RHDV-1) (heterologous protection).

[0247] The primary aim of this study was to assess whether administration of vaccine compositions comprising the VP60 capsid protein of rabbit hemorrhagic disease virus 2 (RHDV-2) as obtained in Example 1 induced a serological immune response against classic rabbit hemorrhagic disease virus (RHDV, also known as RHDV-1 ) and whether this immune response was protective against a heterologous challenge after a single dose administration.

[0248] A total of 30 New Zealand white rabbits (Oryctolagus cuniculus) of 10 weeks of age were included in the study. All rabbits were free (seronegative) for rabbit hemorrhagic disease virus antibodies. Rabbits were randomly allocated into two treatment groups which received a different vaccine composition comprising recombinant VP60 protein of RHDV-2 as obtained in Example 1 and one strict control group as detailed below.

[0249] On Day 0 of the study, animals were vaccinated with a single dose of 0.5 ml of one of the following vaccine compositions formulated to be administered by subcutaneous (SC) route.

[0250] Table 4. Summary of vaccination and challenge study

[0251] - Group 1 (G1): animals were vaccinated with 0.5 ml of a vaccine composition comprising 21586HU (hemagglutination units) of recombinant VP60 protein of RHDV-2 formulated with 20% (w / v) of mineral oil (liquid paraffin).

[0252] - Group 2 (G2): animals were vaccinated with 0.5 ml of a vaccine composition comprising 21586HU (hemagglutination units) of recombinant VP60 protein of RHDV-2 formulated with 10% (w / v) of mineral oil (liquid paraffin).

[0253] - Group 3 (G3 or Control Group): animals were vaccinated with 0.5 ml of a mockvaccine comprising PBS (phosphate buffered saline) solution and served as a strict control.

[0254] The recombinant VP60 protein of RHDV-2 was produced as described in Example 1.

[0255] The different vaccine compositions were formulated by mixing the aqueous phase comprising the recombinant VP60 protein of RHDV-2 with the oily phase in a ratio of 66%:33% under agitation and performing a final homogenization under high-pressure to obtain an oil-in- water emulsion.

[0256] Blood samples were collected from the rabbits periodically throughout the vaccination period and assayed for evidence of seroconversion. Assessment of clinical signs were checked every day after vaccination and throughout the end of study. Local and general systemic clinical signs such as depression, body condition, dyspnea, nasal discharge, ocular discharge and mortality, were carefully monitored daily (two times a day). Any other abnormal clinical sign such as diarrhoea, respiratory signs, among other were also monitored and recorded.

[0257] Seven days after vaccination (Day 7) animals were experimentally infected (challenged) by intramuscular (IM) route to determine the correlation between serological response and protection. Animals were challenged with 1 ml solution of a virulent heterologous RHDV-1 strain (highly virulent field strain isolated from clinically infected animals in 1997) V- 4764 at a titer of 212HU (hemagglutination units).

[0258] To assess the serological immune response and protection induced by the different vaccine compositions based on recombinant VP60 protein of RHDV-2 against classic RHDV infection (RHDV-1 ) i.e. against a heterologous RHDV variant, clinical signs as described above, and mortality caused by the RHDV-1 challenge were monitored on a daily basis from Day 7 to Day 12 post-challenge.

[0259] To assess the serological response, blood samples from all animals were extracted on Day -3 (3 days before the start of the study vaccination), Day 7 and Day 12 of the study. The serological response against RHDV-1 was measured by Hemagglutination Inhibition Assay (HIA), expressed as Iog2 HI / 50pl antibodies against RHDV-1.

[0260] Efficacy of the vaccine compositions were evaluated following these criteria:

[0261] Serology: Seroconversion against both RHDV-1 and RHDV-2 was tested in serum samples by HIA as described above. The validated cut-off value to consider animals seropositive for antibodies against RHDV-2 was Iog2 HI / 50pl > 3, and Iog2 HI / 50pl > 4.5 for antibodies against RHDV-1.

[0262] Mortality: The main evaluated parameter was mortality caused by RHDV-1 virus challenge. The vaccine compositions were considered effective when a percentage equal or higher than 85% of vaccinated animals showed no signs of rabbit hemorrhagic disease (RHD) mortality.

[0263] Clinical signs: Secondary parameters to assess efficacy of the vaccine compositions tested was the presence or absence of typical clinic symptomatology of RHD caused by RHDV-1 . The disease is well characterized for causing a rapid mortality so that clinical signs are usually less evident.

[0264] Results from the serological response and protection after a challenge with a virulent RHDV-1 strain:

[0265] Prior to vaccination, there was no detection of antibodies against either RHDV-1 or RHDV-2 indicating a valid study. In contrast, a clear seroconversion was observed in all vaccinated groups, seven days after the vaccination (Day 7) (Table 5) with an increase in antibodies against RHDV-2 post-challenge. On Day 7 (D7) all vaccinated groups seroconverted and the observed serological response was significant different from the nonvaccinated group (Control Group). On the contrary, no seroconversion was observed in animals of the Control Group (Group 4). Thus, the vaccine compositions comprising recombinant VP60 protein of RHDV-2 were able to elicit an antibody response against RHDV- 2. Unexpectedly, no seroconversion was seen in the vaccinated animals against RHDV-1 challenge, being all antibody titres below the cut-off to be considered seropositive.

[0266] Table 5. Mean antibody levels against RHDV-2 (mean log? HI / 50 l)

[0267] 1vaccination n. a.: no sample available for testing since no animal survived

[0268] 2challenge

[0269] However, the results surprisingly showed a heterologous protection and / or significant reduction of mortality in all vaccinated groups (Group 1 and Group 2), with a 10% and 0% of mortality, respectively.

[0270] In contrast, 100% of mortality was observed in the non-vaccinated Control Group (Group 3)

[0271] Table 6. Overall mortality rate of the different groups

[0272] GROUP MORTALITY (%)

[0273] The results clearly showed that the vaccine compositions comprising the recombinant VP60 protein of RHDV-2 were effective in conferring heterologous protection in challenged animals against RHDV-1 infection. Altogether, the data showed that the vaccine compositions were not only capable to protect against mortality caused by classic RHDV-1 (Group 2) but also to prevent it (Group 2 of Table 6).

[0274] This example together with Example 2 demonstrated that there was a direct correlation between serological response and protection against RHDV-2 and that surprisingly, all vaccine compositions based on recombinant VP60 protein of RHDV-2 induced a protective response in challenged animals with a heterologous RHDV-1 strain, regardless of being serologically irresponsive. This could be explained by a heterologous protection, also known as cross-protection, which is achieved thanks to existing conservative epitopes between both variants, classic RHDV-1 and RHDV-2, which may be present in the immunogenic and vaccine compositions of the invention.

[0275] EXAMPLE 4: Study of the efficacy of vaccine compositions comprising recombinant VP60 protein of RHDV-2 in the presence of circulating antibodies against rabbit hemorrhagic disease virus.

[0276] The aim of this study was to assess the efficacy of vaccine compositions in young rabbits (30-day old rabbits) after receiving an RHD virus challenge (rabbit hemorrhagic disease virus 1 or RHDV-1 ) in the presence of circulating antibodies, particularly maternally derived antibodies (MDAs), against rabbit hemorrhagic disease virus (RHDV) on.

[0277] Considering that the minimum age recommended for vaccination is 30 days of age, circulating antibodies against RHDV, and particularly maternally acquired immunity, may still be present at the time of vaccination and consequently, they may interfere with active immunity development after vaccination. Thus, to determine whether a possible impact of circulating antibodies against RHDV, mainly maternally derived antibodies, on vaccine efficacy in young animals occurs, the present study was performed.

[0278] A total of 38 New Zealand white rabbits (Oryctolagus cuniculus) of 30 days of age were included in this study (Table 7). Animals were randomly allocated into 3 different groups according to their immunological status against classic RHDV antibodies. Groups A and B were vaccinated with 0.50 ml of a vaccine composition comprising recombinant VP60 protein of rabbit hemorrhagic disease virus 2 (RHDV-2) as obtained in Example 1 and Group 3 served as strict control group as detailed below.

[0279] Group AMO rabbits seronegative for antibodies against RHDV-1 and for antibodies against RHDV-2.

[0280] Group B: 14 rabbits seropositive for antibodies against RHDV-1 and for antibodies against RHDV-2.

[0281] - Group C: 14 rabbits seropositive for antibodies against RHDV-1 and for antibodies against RHDV-2.

[0282] Table 7. Summary of vaccination and challenge study

[0283] The antibodies titers against RHDV-1 and RHDV-2 of Group B and Group C were representatives of the titers found in animals of 30 days of age (Day 0 of the study) under field conditions.

[0284] On Day 0 of the study, animals in Group A and Group B were vaccinated with a single dose of 0.5 ml of a vaccine composition comprising 21644HU / dose (hemagglutination units, HU) of recombinant VP60 protein of RHDV-2 formulated in 20% (w / v) mineral oil (liquid paraffin) as adjuvant to be administered by subcutaneous (SC) route.

[0285] The recombinant VP60 protein of RHDV-2 was produced as described in Example 1.

[0286] The vaccine compositions were formulated by mixing the aqueous phase comprising the recombinant VP60 protein of RHDV-2 with the oily phase in a ratio of 66%:33% under agitation and performing a final homogenization under high-pressure to obtain an oil-in-water emulsion.

[0287] Group C was used as a control group and received a mock-vaccine comprising PBS (phosphate buffered saline) solution through the same route (SC) and with the same administration scheme (one single dose).

[0288] In order to demonstrate the absence of interference of circulating RHDV antibodies on the vaccine take and thus with the efficacy of the vaccine composition to be tested, all animals were challenged with a RHDV-1 virulent strain (V-4764 strain, highly virulent field strain isolated from clinically infected animals in 1997) at age of 10 weeks, at the time when the antibody titres in Group C (Control Group) were not detected.

[0289] Experimental infection (challenge) was performed on Day 43 (D43) of the study by administering a 1 ml of a solution of virulent RHDV-1 strain V-4764, at a dose of 212HU / ml by intramuscular (IM) route.

[0290] General clinical signs of all animals and mortality were recorded throughout all the study, from Day 0 until 14 days post-challenge (Day 57 of the study). Fourteen days after challenge, rabbits were euthanized.

[0291] After challenge, mortality rates were followed up to support the efficacy of the vaccine compositions in the presence of circulating antibodies against RHDV. The observation period of all the animals were prolonged up to 14 days after challenge. Blood samples were collected from all animals at the day of vaccination (DO) in all groups and at day of the challenge (D43) only for group C in order to monitor the decay of antibodies against RHDV. Assessment of clinical signs were checked every day after vaccination and throughout the end of study. Local and general systemic clinical signs such as depression, body condition, dyspnea, nasal discharge, ocular discharge and mortality, were carefully monitored daily two times a day. Any other abnormal clinical sign such as diarrhoea, respiratory signs, among other were also monitored and recorded.

[0292] The efficacy parameters assessed in this study were:

[0293] Mortality: The main parameter assessed was the mortality caused by RHDV-1 virus. Mortality was monitored and recorded up to 14 days after challenge (Day 57 of the study).

[0294] Clinical signs: Secondary parameters to assess efficacy was the presence or absence of typical clinical signs of Rabbit Hemorrhagic Disease (RHD). The disease is characterized for causing mainly rapid mortality so that clinical signs are usually less evident.

[0295] Serology: As secondary parameter, serological response against RHDV-1 and RHDV- 2 (as Iog2 HI / 50pl) were measured by hemagglutination inhibition assay (HIA) according to standard protocols. The validated cut-off value to consider animals seropositive for antibodies against RHDV-2 was Iog2 HI / 50pl > 3, and Iog2 HI / 50pl > 4.5 for antibodies against RHDV-1.

[0296] At the day of vaccination (DO) rabbits in Group A were all seronegative to RHDV-1 and RHDV-2 antibodies; whereas rabbits in Group B and Group C were all seropositive against both RHDV-1 and RHDV-2. The antibody titers in groups B and C (seropositive animals) were not significantly different between them.

[0297] Results on mortality are summarized in Table 8.

[0298] Table 8: Overall mortality rate of the different groups

[0299] GROUP MORTALITY (%)

[0300] The results obtained showed that the survival rate of both vaccinated groups, i.e. Group A and Group B, was 100%. Thus, a complete prevention of rabbit hemorrhagic disease (RHD) was achieved. In contrast, mortality in Group C (Control Group) were mainly reported between 48 and 72 hours after challenge. A mortality rate of 92% was recorded in Group C, which was significantly different than that of vaccinated groups A and B (with no observed mortality at all).

[0301] According to the results, all vaccinated groups were equally protected against virulent RHDV-1 challenge regardless of the presence of circulating antibodies against RHDV. These results unexpectedly demonstrated that the vaccine compositions comprising recombinant VP60 protein of RHDV-2 were effective in conferring heterologous protection (also known as cross-protection) against RHDV-1 infection, even in the presence of circulating RHDV antibodies, particularly of maternal origin (maternally derived antibodies or MDAs).

[0302] Necropsy was conducted for each animal found dead. Liver samples were collected and tested for the presence of RHDV by hemagglutination assay (HA). In the Control Group, all dead animals were confirmed as positive to RHDV. These results concluded that 92% of the control animals died due to RHDV, which was significantly greater than the 0% of mortality observed in both vaccinated Groups A and B.

[0303] The 100% survival rate observed in the vaccinated groups A and B (MDA- and MDA+, respectively) confirms that circulating antibodies, such as maternally derived antibodies, did not interfere in the efficacy of the vaccine compositions based on the recombinant VP60 protein of RHDV-2 tested in this study.

[0304] On the other hand, the mortality rate obtained in the Group C (92%) confirms that the infection was properly performed and the study well designed.

[0305] Overall, the results from this efficacy study support that no relevant differences in the response to an experimental RHDV infection (challenge) were observed between vaccinated animals in presence or in absence of circulating antibodies particularly maternally derived antibodies (MDAs), against RHDV. Thus, the presence of pre-existing circulating antibodies in rabbits up to 30 days of age do not interfere on the development of the active immune response after vaccination with the vaccine compositions of the present invention. The efficacy claims of the vaccine compositions remained the same regardless of the level of circulating antibodies at the time of vaccination. All vaccinated groups were cross-protected against RHDV-1 and mortality was fully prevented by the administration of vaccine compositions based on the recombinant VP60 protein of RHDV-2 as obtained in Example 1 and formulated to be administered in a single dose administration.

[0306] EXAMPLE 5: Study on the duration of immunity (DOI) of single dose administration of a vaccine composition comprising recombinant VP60 protein of RHDV-2 against classic rabbit hemorrhagic disease virus (also known as RHDV-1) in rabbits. In previous examples, it was demonstrated that a recombinant vaccine based on the VP60 capsid protein of rabbit hemorrhagic disease virus 2 (RHDV-2) reduced or even fully prevented mortality caused by both, the classic rabbit hemorrhagic disease virus ( RHDV-1 ) and the new variant RHDV-2 of Rabbit Hemorrhagic Disease, at the same time and with a single dose administration.

[0307] This aim of this study was to assess the duration of immunity (DOI) of the administration in young rabbits of a single dose of the vaccine composition comprising the recombinant VP60 protein of RHDV-2 produced as described in Example 1 in young rabbits against new variant of Rabbit Hemorrhagic Disease virus (RHDV-1) by means of a challenge at approximately one year after the vaccination.

[0308] A total of 30 New Zealand white rabbits (Oryctolagus cuniculus) of 30 days old and free from antibodies against RHDV-1 and RHDV-2 were enrolled for this study. Rabbits were equally and randomly distributed into two different groups, i.e. Group 1 (vaccinated group or G1) and Group 2 (non-vaccinated group, G2 or Control Group) of 15 animals per group.

[0309] On Day 0 (DO) of the study, the animals in Group 1 received a single dose of a 0.5 ml vaccine composition comprising 21644HU / dose (hemagglutination units) of the recombinant VP60 protein of RHDV-2 in 20% (w / v) mineral oil (liquid paraffin) as adjuvant to be administered by subcutaneous (SC) route. The recombinant VP60 protein of RHDV-2 was produced as described in Example 1. The vaccine composition was formulated by mixing the aqueous phase comprising the recombinant VP60 protein of RHDV-2 with the oily phase in a ratio of 66%:33% under agitation and performing a final homogenization under high-pressure to obtain an oil-in-water emulsion. The animals in Group 2 served as control group and received a mock-vaccine comprising exclusively PBS (phosphate buffered saline) solution without antigen. All animals were vaccinated by the SC route and according to the proposed schedule for vaccination (one single dose) with the corresponding vaccination plan. At approximately 12 months (365 days) post-vaccination, all the animals were challenged by intramuscular (IM) route with 1 ml of a solution of a virulent RHDV-1 strain V-4764 (highly virulent field strain isolated from clinically infected animals in 1997) at a titer of 212HU (hemagglutination units) in order to assess the duration of immunity (DOI) of the tested vaccine compositions.

[0310] Afterwards, all the animals were daily observed for 14 days after challenge (until day 379 of the study). Fourteen days after challenge, all rabbits were euthanized and liver samples from dead animals were analyzed to assess the presence of RHDV-1 in order to confirm the cause of death.

[0311] Blood samples were collected from the rabbits periodically through the vaccination period to obtain serum and assayed for evidence of seroconversion. Assessment of clinical signs were checked every day after vaccination and throughout the end of the study. Local and general systemic clinical signs such as depression, body condition, dyspnea, nasal discharge and ocular discharge, and mortality, were carefully monitored daily (two times a day). Any other abnormal clinical sign such as diarrhoea, respiratory signs, among others were also monitored and recorded.

[0312] To assess the DOI of the single dose vaccine composition, the serological response against RHDV-2 and RHDV-1 was measured by hemagglutination inhibition assay (HIA), expressed as logz HI / 50pl antibodies. The anti-RHDV-2 and anti-RHDV-1 antibody titers were monitored at different days post-vaccination (Day 42, Day 84, Day 126, Day 168, Day 210, Day 252, Day 294 and Day 336), and at Day 379 (also 14 days post-challenge). Blood serum samples were taken on those specific days, accordingly. The validated cut-off value to consider an animal seropositive for antibodies against RHDV-2 was Iog2 HI / 50p I > 3, and Iog2 HI / 50pl > 4.5 for antibodies against RHDV-1 .

[0313] The mean serological anti-RHDV-2 antibody response of the animals in the vaccinated group (Group 1 ) and in the Control Group (Group 2) are shown in Figure 2. Unexpectedly, no seroconversion was seen against classic RHDV-1 in the vaccinated group (Group 1), since all antibody titres were below the cut-off to be considered seropositive, confirming once again the results obtained in previous Example 3.

[0314] As shown in Figure 2, on Day 41 post-vaccination (D41), the animals in the vaccinated group (Group 1 ) showed a significantly higher antibody titre compared with that observed in the Control Group (Group 2). Additionally, the antibody titres of the vaccinated animals of Group 1 were maintained at high levels through all the study. It was observed that antibody titres against RHDV-2 was maintained at least until 336 days post-vaccination (D336). Also, as shown in Figure 2, the high mean antibody titres were maintained at 379 days postvaccination (14 days post-challenge) (D379).

[0315] The results on mortality given in Table 9 surprisingly showed a heterologous protection and / or significant reduction of mortality in the vaccinated group (Group 1). Vaccinated group (Group 1) had a significantly reduced mortality rate, i.e. 10%, compared to the Control Group (Group 2) wherein the mortality rate was 80%.

[0316] Table 9: Overall mortality rate at D379 of the different groups.

[0317] GROUP MORTALITY (%)

[0318] 2 I

[0319] 80%

[0320] (Control)

[0321] According to the results obtained in this study, the vaccine composition based on recombinant VP60 protein of RHDV-2 surprisingly induced a protective response in challenged animals with a heterologous RHDV-1 strain, regardless of being serologically irresponsive, as also observed in Example 3.

[0322] Thus, it was demonstrated, on the one hand, that the duration of immunity (DOI) of the vaccine composition comprising the recombinant VP60 protein of RHDV-2 was up to one-year post-vaccination; and on the other hand, that the vaccine composition was able to protect from mortality caused by an RHDV-1 infection still one year after a single dose administration, demonstrating its efficacy regardless of being serologically irresponsive to RHDV-1 , and demonstrating again the unexpected heterologous protection of the vaccine composition based on recombinant VP60 protein of RHDV-2 observed in Example 3.

[0323] EXAMPLE 6: Study on the duration of immunity (DOI) of a single dose administration of a vaccine composition comprising recombinant VP60 protein of RHDV-2 against rabbit hemorrhagic disease virus 2 (RHDV-2) in rabbits.

[0324] In previous examples, it was demonstrated that a recombinant vaccine based on the VP60 capsid protein of RHDV-2 reduces or even fully prevents mortality caused by both, the classic rabbit hemorrhagic disease virus (also known as RHDV-1 ) and the new variant RHDV- 2 of rabbit hemorrhagic disease (RHD), at the same time and with only a single dose administration.

[0325] The aim of this new study was to assess the duration of immunity (DOI) of the administration of one single dose of the vaccine composition comprising the recombinant VP60 protein of RHDV-2 produced as described in Example 1, in young rabbits against new variant of rabbit hemorrhagic disease virus 2 (RHDV-2) by means of a challenge at approximately one year after vaccination.

[0326] A total of 52 New Zealand white rabbits (Oryctolagus cuniculus) of 30 days of age and free from antibodies against RHDV-1 and RHDV-2 were include in this study. Rabbits were equally and randomly distributed into two different groups, Group 1 (G1 or vaccinated group) and Group 2 (non-vaccinated group, G2 or Control Group) of 26 animals per group.

[0327] On Day 0 (DO) of the study, the animals in Group 1 received a single dose of a 0.5 ml vaccine composition comprising 21644HU / dose (hemagglutination units) of the recombinant VP60 protein of RHDV-2 formulated in 20% (w / v) of mineral oil (liquid paraffin) as adjuvant to be administered by subcutaneous (SC) route. The recombinant VP60 protein of RHDV-2 was produced as described in Example 1. The vaccine composition was formulated by mixing the aqueous phase comprising the recombinant VP60 protein of RHDV-2 with the oily phase in a ratio of 66%:33% under agitation and performing a final homogenization under high-pressure to obtain an oil-in-water emulsion. The animals in Group 2 served as a control group and received a mock-vaccine comprising exclusively a PBS (phosphate buffered saline) solution without antigen. All animals were vaccinated by the SC route and according to the proposed schedule for vaccination (one single dose) with the corresponding vaccination plan. At approximately 12 months (361 days) post-vaccination, all the animals were challenged by intramuscular (IM) route with 1 ml of a solution of a virulent RHDV-2 strain V-1037 (highly virulent strain isolated from clinically infected animals in 2013) at a titer of 2145HU (hemagglutination units) in order to assess the duration of immunity (DOI) of the vaccine.

[0328] Afterwards, all the animals were observed daily for 14 days after challenge (until Day 375 of the study). Fourteen days after challenge, all rabbits were euthanized and liver samples from dead animals were analyzed to assess the presence of RHDV-2, in order to confirm the cause of death.

[0329] For the vaccine to be considered efficacious, significant differences in mortality had to be observed between control and vaccinated groups.

[0330] Blood samples were collected from the rabbits periodically throughout the vaccination period to obtain serum and assayed for evidence of seroconversion. Assessment of clinical signs were checked every day after vaccination and throughout the end of study. Local and general systemic clinical signs such as depression, body condition, dyspnea, nasal discharge and ocular discharge, and mortality, were carefully monitored daily (two times a day). Any other abnormal clinical sign such as diarrhoea, respiratory signs, among others were also monitored and recorded.

[0331] To assess the DOI of the single dose vaccine composition, the serological response against RHDV-2 was measured by hemagglutination inhibition assay (HIA), expressed as Iog2 HI / 50pl antibodies against RHDV-2. The anti-RHDV-2 antibody titer was monitored at different days post-vaccination (D39, D81 , D123, D159, D215, D257, D292, D326, D361), and at D375 (14 days post-challenge). Blood serum samples were taken on those specific days, accordingly. The validated cut-off value to consider an animal seropositive for antibodies against RHDV-2 was Iog2 HI / 50p I > 3.

[0332] The mean serological response of the animals in the vaccinated group (Group 1) and in the Control Group (Group 2) are shown in Figure 3.

[0333] As shown in Figure 3, on Day 39 post-vaccination (D39), the animals in the vaccinated group (Group 1) showed a significantly higher antibody titre compared to that of observed in the Control Group (Group 2). Additionally, the antibody titres of the vaccinated animals of Group 1 were maintained at high levels through all the study. It was observed that antibody titres against RHDV-2 was maintained at least until 361 days post-vaccination (D361). Also, as shown in Figure 3, the high mean antibody titer was maintained and even increased at D375 days post-vaccination (14 days post-challenge) (D375).

[0334] According to the results obtained in this study, it was demonstrated that the duration of immunity (DOI) of the vaccine composition comprising the recombinant VP60 protein of RHDV-2 was up to one-year post-vaccination. The vaccine composition was able to elicit a protective immune response against RHDV-2 still one year after a single dose administration.

[0335] EXAMPLE 7: Study of the efficacy of vaccine compositions comprising recombinant VP60 protein of RHDV-2 against a highly virulent strain of rabbit hemorrhagic disease virus 2 (RHDV-2)

[0336] The aim of this study was to assess the efficacy of vaccine compositions comprising recombinant VP60 protein of a RHDV-2 variant strain in young rabbits (30-day old rabbits) against a new highly virulent RHDV-2 variant strain. Accordingly, vaccine efficacy was tested by means of an experimental challenge with a RHDV-2 variant strain, namely V-1247 strain, corresponding to a highly virulent field isolate of RHDV-2 isolated in The Netherlands in 2022.

[0337] A total of 38 New Zealand white rabbits (Oryctolagus cuniculus) of 30 days of age were included in this study. All rabbits were free (seronegative) for rabbit hemorrhagic disease virus antibodies. Animals were randomly allocated into 3 different groups, two of them with 15 animals and one of them with 8 animals, which received either a vaccine composition comprising the recombinant VP60 of a RHDV-2 variant strain as obtained in Example 1 or a mock-vaccine (PBS). Two different vaccine batches were tested and included in the study together with a single strict control group, as detailed in Table 10 below.

[0338] Table 10. Study design

[0339] SC: subcutaneous, pv: post-vaccination

[0340] On Day 0 of the study, animals in Groups A and B were vaccinated with a single dose of 0.50 ml of a vaccine composition comprising 21644HU / dose (hemagglutination units, HU) of the recombinant VP60 protein of rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain as obtained in Example 1 and coming from different batch productions (batch 1 and batch 2), formulated in 20% (w / v) mineral oil (liquid paraffin) as adjuvant by mixing the aqueous phase comprising the recombinant VP60 protein of RHDV-2 with the oily phase (ratio 66%:33%) under agitation and performing a final homogenization under high-pressure to obtain an oil-in- water emulsion. The vaccine compositions were administered by subcutaneous (SC) route as a single dose. Whereas animals in Group C (control group) received 0.50 ml of a mock-vaccine comprising PBS (phosphate buffered saline) solution through the same route (SC) and with the same administration scheme (one single dose). Group C (or Control Group) served as a strict control group.

[0341] The experimental infection (challenge) was performed 14 days post-vaccination by administering a 1 ml of a solution of a highly virulent RHDV-2 variant strain, namely V-1247, at a dose of 24HU / 50pl by intramuscular (IM) route. This challenge strain corresponded to a highly virulent RHDV-2 variant strain isolated from the field in The Netherlands in 2022.

[0342] After challenge, mortality rates were followed up as a main parameter to assess the efficacy of the administered vaccine compositions. The observation period of all the animals were prolonged up to 14 days after challenge where different local and general clinical signs were also monitored and assessed.

[0343] Clinical signs such as depression, body condition, dyspnea, nasal discharge and ocular discharge of all animals was performed and the data recorded throughout all the study, since Day 0 until 14 days post-challenge (Day 28 of the study). Abnormal clinical signs such as diarrhea and respiratory signs, among others, were also monitored and recorded.

[0344] Fourteen days after challenge, all rabbits that survived the study were humanely euthanized. All animals that died during the study were necropsied the same day and liver samples were collected to assess the presence of RHDV-2 in liver in order to confirm the cause of death.

[0345] Results on mortality after a challenge with a highly virulent RHDV-2 strain as the main parameter to assess the vaccine efficacy are summarized in Table 11 .

[0346] Table 11 : Overall mortality rate of the different tested groups

[0347] The results of the study clearly demonstrated that mortality in both vaccinated groups, i.e. Group A and Group B, was prevented since none animal died after the challenge with a highly virulent RHDV-2 variant strain, as seen in Table 11 , the mortality rate in the vaccinated Groups A and B was 0%. In contrast, all animals in Group C (Control group) did not survive the challenge against a highly virulent RHDV-2 variant strain, where a mortality rate of 100% was recorded in Group C.

[0348] The 100% survival rate observed in the vaccinated Groups A and B confirmed that the vaccine compositions of the invention comprising a recombinant VP60 protein of a RHDV-2 variant strain were able to confer complete protection after a single dose administration, achieving the prevention of the disease, against a RHDV-2 infection, even when the RHDV-2 infection was caused by a highly virulent strain.

[0349] Overall, the results from this efficacy study support that the recombinant VP60 protein of a RHDV-2 variant strain as a vaccine composition is also effective against infections caused by highly virulent RHDV-2 variant strains. All vaccinated groups were protected against infections caused by highly virulent RHDV-2 variant strains and mortality was fully prevented by the administration of the vaccine compositions of the invention administered in a single dose.

Claims

CLAIMS1 . An immunogenic or vaccine composition comprising a recombinant VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain, wherein the composition is capable of a) inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2) and b) inducing heterologous protection against RHD caused by rabbit hemorrhagic disease virus 1 (RHDV-1), wherein the immunogenic or vaccine composition is devoid of immunogens derived from RHDV-1 variant strains and wherein said protection is induced after the administration of a single dose of the composition.

2. The immunogenic or vaccine composition according to claim 1 , wherein the recombinant VP60 protein comprises the sequence according to SEQ ID NO: 1 or a functionally equivalent variant thereof.

3. The immunogenic or vaccine composition according to any one of claims 1 or 2, wherein the immunogenic or vaccine composition comprises between about 210hemagglutination units (HU) to about 218HU per dose of recombinant VP60 protein of RHDV-2 variant strain.

4. The immunogenic or vaccine composition according to any one of claims 1 to 3, wherein the composition is formulated to be administered by subcutaneous, intradermal, or intramuscular route, preferably by subcutaneous route.

5. The immunogenic or vaccine composition according to any one of claims 1 to 4 further comprising virus-like particles assembled of the recombinant VP60 protein of a RHDV- 2 variant strain.

6. The immunogenic or vaccine composition according to any one of claims 1 to 5, wherein the composition is capable of inducing homologous protection against rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus 2 (RHDV-2), wherein the RHDV-2 is a highly virulent RHDV-2 variant strain.

7. The immunogenic or vaccine composition according to any one of the claims 1 to 6 further comprising at least one additional antigen of other pathogens causing disease in rabbits.

8. The immunogenic or vaccine composition according to claim 7, wherein the at least one additional antigen is selected from a group of microorganisms consisting of: myxoma virus, shope fibroma virus (SFV), Pasteurella multocida, Staphylococcus aureus, E. coli, Salmonella enterica, Salmonella typhimurium, Clostridium spiroforme, Clostridium perfringens, Clostridium cuniculi, Eimeria sp., Encephalitozoon cuniculi and Trichophyton mentagrophytes.

9. The immunogenic or vaccine composition according to any one of claims 1 to 8, further comprising an acceptable pharmaceutical adjuvant.

10. The immunogenic or vaccine composition according to claim 9, wherein the acceptable pharmaceutical adjuvant is light mineral oil, preferably is liquid paraffin.

11. The immunogenic or vaccine composition according to claim 9 or 10, wherein the acceptable pharmaceutical adjuvant is at a concentration of between about 3% w / v to about 30% w / v, preferably 20% w / v.

12. The immunogenic or vaccine composition according to any one of claims 1 to 11 , wherein the composition is obtained by a method comprising: a) culturing a yeast host cell comprising a nucleotide sequence encoding a VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain to obtain the recombinant VP60 protein; b) separating the recombinant VP60 protein of a RHDV-2 variant strain from the yeast host cell non-soluble components, and c) obtaining the composition comprising the recombinant VP60 protein of a RHDV-2 variant strain and the yeast host cell soluble components, and discarding the yeast host cell non-soluble components.

13. The immunogenic or vaccine composition according to claim 12, wherein the yeast host cell belongs to the species Komagataella phaffii.

14. The immunogenic or vaccine composition according to claim 12 or 13, wherein the method does not comprise a step of isolating the recombinant VP60 protein of a RHDV-2 variant strain from the immunogenic composition obtained in step c).

15. The immunogenic or vaccine composition according to any one of claims 12 to 14, wherein step c) further comprises a step of sterilization of the composition comprising the recombinant VP60 protein of a RHDV-2 variant strain and the yeast host cellsoluble components, preferably wherein said sterilization is carried out by filtration through a 0.22 pm filter.

16. The immunogenic or vaccine composition as defined in any one of claims 1 to 15 for use as a medicament.

17. The immunogenic or vaccine composition as defined in any one of claims 1 to 15 for use in the treatment and / or prophylaxis of rabbit hemorrhagic disease (RHD) and associated diseases in a subject.

18. The immunogenic or vaccine composition as defined in any one of claims 1 to 15 for use in a method for inducing an immune response against rabbit hemorrhagic disease virus in a subject.

19. The immunogenic or vaccine composition for use according to claim 18, in a method for inducing a homologous immune response against RHDV-2 and a heterologous immune response against RHDV-1.

20. The immunogenic or vaccine composition for use according to claim 18 or 19, wherein the immune response confers protection against RHD in a subject.21 . The immunogenic or vaccine composition for use according to claim 20, wherein the RHD is caused by infection with a RHDV-1 variant strain and / or a RHDV-2 variant strain.

22. The immunogenic or vaccine composition for use according to claim 21 , wherein the RHDV-2 variant strain is a highly virulent RHDV-2 variant strain.

23. The immunogenic or vaccine composition for use according to any one of claims 20 to 22, wherein the protection against RHD is measured as survival rate or mortality rate of naturally or experimentally infected subjects.

24. The immunogenic or vaccine composition for use according to claim 23, wherein the survival rate of subjects infected with RHDV-1 is of at least about 60%, of at least about 70%, at least about 80%, at least about 90%, about 100% of naturally or experimentally infected subjects.

25. The immunogenic or vaccine composition for use according to claim 23 or 24, wherein the survival rate of subjects infected with RHDV-2 is of at least about 60%, at least about 70%, at least about 80%, at least about 90%, about 100% of naturally or experimentally infected subjects.

26. The immunogenic or vaccine composition for use according to any one of claims 23 to 25, wherein the survival rate of subjects infected with RHDV-1 and RHDV-2 is of at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100% of naturally or experimentally infected subjects27. The immunogenic or vaccine composition for use according to any one of claims 17 to 26, wherein the immunogenic or vaccine composition is administered by subcutaneous, intradermal, or intramuscular route, preferably by subcutaneous route.

28. The immunogenic or vaccine composition for use according to any one of claims 17 to 27, wherein the immunogenic or vaccine composition is administered in a single dose.

29. The immunogenic or vaccine composition for use according to any one of claims 17 to 28 wherein the immunogenic or vaccine composition is administrated at a dose of between about 210hemagglutination units (HU) to about 218HU per dose of recombinant VP60 protein of RHDV-2 variant strain.

30. The immunogenic or vaccine composition for use according to any one of claims 17 to 29, wherein the subject is a mammal from the Lagomorpha order, preferably from the Leporidae family.

31. The immunogenic or vaccine composition for use according to any one of claims 17 to 30, wherein the subject has circulating anti-RHDV-1 and / or anti-RHDV-2 antibodies.

32. The immunogenic or vaccine composition for use according to claim 31 , wherein the circulating anti-RHDV-1 and / or anti-RHDV-2 antibodies are maternally-derived antibodies (MDAs).

33. The immunogenic or vaccine composition for use according to any one of claims 17 to 32, wherein the treatment and / or prophylaxis of RHD or the homologous immuneresponse against RHDV-2 and the heterologous immune response against RHDV-1 or the protection against RHD is conferred for at least one year after administration of a single dose.

34. The immunogenic or vaccine composition for use according to any one of claims 17 to 32, wherein the treatment and / or prophylaxis of RHD or the homologous immune response against RHDV-2 and the heterologous immune response against RHDV-1 or the protection against RHD is conferred by virus-like particles assembled of the recombinant VP60 protein of a RHDV-2 variant strain.

35. The immunogenic or vaccine composition for use according to claim 33 or 34, wherein the RHDV-2 variant strain is a highly virulent RHDV-2 variant strain.

36. A pre-filled vaccine delivery device comprising an immunogenic or vaccine composition as defined in any one of claims 1 to 15.

37. A method for the production of the immunogenic or vaccine composition as defined in any one of claims 1 to 15, comprising: a) culturing a yeast host cell comprising a nucleotide sequence encoding a VP60 protein of a rabbit hemorrhagic disease virus 2 (RHDV-2) variant strain to obtain the recombinant VP60 protein; b) separating the recombinant VP60 protein of a RHDV-2 variant strain from the yeast host cell non-soluble components; and c) obtaining the composition comprising the recombinant VP60 protein of a RHDV-2 variant strain and the yeast host cell soluble components and discarding the yeast host cell non-soluble components.

38. The method according to claim 37, wherein the method does not comprise a step of isolating the recombinant VP60 protein of a RHDV-2 variant strain from the composition obtained in step c).

39. The method according to claim 37 or 38, further comprising the step of mixing the composition obtained in step c) with an acceptable pharmaceutical adjuvant.

40. The method according to claim 39, wherein the acceptable pharmaceutical adjuvant is light mineral oil, preferably is liquid paraffin.

41. The method according to claim 38 or 40, wherein the acceptable pharmaceutical adjuvant is at a concentration of between about 3% w / v to about 30% w / v, preferably 20% w / v.

42. The method according to any one of claims 37 to 41 , wherein the yeast host cell belongs to the species Komagataella phaffi.

43. The method according to any one of claims 37 to 42, wherein step c) further comprises a step of sterilization of the composition comprising the recombinant VP60 protein of a RHDV-2 variant strain and the yeast host cell soluble components, preferably wherein said sterilization is carried out by filtration through a 0.22 pm filter.

44. The method according to any one of claims 37 to 43 wherein the composition of step c) comprises virus-like particles assembled of the recombinant VP60 protein of a RHDV-2 variant strain.

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