Vaccine against bovine viral diarrohea virus and bovine respiratory disease

Abstract

The present invention relates to an immunogenic or vaccine composition for use in the treatment and / or prevention of Bovine Viral Diarrohea Virus type 1 (BVDV-1) and / or Bovine Viral Diarrohea Virus type 2 (BVDV-2) infection in cattle and associated Bovine Respiratory Disease.

Description

[0001] VACCINE AGAINST BOVINE VIRAL DIARROHEA VIRUS AND BOVINE RESPIRATORY DISEASE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of veterinary medicine, more particularly to an immunogenic or vaccine composition for the protection of bovine animals from Bovine Respiratory disease (BRD) caused by infection of Bovine Viral Diarrohea Virus type 1 and type 2 (BVDV-1 and BVDV-2).

[0004] BACKGROUND OF THE INVENTION

[0005] Bovine Respiratory Disease (BRD) leads to severe pneumonia and is a major cause of mortality in cattle worldwide in both beef and dairy production thereby causing a huge economic loss annually due to calf mortality, reduced performance and use of antibiotics. This disease is a multifactorial syndrome, where several bacterial and viral pathogens are involved. Most important bacterial pathogens include Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis, while the viral counterpart involves Bovine Viral Diarrhea Virus (BVDV), Bovine Respiratory Syncytial Virus (BRSV), Bovine Herpesvirus type 1 (BoHV-1 ), Parainfluenza 3 Virus (PI-3) and others such as Adenovirus and more recently, Bovine Respiratory Coronavirus. All of them are known to be involved alone or in synergism with each other in the pathogenesis of BRD. BRD is characterized by one or more of the following clinical signs or symptoms: fever of over 39.5°C, difficulty of breathing at several degree of severity, nasal discharge, varying degrees of depression (including drooping ears, an extended head, a bowed back, and / or self-isolation from other cattle), reduction or loss of appetite, rapid and shallow breathing, increased lung sounds, coughing, and pneumonia.

[0006] Bovine Viral Diarrhea Virus (BVDV), is a single stranded positive-sense RNA pestivirus of the family Flaviviridae, including two genotypes, namely BVDV-1 and BVDV-2, based on their significant differences at genomic level and limited cross neutralization. Several conventional attenuated BVDV strains are known in the art, which have been disclosed for vaccine development, for instance attenuated BVDV viruses KE-9 and NY-93 from the commercially available vaccine Bovela (Boehringer Ingelheim Vetmedica GmbH) and attenuated BVDV strains from the commercially available vaccine Bovi-Shield GOLD® 5 (Zoetis Inc.).

[0007] However, current BVDV vaccines do not confer broad protection, have immunosuppressive traits, and can induce alloreactive pancytopenia in neonates. Congenital infections may cause resorption, abortion, stillbirth, or live-birth. Congenitally infected fetuses that survive in utero infection (i.e., the live-births) may be born as BVDV- infected calves. The BVDV infection in these calves will persist during the entire life of the calf, and they will shed BVDV continuously in the farm environment, thus being a major concern for cattle producers because they are the primary source for spread of BVDV within and among cattle herds known as persistently infected (PI) calves. Symptoms of BVDV infection can be variable in adult cattle. Signs of acute infection include fever, lethargy, loss of appetite, ocular discharge, nasal discharge, oral lesions, diarrhea and decreasing milk production. Chronic infection may lead to signs of mucosal disease. In calves, the most commonly recognized birth defect is cerebellar hypoplasia. The signs of this include ataxia / lack of voluntary coordination of muscle movements, tremors, a wide stance, stumbling, failure to nurse, and any combination thereof. In severe cases the calf may die. Transient infections include diarrhea, calf pneumonia, decreased milk production, reproductive disorders, increased occurrence of other diseases, and death. The losses from fetal infection include abortions, congenital defects, weak and abnormally small calves, unthrifty, persistently infected (PI) animals, and death among PI animals. New tools are needed to fight against BVDV for both respiratory and reproductive parameters.

[0008] Bovine Respiratory Syncytial Virus (BRSV) is the major cause of Bovine Respiratory Disease (BRD) in calves during their first year of life. Their exposure to the disease is estimated at above 50% worldwide in both dairy cattle and beef cattle herds.

[0009] BRSV belongs to the genus Pneumovirus, a single-strand RNA virus. This virus can act as a primary pathogen or predispose the calf to secondary bacterial infections such as Mannheimia haemolytica, Histophilus somni, Pasteurella multocida and Mycoplasma bovis. Transmission can occur directly, via aerosol droplets or direct contact with an infected animal, or indirectly through contaminated surfaces. The incubation time is usually between 2 and 5 days and the infection involve the upper respiratory tract, or both the lower and upper ones. Depending on the virulence of the virus, clinical signs can range from mild to severe. The most common are: tachypnea, serous ocular secretions, dry mouth, reduced activity, anorexia, fever up to 40°C, dyspnoea and death. Signs appear at the time of infection and continue for about 7-10 days post-infection. It can also occur in a subclinical form, in which case the animals usually experience loss of appetite, reduced activity and decreased weight gain. Humoral immunity plays an important role in the defence of calves against BRSV. To prevent the disease, it is necessary to combine good management of the herd to prevent stress factors with effective vaccines against BRSV. Modified-live vaccines have shown efficacy in the prevention of this virus. However, there have been cases in which inactivated BRSV vaccines not only failed to protect against the disease, but also induced an exacerbated response to BRSV.

[0010] Bovine Herpesvirus type-1 (BoHV-1 ), also called Infectious Bovine Rhinotracheitis Virus (IBRv), is an enveloped virus, double-stranded DNA virus classified in the Herpesvi dae family. Bovine herpesvirus-1 (BoHV-1 ) was first observed in the United States as an acute upper respiratory tract disease in cattle. However, the first description of the disease was from Europe and was a vulvovaginitis in females. Infection by BoHV-1 is transmitted by direct contact with upper respiratory, conjunctival, or genital tract mucous membranes. Infected animals shed virus from respiratory mucous membranes and secretions, or genital mucous membranes and secretions, after exposure. The virus is present in all fetuses aborted as the result of BoHV-1 infection, and these fetuses can serve as a source for transmission of disease. Manifestations of the BoHV-1 clinical disease include respiratory tract disease, genital tract disease of the superficial surfaces, conjunctivitis, abortion, encephalitis, and generalized disease in the neonate. Researchers readily propagated the virus in cell culture, which led to development of vaccines relatively soon after the disease was characterized in the United States. Because of potential losses for BoHV-1-induced respiratory disease and abortions, BoHV-1 vaccination programs are common in beef and dairy practice. The modified live virus (MLV) vaccines were the initially licensed for use in cattle for protection against BoHV-1 , for instance HIPRABOVIS® IBR MARKER LIVE (Laboratorios HIPRA, S.A.), BOVILIS® IBR MARKER LIVE (Merck Sharp & Dohme Animal Health, S.L.), or Rispoval® IBR-Marker Live (Zoetis Spain, S.L.). Vaccines are usually attenuated by multiple passages in cell culture and / or in heterologous species’ cell cultures or by genetic modification and often retain their ability to replicate in a susceptible animal, possibly causing a viremia. MLV parenteral vaccines are relatively inexpensive, offer a convenient route of administration, and stimulate a rapid onset of immunity. The MLV parenteral vaccines may cross the placenta and infect the fetus, causing abortion. Most MLV BoHV-1 parenteral vaccines are not approved for use in pregnant heifers / cows or nursing calves. Although, recently some companies have received label claims for BoHV-1 MLV vaccine use in pregnant cows, it is needed that they are always vaccinated with that line of vaccines within 12 months and to nursing calves provided their dams were vaccinated within 12 months. Otherwise, they cannot be used. Therefore, new solutions are also needed in terms of improving efficacy and safety of vaccines against IBR caused by BoHV-1.

[0011] Bovine Parainfluenza-3 (PI-3) virus is an enveloped, negative-sense, single-strand RNA virus classified in the Paramyxoviridae family. PI-3 includes three genotypes, Pl-3a, Pl-3b and Pl-3c. Infections caused by PI-3 are common in cattle. Replication of PI-3 virus in the respiratory tract has been shown to interfere with the defence mechanisms such as the function of the mucociliary apparatus and alveolar macrophages and lymphocytes. The most important role of PI-3 is to serve as an initiator that may lead to the development of secondary bacterial pneumonia. Clinical signs include pyrexia, cough, serous nasal and lacrimal discharge, increased respiratory rate, and increased breath sounds. The severity of clinical signs worsens with the onset of bacterial pneumonia. Lesions include cranioventral lung consolidation, bronchiolitis, and alveolitis with marked congestion and haemorrhage. Inclusion bodies may be identified. Most fatal cases will also have a concurrent bacterial bronchopneumonia. As with BVDV, the current commercial PI-3 vaccines, mostly based on a PI-3 inactivated strains, are only effective against homologous strains and closely related variants.

[0012] Because BVDV, among other viruses associated with BRD, have serious implications not only for the respiratory but also for the reproductive health in cattle it is crucial preventing and controlling these viral infections in cattle herds, not only to reduce the impact on respiratory health but also to safeguard reproductive performance. Bovine Respiratory Disease has challenged the cattle industries for decades. While several vaccines increased awareness of the need for healthy immune systems, and innovative antibiotics have all helped with the control of BRD, there is still significant need to reduce the negative impacts of the BRD disease. Accurate diagnosis continued antimicrobial stewardship, and increased awareness of antimicrobial resistance require development and adoption of new, modern technologies to help lessen the burden of this disease. Vaccination with new formulations with a broad spectrum of effectiveness in this disease, both at respiratory and reproductive level, is needed in the art. SUMMARY OF THE INVENTION

[0013] The present invention is based on the finding that a composition comprising recombinant E2 proteins derived from the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and from the Bovine Viral Diarrhoea Virus type 2 (BVDV-2) provides excellent protection against BVDV infection both in terms of treating and / or preventing clinical signs and disease, including complete fetal protection against transfer of BVDV as well as prevention of birth of persistently BVDV infected calves.

[0014] As such, the present invention relates to an immunogenic or vaccine composition for use in a method of treatment and / or prevention of Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or Bovine Viral Diarrhoea Virus type 2 (BVDV-2) infection in cattle, where the immunogenic or vaccine composition comprises a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain.

[0015] DESCRIPTION OF THE FIGURES

[0016] Figure 1. Titers of total antibodies against BVDV quantified by ELISA for each animal in each Group of treatment. The S / P ratio of ELISA titers of total antibodies against BVDV is represented on the ordinates for each animal (1 to 7) in each Group of treatment (1 to 5) represented on the abscissas. Titers on day 14 (D14) of the study are represented in grey columns and titers on day 35 (D35) of the study are represented in black columns.

[0017] Figure 2. Titers of neutralizing antibodies against BVDV-1 (Groups 1 , 3 and 5) and of neutralizing antibodies against BVDV-2 (Groups 2, 4 and 5) quantified with a seroneutralization assay for each animal in each Group of treatment. The log? SN titers of neutralizing antibodies against BVDV-1 and BVDV-2 is represented on the ordinates for each animal (1 to 7) in each Group of treatment (1 to 5) represented on the abscissas. Titers on day 14 (D14) of the study are represented in grey column and titers on day 35 (D35) of the study are represented in black columns.

[0018] Figure 3. Titers of total antibodies against BVDV quantified by ELISA per group (Groups A to C) from day-2 (D-2) to day 70 (D70) of the study. The S / P ratio of ELISA titers of total antibodies against BVDV is represented on the ordinates for each day of the study represented on the abscissas per group. Figure 4. Neutralizing anti-BVDV-1 antibody titres (seroneutralization assay) per group (Groups A to C) from day -2 (D-2) to day 70 (D70) of the study. The log? SN titers of neutralizing antibodies against BVDV-1 is represented on the ordinates, for each day of the study on the abscissas per group.

[0019] Figure 5. BVDV-1 viremia (total virus titre, as average titre) per group (Groups A to C) from day 3 (D3) to day 12 (D12) after the experimental infection. The average virus titre (total virus) is represented on the ordinates for each group.

[0020] Figure 6. Neutralizing anti-BVDV-2 antibody titres (seroneutralization assay) per group from day -1 (D-1 ) to day 63 (D63) of the study. The log? SN titers of neutralizing antibodies against BVDV-2 is represented on the ordinates, for each day of the study represented on the abscissas per group.

[0021] Figure 7. Percentage of positive animals to anti-BVDV-2 neutralizing antibodies per group from day -1 (D-1 ) to day 63 (D63) of the study. The percentage of positive animals for neutralizing antibodies against BVDV-2 is represented on the ordinates, for each day of the study represented on the abscissas per group.

[0022] Figure 8. Nasal shedding for BVDV-2 per group (Groups A to D) from the day of the experimental infection (DO) to 14 days after the experimental infection (D14). The average virus titre (total virus) is represented on the ordinates for each day after the experimental infection represented on the abscissas per group.

[0023] Figure 9. BVDV-2 viremia (total virus titer, as average titre) per group (Groups A to D) from the day of the experimental infection (DO) to 21 days after the experimental infection (D21 ). The average virus titre (total virus) is represented on the ordinates for each day after the experimental infection represented on the abscissas per group.

[0024] Figure 10. Nasal shedding for BRSV per group in Sub-study 1 (Groups A to D) from day of the experimental infection (DO) to 14 days after the experimental infection (D14). Virus titer (mean log? total virus / ml) is represented on the ordinates for each day after the experimental infection, represented on the abscissas per group.

[0025] Figure 11. Daily rectal temperatures per group in Sub-study 1 (Groups A to D) from one day before the experimental infection (D-1 ) until 14 days after the experimental infection (D14). Rectal temperature (°C) is represented on the ordinates for each day after the experimental infection, represented on the abscissas per group.

[0026] Figure 12. Neutralizing antibody titers against BRSV (seroneutralization assay) per group in Sub-study 1 (Groups Ato D) from day 0 (DO) to day 56 (D56) of the study. The log? SN titers of neutralizing antibodies against BRSV is represented on the ordinates, for each day of the study on the abscissas per group.

[0027] Figure 13: Nasal shedding for PI-3 per group in Sub-study 2 (Groups A to C) from day of the experimental infection (DO) to 14 days after the experimental infection (D14). The average virus titer (log CCID5o / ml) is represented on the ordinates for each day after the experimental infection, represented on the abscissas per group.

[0028] Figure 14. Daily rectal temperatures per group in Sub-study 2 (Groups A to C) from one day before the experimental infection (D-1 ) until 14 days after the experimental infection (D14). Rectal temperature (°C) is represented on the ordinates for each day after the experimental infection, represented on the abscissas per group.

[0029] Figure 15. Neutralizing antibody titres against PI-3 (seroneutralization assay) per group in Sub-study 2 (Groups A to C) on day 0 (DO), 21 (D21 ), 42 (D42) and 56 (D56) of the study. The log? SN titers of neutralizing antibodies against PI-3 is represented on the ordinates, for each day of the study on the abscissas per group.

[0030] Figure 16. Nasal shedding for BoHV-1 per group in Sub-study 3 (Groups A to C) from day of the experimental infection (D66) to 15 days after the experimental infection (D81 ). The average virus titer (log CCID5o / ml) is represented on the ordinates for each day after the experimental infection, represented on the abscissas per group.

[0031] Figure 17. Daily rectal temperatures per group in Sub-study 3 (Groups A to C) from one day before the experimental infection (D-1 ) until 21 days after the experimental infection (D21 ). Rectal temperature (°C) is represented on the ordinates for each day after the experimental infection, represented on the abscissas per group.

[0032] Figure 18. Neutralizing antibody titres against BoHV-1 (seroneutralization assay) per group in Sub-study 3 (Groups A to C) on day 0 (DO), 21 (D21 ), 66 (D66) and 87 (D87) of the study. The log? SN titers of neutralizing antibodies against BoHV-1 is represented on the ordinates, for each day of the study on the abscissas per group.

[0033] Figure 19. Titers of total anti-BVDV-1 antibodies quantified by ELISA per group (Groups A and B) from day -1 (D-1 ) to day 407 (D407) of the study. The S / P ratio of ELISA titers of total antibodies against BVDV-1 is represented on the ordinates for each day of the study represented on the abscissas.

[0034] Figure 20. Titers of neutralizing anti-BVDV-1 antibodies (seroneutralization assay) per group (Groups A and B) from day -1 (D-1 ) to day 407 (D407) of the study. The log? SN titers of neutralizing antibodies against BVDV-1 is represented on the ordinates for each day of the study on the abscissas.

[0035] Figure 21. Average titer of I RPC for interferon gamma (I RPC, relative index per cent) on the day of challenge (D344) and seven days after the challenge (D351 ). The average I RPC for interferon gamma is represented on the ordinates for each day of the study on the abscissas per group (Groups A and B).

[0036] Figure 22. BVDV-1 viremia (total virus titer, as average titer) per group from the day of the experimental infection (DO) to 21 days after the experimental infection (D21 ). The average virus titer (total virus) is represented on the ordinates for each day after the experimental infection represented on the abscissas per group (Groups A and B). DO corresponds to D344 of the study and D21 to D365 of the study.

[0037] Figure 23. Average of BVDV-1 virus titre (CCID5o / ml) per Group (A and B) and per tissue sample collected from fetuses, brain, thymus gland, liver, Peyer’s patch and spleen. The average of BVDV-1 virus titer (CCID5o / ml) is represented on the ordinates for each fetus tissue represented on the abscissas per group (Groups A and B).

[0038] Figure 24. Percentage (%) of positive samples to BVDV-1 per tissue sample collected from fetuses (brain, thymus gland, liver, Peyer’s patch and spleen) and per group (Groups A and B). The percentage of positive fetal samples to BVDV-1 virus is represented on the ordinates for each fetus tissue represented on the abscissas per group (Groups A and B).

[0039] Figure 25: Titers of total anti-BVDV-2 antibodies quantified by ELISA per group (Groups A and B) from day 0 (DO) to day 442 (D442) of the study. The S / P ratio of ELISA titers of total antibodies against BVDV-2 is represented on the ordinates for each day of the study represented on the abscissas.

[0040] Figure 36. Neutralizing anti-BVDV-2 antibody titers (seroneutralization assay) per group (Groups A and B) from day 0 (DO) to day 442 (D442) of the study. The log? SN titers of neutralizing antibodies against BVDV-2 is represented on the ordinates, for each day of the study on the abscissas.

[0041] Figure 27. Average titer of I RPC for interferon gamma (I RPC, relative index per cent), on the day of challenge (D347) and seven days after the challenge (D354). The average I RPC for interferon gamma is represented on the ordinates for each day of the study on the abscissas per group (Groups A and B).

[0042] Figure 28. BVDV-2 viremia (total virus titer, as average titer) per group from the day of the experimental infection (DO) to 21 days after the experimental infection (D21 ), which correspond from day 347 (D347) to day 368 (D368) of the study. The average virus titer (total virus) is represented on the ordinates for each day after the experimental infection represented on the abscissas per group (Groups A and B).

[0043] Figure 29. Average of BVDV-2 virus titer (CCID5o / ml) per Group (A and B) and per tissue sample collected from fetuses, brain, spleen, thymus, Peyer’s patch, and liver. The average of BVDV-2 virus titer (CCID5o / ml) is represented on the ordinates, for reach fetus tissue represented on the abscissas per group (Groups A and B).

[0044] Figure 30. Percentage (%) of positive samples to BVDV-2 per tissue sample collected from fetuses (brain, spleen, thymus gland, Peyer’s patch, and liver) per group (Groups A and B). The percentage of positive fetal samples to BVDV-2 virus is represented on the ordinates, for each fetus tissue represented on the abscissas per group (Groups A and B).

[0045] Figure 31. BVDV and IBR total antibody titers in colostrum samples. The S / P ratio of ELISA titers of total antibodies against BVDV is represented on the ordinates for each study group represented on the abscissas (left panel). IRPC (relative index per cent) for IBR is represented on the ordinates for each study group represented in the abscissas (right panel). * indicates statistically significant differences (p<0.05).

[0046] Figure 32. BVDV total antibody titers and IBR gB (glycoprotein B) antibody titers in serum samples. (A) The S / P ratio of ELISA titers of total antibodies against BVDV is represented on the ordinates for each study group represented on the abscissas. (B) ELISA titers are represented as % IN on the ordinates for each study group represented in the abscissas * indicates statistically significant differences (p<0.05).

[0047] Figure 33. BVDV-1 , BVDV-2 and IBR neutralizing antibodies titers in newborn serum samples. The log? titers of neutralizing (SN) antibodies is represented on the ordinates for each study group on the abscissas.* indicate statistically significant differences (* p<0.05, ** p<0.01 ).

[0048] DETAILED DESCRIPTION OF THE INVENTION

[0049] 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.

[0050] The terms “about” and “approximately” shall generally mean an acceptable degree of variation for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of variation 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.

[0051] As such, the present invention relates to an immunogenic or vaccine composition for use in a method of treatment and / or prevention of Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or Bovine Viral Diarrhoea Virus type 2 (BVDV-2) infection in cattle, from here onwards the medical use of the invention, where the immunogenic or vaccine composition comprises a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain.

[0052] The terms “immunogenic”, “immunogenic component” or “immunogenic composition” refer to a component that is capable of eliciting, establishing, inducing or improving an immune response in a subject of a cellular or antibody-mediated immune response type upon its administration to the subject. An “immunogenic composition” comprises molecules with antigenic properties, such as killed, inactivated 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. An “antigenic” portion of a polypeptide, also referred herein to as an epitope, can be that portion that is immunodominant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conjugating the “antigenic” portion to a carrier polypeptide for immunization. It is to be understood, that in the present invention, the immunogenic composition for use according to the invention comprises at least two immunogenic polypeptides, a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain, and said immunogenic components elicit an immune response against bovine respiratory disease (BRD) caused by BVDV-1 and / or BVDV-2 in a subject. The immunogenic composition of the medical use according to the present invention also comprises further immunogenic components capable of eliciting, establishing, inducing or improving an immune response in a subject, either of cellular or antibody-mediated immune response upon administration to the subject. The immunogenic composition of the medical use according to the present invention also comprises further immunogenic components against bovine respiratory disease (BRD) capable of eliciting, establishing, inducing or improving an immune response in a subject, either of cellular or antibody-mediated immune response upon administration to the subject.

[0053] The term “Bovine Respiratory Disease”, or its acronym “BRD”, as used herein, refers to a multifactorial syndrome, where several bacterial and viral pathogens are involved. Although referred as a respiratory disease, BRD clinical signs can affect the respiratory system, the digestive track, the reproductive system, the neurological system, the immune system, cardiovascular system, circulatory system, amongst others. In the context of the present invention, the Bovine Respiratory Disease (BRD) encompasses all possible clinical signs and / or symptoms which are a cause or a consequence of the disease, in particular it encompasses the clinical signs and / or symptoms in the respiratory system, the reproductive system, the immune system and the neurological system, in subjects from any age and any gender. Examples of clinical signs which can be caused by BRD or are a consequence of BRD include, without limitation, fever of over 39.5°C, difficulty of breathing at several degree of severity, nasal discharge, varying degrees of depression, including drooping ears, an extended head, a bowed back, and / or self-isolation from other cattle, reduction or loss of appetite, rapid, shallow breathing, increased lung sounds, coughing, and pneumonia, ataxia / lack of voluntary coordination of muscle movements, tremors, a wide stance, stumbling, failure to nurse, and any combination thereof. In severe cases the calf may die, thus, mortality is also associated to BRD. Transient infections include diarrhea, calf pneumonia, decreased milk production, reproductive disorders, increased occurrence of other diseases, and death. The losses from fetal infection include abortions, congenital defects, weak and abnormally small calves, unthrifty, persistently infected (PI) animals, and death among PI animals. Other clinical signs and / or conditions associated to infection are viremia, virus shedding, leukopenia, hyperthermia, lung lesions, fetal or transplacental infection, and birth of persistently infected (PI) calves. In a particular embodiment of the medical use of the invention, the immunogenic or vaccine composition treats and / or prevents the clinical signs associated with Bovine Respiratory Disease (BRD), wherein the clinical signs are selected from a group consisting of: viremia, virus shedding, leukopenia, lung lesions, hyperthermia and any combination thereof.

[0054] In a particular embodiment of the medical use of the invention, the immunogenic or vaccine composition treats and / or prevents the clinical signs associated with Bovine Respiratory Disease (BRD), wherein the clinical signs are selected from a group consisting of: abortions, congenital defects, cerebellar hypoplasia, encephalitis, weak and abnormally small calves, unthrifty, persistently infected (PI) calves, mortality, failure to nurse, early embryonic death, fetal mummification, fetal malformations, fetal transfer, transplacental fetal infection, and transient infertility.

[0055] The term “vaccine” or “vaccine composition”, as used herein, refers to an immunogenic composition that when administered to the subject, elicits or is able to elicit, establish, induce or improve, directly or indirectly, an immune response in a subject to a particular disease of cellular or antibody-mediated type that is protective. A vaccine or vaccine composition typically contains an immunogenic component 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 bovine respiratory disease-causing microorganism. It is to be understood, that in the present invention, in the vaccine composition of the medical use according to the invention, the immunogenic component which is able to elicit an immune response to a particular disease is a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain and said immunogenic component elicits an immune response towards the disease-causing microorganism BVDV-1 and / or BVDV-2 whose infection causes bovine respiratory disease (BVD). Particularly, the vaccine compositions of the present invention elicit an immunological response in the host of a cellular or antibody-mediated type upon administration to the subject that it is protective against BVDV-1 and / or BVDV-2. The term “combination vaccine” means that the vaccine compositions of the present invention contains various immunogenic components (antigens) in a single preparation, protecting against at least two or more diseases or against one disease caused by two or more microorganisms. Thus, the vaccine compositions of the invention comprise as “active principle” an “immunogenic composition”, according to the invention.

[0056] The immunogenic or vaccine composition of the medical use 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”, “immunogenic 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 immunogenic or vaccine compositions of the medical use of the invention are effective for use in a method of treatment and / or prevention of Bovine Respiratory Disease (BRD) caused by Bovine Viral Diarrhoea Virus type 1 (BVDV- 1 ) and / or Bovine Viral Diarrhoea Virus type 2 (BVDV-2) infection in cattle. In a particular embodiment of the immunogenic or vaccine composition of the medical use of the invention, the immunological response is protective. In another particular embodiment, the immunological response is protective against bovine respiratory disease (BRD). In a further embodiment of the immunogenic or vaccine composition of the medical use of the invention, the vaccine compositions of the invention are for use in a method of treatment and / or prevention of Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or Bovine Viral Diarrhoea Virus type 2 (BVDV-2) infection in cattle. In another embodiment of the immunogenic or vaccine composition of the medical use of the invention, the vaccine compositions of the invention are for use in a method for reducing the incidence, severity, frequency and / or duration of clinical signs and disease caused by Bovine Viral Diarrhoea Virus type 1 (BVDV-

[0057] 1 ) and / or Bovine Viral Diarrhoea Virus type 2 (BVDV-2) in cattle.

[0058] The medical use of the invention refers to the treatment and / or prevention of Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or Bovine Viral Diarrhoea Virus type 2 (BVDV-

[0059] 2) infection in cattle. The terms “Bovine Viral Diarrhoea Virus” or its acronym “BVDV” or “Bovine Viral Diarrhea Virus” are used interchangeably herein and refer to all viruses belonging to genotypes BVDV-1 and BVDV-2 in the genus Pestivirus within the family Flaviviridae. The classical BVDV type 1 strains and the more recently recognized BVDV type 2 strains display some limited but distinctive differences in nucleotide and amino acid sequences. These differences are immunologically distinguishable by antibodies. 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 BVDV virus. According to the proposed classification and nomenclature there are two genotypes among Bovine Viral Diarrhoea Viruses (BVDV), BVDV-1 and BVDV-2 (Ridpath, J. F. et al, Virology 1994, 205, 66-74). The BVDV genome is approximately 12.5 kb in length and contains one large open reading frame (ORF). The ORF codes for a large polyprotein of approximately 450 kDa which is processed co- and post-translationally by both host and viral proteases. The N-terminal end of standard BVDV polyprotein results in a non-structural protein p20 (Npro), capsid protein p14 (C); envelope glycoproteins gp48 (E0), gp25 (E1 ), gp53 (E2); non-structural proteins p125 (NS23), p10 (NS4A), p32 (NS4B), p58 (NS5A) and p75 (NS5B). BVDV may exist in two biotypes, cytopathic and non-cytopathic. The two biotypes differ by the production of an 80 kDa polypeptide (non-structural protein p80, NS3) by the cytopathic BVDV strains.

[0060] The term “Bovine Viral Diarrhea Virus type 1 ” or its acronym “BVDV-1” as used herein refers to Bovine Viral Diarrhea Virus of type 1 including non-cytopathic (nep) and cytopathic (cp) biotypes, classified according to whether they produce visible change in cell cultures, as described in Mosena A.C.S. et al. (2.022, 299, 114328). The BVDV-1 can be classified into at least 22 subgenotypes (1 a to 1 u) due to the genetic diversity among pestiviruses. The term “BVDV-1 ” includes any sub-species associated to the genetic diversity, namely from subgentoype 1 a to subgenotype 1 u.

[0061] The term “Bovine Viral Diarrhea Virus type 2” or its acronym “BVDV-2” as used herein refers to Bovine Viral Diarrhea Virus of type 2 including non-cytopathic (nep) and cytopathic (cp) biotypes, classified according to whether they produce visible change in cell cultures, as described in Mosena A.C.S. et al. (2022, 299, 114328). The BVDV-2 can be classified into at least 4 subgenotypes (2a to 2d) due to the genetic diversity among pestiviruses. The term “BVDV-2” includes any sub-species associated to the genetic diversity, namely from subgenotype 2a to subgenotype 2d.

[0062] As used herein, the terms "to treat", “treating” or "treatment" refer, and include without limitation, therapeutic treatment, the purpose of which is to reverse, restraining, slowing, reduce, suppress, delay or stop the progression or severity of an existing symptom, clinical sign, disorder, or 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 and the severity of said condition associated to the disease or disorder. Treatment is usually "effective" when at least 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 associated to the disease, 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 at least one or more symptoms or clinical signs, a reduction in the extent of the disease, a reduction in the duration or frequency of the condition associated to the disease, a stabilization (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), including protection from the disease. The term "treatment" of a disease also includes providing relief from symptoms, clinical signs or side effects of the disease (including symptomatic treatment). The immunogenic and vaccine compositions of the medical use of the invention are for use in a method of treatment of Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or Bovine Viral Diarrhoea Virus type 2 (BVDV-2) infection in cattle.

[0063] The terms “prevention”, “to prevent”, “preventing” or “prophylaxis”, as used herein, include, without limitation, the capacity to prevent, decrease, reduce, minimize, or delay the onset or development of a disease or condition before its onset. It also refers ameliorating the risk of a symptom, clinical sign, disorder, condition, or disease. The term prevention also includes protecting the subject from a symptom, disorder, condition, or disease. The immunogenic and vaccine compositions of the medical use of the invention are for use in a method of prevention and may be applied or administered prophylactically. The immunogenic and vaccine compositions of the medical use of the invention are for use in a method of prevention of Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or Bovine Viral Diarrhoea Virus type 2 (BVDV-2) infection in cattle.

[0064] In the present context the disease is bovine respiratory disease (BRD), both respiratory and reproductive disease. The term “prevention”, as used herein, also relates to resistance to new infection, where thanks to the administration of the immunogenic or vaccine 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 BVDV-1 and / or BVDV-2, a delay in the onset of viremia, reduced viral persistence, a reduction of virus shedding, a reduction in the overall viral load and / or a reduction of viral excretion, and reduction or prevention of mortality.

[0065] The term “prevention” as used herein is also meant to include the term “protection”. In a particular embodiment of the medical use of the invention the treatment and / or prevention of BVDV-1 and / or BVDV-2 infection in cattle confers protection against bovine respiratory disease (BRD) in the cattle. In another embodiment of the medical use of the invention the treatment and / or prevention confers protection against Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or Bovine Viral Diarrhoea Virus type 2 (BVDV-2) infection in cattle.

[0066] The term “confers” is also meant to include terms such as provides, elicits, induces, furnishes, and other equivalent terms. The expression “confers protection against bovine respiratory disease in the cattle” and “confers protection against Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or Bovine Viral Diarrhoea Virus type 2 (BVDV-2) infection in cattle” in the present context refers to the protective immunological (memory) response, such that resistance to new infection by BVDV strains, either BVDV-1 or BVDV-2, or other virus causing BRD disease, is enhanced and / or the clinical severity of the BRD disease reduced or prevented. In a particular embodiment of the medical use of the invention, the BRD is caused by infection with a virus selected from a group consisting of: Bovine Respiratory Syncytial Virus (BRSV), Parainfluenza-3 virus (PI-3), Bovine Herpesvirus type 1 (BoHV-1 ) and any combination thereof.

[0067] In a particular embodiment of the medical use of the invention, the treatment and / or prevention of BVDV-1 and / or BVDV-2 infection is the reduction of the clinical signs and disease associated with BVDV-1 and / or BVDV-2 infection. In another particular embodiment of the medical use of the invention, the treatment and / or prevention of BVDV-1 and / or BVDV-2 is the reduction of the clinical sings and disease associated with the respiratory and reproductive disease.

[0068] The term “clinical sign” or “clinical symptom” or “clinical disease” as used herein refers to objective evidence of disease or condition caused by an infection or effects which promote the infection caused by a virus associated with Bovine Respiratory Disease (BRD), affecting respiratory and / or reproductive parameters. Symptoms and / or clinical signs associated with an infection and the evaluations of such symptoms vary depending upon the infectious agent, and are routine and known in the art. For instance, examples of conditions and / or clinical signs associated with BVDV-1 and / or BVDV-2 infection, and infection by other agents causing BRD, such as PI-3, BRSV and BoHV-1 , are amongst others, fever of over 39.5°C, difficulty of breathing at several degree of severity, nasal discharge, varying degrees of depression, including drooping ears, an extended head, a bowed back, and / or self-isolation from other cattle, reduction or loss of appetite, rapid, shallow breathing, increased lung sounds, coughing, and pneumonia, ataxia / lack of voluntary coordination of muscle movements, tremors, a wide stance, stumbling, failure to nurse, and any combination thereof. In severe cases the calf may die, thus mortality is also a clinical sign associated to BRD. Transient infections include diarrhea, calf pneumonia, decreased milk production, reproductive disorders, increased occurrence of other diseases, and death. The losses from fetal infection include abortions, congenital defects, weak and abnormally small calves, unthrifty, persistently infected (PI) animals, and death among PI animals. Other clinical signs and / or conditions associated to infection are viremia, virus shedding, leukopenia, hyperthermia, lung lesions, fetal or transplacental infection, and birth of persistently infected (PI) calves. The expression “associated with BVDV-1 and / or BVDV- 2 infection” as used herein refers to clinical signs and / or conditions which are caused or a consequence, directly or indirectly, of the infection. In a particular embodiment of the medical use of the invention, the treatment and / or prevention of BVDV-1 and / or BVDV-2 infection is the reduction of the clinical signs associated with BVDV-1 and / or BVDV-2 infection, wherein the clinical signs and / or conditions associated with BVDV-1 and / or BVDV- 2 infection are selected from a group consisting of: viremia, viral shedding, viremia and viral shedding, viral excretion, viremia and viral excretion, viral shedding and viral excretion, viremia and viral shedding and viral excretion, leukopenia, viremia and leukopenia, viral shedding and leukopenia, viremia and viral shedding and leukopenia, viral excretion and leukopenia, viremia and viral excretion and leukopenia, viral shedding and viral excretion and leukopenia, viremia and viral shedding and viral excretion and leukopenia, hyperthermia, viremia and hyperthermia, viral shedding and hyperthermia, viremia and viral shedding and hyperthermia, viral excretion and hyperthermia, viremia and viral excretion and hyperthermia, viral shedding and viral excretion and hyperthermia, viremia and viral shedding and viral excretion and hyperthermia, leukopenia and hyperthermia, viremia and leukopenia and hyperthermia, viral shedding and leukopenia and hyperthermia, viremia and viral shedding and leukopenia and hyperthermia, viral excretion and leukopenia and hyperthermia, viremia and viral excretion and leukopenia and hyperthermia, viral shedding and viral excretion and leukopenia and hyperthermia, viremia and viral shedding and viral excretion and leukopenia and hyperthermia, lung lesions, viremia and lung lesions, viral shedding and lung lesions, viremia and viral shedding and lung lesions, viral excretion and lung lesions, viremia and viral excretion and lung lesions, viral shedding and viral excretion and lung lesions, viremia and viral shedding and viral excretion and lung lesions, leukopenia and lung lesions, viremia and leukopenia and lung lesions, viral shedding and leukopenia and lung lesions, viremia and viral shedding and leukopenia and lung lesions, viral excretion and leukopenia and lung lesions, viremia and viral excretion and leukopenia and lung lesions, viral shedding and viral excretion and leukopenia and lung lesions, viremia and viral shedding and viral excretion and leukopenia and lung lesions, hyperthermia and lung lesions, viremia and hyperthermia and lung lesions, viral shedding and hyperthermia and lung lesions, viremia and viral shedding and hyperthermia and lung lesions, viral excretion and hyperthermia and lung lesions, viremia and viral excretion and hyperthermia and lung lesions, viral shedding and viral excretion and hyperthermia and lung lesions, viremia and viral shedding and viral excretion and hyperthermia and lung lesions, leukopenia and hyperthermia and lung lesions, viremia and leukopenia and hyperthermia and lung lesions, viral shedding and leukopenia and hyperthermia and lung lesions, viremia and viral shedding and leukopenia and hyperthermia and lung lesions, viral excretion and leukopenia and hyperthermia and lung lesions, viremia and viral excretion and leukopenia and hyperthermia and lung lesions, viral shedding and viral excretion and leukopenia and hyperthermia and lung lesions, viremia and viral shedding and viral excretion and leukopenia and hyperthermia and lung lesions.

[0069] In a particular embodiment of the medical use of the invention, the treatment and / or prevention of BVDV-1 and / or BVDV-2 infection is the reduction of the clinical signs caused by BVDV-1 and / or BVDV-2 infection, wherein the clinical signs associated with BVDV-1 and / or BVDV-2 infection are selected from a group consisting of: viremia, virus shedding, virus excretion, leukopenia, hyperthermia, lung lesions, and any combination thereof.

[0070] The terms “reproductive condition”, “reproductive disease”, “reproductive parameters” or “reproductive clinical signs” as used herein refer to objective evidence of disease or condition caused by an infection or effects which promote the infection caused by a virus associated with Bovine Respiratory Disease (BRD), affecting the reproductive system in a subject. Examples of reproductive conditions and / or reproductive clinical signs associated with BVDV-1 and / or BVDV-2 infection, and BRD-causing organisms, such as PI-3, BRSV and BoHV-1 , are without limitation, abortions; congenital defects, wherein the most commonly recognized birth defect is cerebellar hypoplasia, encephalitis and generalized disease in the neonate, weak and abnormally small calves, unthrifty, persistently infected (PI) subjects, and death among PI subjects, failure to nurse, early embryonic death, fetal mummification, fetal malformations, fetal transfer, transplacental fetal infection, and transient infertility.

[0071] In a particular embodiment of the medical use of the invention, the treatment and / or prevention of BVDV-1 and / or BVDV-2 infection is the reduction of the clinical signs caused by BVDV-1 and / or BVDV-2 infection, wherein the clinical signs associated with BVDV-1 and / or BVDV-2 infection are selected from a group consisting of: abortions, congenital defects, cerebellar hypoplasia, encephalitis, weak and abnormally small calves, unthrifty, persistently infected (PI) calves, mortality, failure to nurse, early embryonic death, fetal mummification, fetal malformations, fetal transfer, transplacental fetal infection, and transient infertility.

[0072] In a particular embodiment of the medical use of the invention, the clinical signs are clinical signs which promote infection or clinical signs which are caused by the infection. The expression “clinical signs which promote infection” refer to clinical signs detected in a subject which not only promote the development of the infection in the subject wherein the clinical signs were detected but further promote the transmission of viruses to other subjects such as viraemia, virus shedding and / or virus excretion, transplacental infection, and persistently infected calves. The expression “clinical signs which are caused by the infection” as used herein refer to clinical signs which are a consequence of the infection, such as leukopenia, hyperthermia, lung lesions, mortality, and persistently infected calves, and any combination thereof.

[0073] In a particular embodiment of the medical us of the invention, the treatment of BVDV- 1 and / or BVDV-2 infection is the reduction of the clinical signs caused by BVDV-1 and / or BVDV-2 infection, wherein the clinical signs associated with BVDV-1 and / or BVDV-2 infection are selected from a group consisting of: leukopenia, hyperthermia, lung lesions and any combination thereof. In a particular embodiment of the medical use of the invention, the treatment of BVDV-1 and / or BVDV-2 infection is the reduction of the clinical signs caused by BVDV-1 and / or BVDV-2 infection, wherein the clinical signs associated with BVDV-1 and / or BVDV-2 infection are selected from a group consisting of abortions, congenital defects, cerebellar hypoplasia, encephalitis, weak and abnormally small calves, unthrifty, persistently infected (PI) calves, mortality, failure to nurse, early embryonic death, fetal mummification, fetal malformations, fetal transfer, transplacental fetal infection, and transient infertility. In another particular embodiment of the medical use of the invention, the prevention of BVDV-1 and / or BVDV-2 infection is the reduction of the clinical signs which promote the BVDV-1 and / or BVDV-2 infection, wherein the clinical signs are selected from viremia, virus shedding and / or virus excretion, transplacental infection and / or persistently infected calves. In another particular embodiment of the medical use of the invention, the prevention of BVDV-1 and / or BVDV-2 infection is the reduction of the clinical signs which promote the BVDV-1 and / or BVDV-2 infection, wherein the clinical signs are selected from the group consisting of abortions, congenital defects, cerebellar hypoplasia, encephalitis, weak and abnormally small calves, unthrifty, persistently infected (PI) calves, mortality, failure to nurse, early embryonic death, fetal mummification, fetal malformations, fetal transfer, transplacental fetal infection, and transient infertility.

[0074] The expression “reduction of clinical signs” as used herein refers to the improvement or alleviation of an objective symptom attributable to the infection caused by BVDV-1 and / or BVDV-2 in a subject receiving or having received the immunogenic or vaccine composition comprising a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain in comparison with a subject infected by BVDV-1 and / or BVDV-2 who did not received the immunogenic or vaccine composition, wherein said comparison can be expressed in percentage or fraction values, without limitation. In a particular embodiment of the medical use of the invention, the reduction of the clinical sign is by at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, 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%, at least 100% compared to subjects not receiving nor having received the immunogenic or vaccine composition. The expression “reduction of clinical signs” is to be understood as comprising the expressions reduction of viremia, reduction of virus shedding, reduction of virus excretion, reduction of leukopenia, reduction of lung lesions, reduction of hyperthermia, reduction of mortality, reduction of transplacental fetal infection, and reduction of persistently infected calves.

[0075] The term "reduction of viremia" as used herein refers to the reduction of virus entering the bloodstream of an animal, wherein the viremia level, i.e. the number of virus DNA or RNA copies per ml_ of blood or the number of plaque forming colonies per deciliter of blood, is reduced in the blood and associated tissues of cattle receiving or having received the immunogenic or vaccine composition comprising a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain of the present invention by at least 10% in comparison to cattle not receiving nor having received the composition. In a particular embodiment of the medical use of the invention, the viremia is reduced in cattle receiving or having received the immunogenic or vaccine composition comprising a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain of the present invention by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably by at least 99.9%, more preferably by at least 99.99%, and even more preferably by at least 99.999% compared to cattle not receiving nor having received the immunogenic or vaccine composition.

[0076] The terms “reduction of virus / viral shedding” and “reduction of virus / viral excretion” are used interchangeably herein, and refer to the reduction of the expulsion and release of virus progeny following successful reproduction during a host cell infection and the reduction of the release of the virus from the subject by excretion of the virus in tissues, biological fluids, such as saliva, urine, feces, sweet, lacrimal fluid, and excretion in exhaled air of cattle receiving or having received the immunogenic or vaccine composition comprising a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain of the present invention by at least 10% in comparison to cattle not receiving nor having received the composition. Virus shedding and virus excretion occurs once the host cell is exhausted of all resources in making viral progeny, after which the viruses will leave the host cell by several methods such as, without limitation, budding, apoptosis of the host cell and exocytosis and be excreted from the body by the tissues or any biological fluid. In a particular embodiment of the medical use of the invention, the virus shedding and virus excretion is reduced in cattle receiving or having received the immunogenic or vaccine composition comprising a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain of the present invention by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably by at least 99.9%, more preferably by at least 99.99%, and even more preferably by at least 99.999% compared to cattle not receiving nor having received the immunogenic or vaccine composition.

[0077] The term “reduction of leukopenia” as used herein refers to reduction in the abnormal decrease of leukocytes, or white blood cells, in the blood of cattle receiving or having received the immunogenic or vaccine composition comprising a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain of the present invention by at least 10% in comparison to cattle not receiving nor having received the composition. Leukocyte is a symptom associated in general with viral infections which places the subject infected at risk of further infections or in a debilitated state unable to fight an existing infection. In a particular embodiment of the medical use of the invention, the leukopenia is reduced in cattle receiving or having received the immunogenic or vaccine composition comprising a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain of the present invention by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably by at least 99.9%, more preferably by at least 99.99%, and even more preferably by at least 99.999% compared to cattle not receiving nor having received the immunogenic or vaccine composition.

[0078] The term “reduction of lung lesion” as used herein refers to reduction in the number of macroscopy lung lesions of cattle receiving or having received the immunogenic or vaccine composition comprising a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain of the present invention by at least 10% in comparison to cattle not receiving nor having received the composition. Lung lesions, also called infectious primary pulmonary lesions, can be classified into suppurative bronchopneumonia, fibrinous, hemorrhagic and / or necrotic bronchopneumonia, bronchointerstitial pneumonia and association of suppurative pneumonia and broncho-interstitial pneumonia (Murray et al., J. Vet. Diagn. Investig. 2017, 29, 20-34). In a particular embodiment of the medical use of the invention, the number of lung lesions is reduced in cattle receiving or having received the immunogenic or vaccine composition comprising a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain of the present invention by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably by at least 99.9%, more preferably by at least 99.99%, and even more preferably by at least 99.999% compared to cattle not receiving nor having received the immunogenic or vaccine composition.

[0079] The term “reduction of hyperthermia” as used herein refers to reduction in the abnormal increase in body temperature, or fever, of cattle receiving or having received the immunogenic or vaccine composition comprising a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain of the present invention by at least 10% in comparison to cattle not receiving nor having received the composition. Hyperthermia is a symptom associated in general with viral infections which is general associated with the host defense mechanisms trying to fight off the infection. In a particular embodiment of the medical use of the invention, the hyperthermia is reduced in cattle receiving or having received the immunogenic or vaccine composition comprising a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain of the present invention by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably by at least 99.9%, more preferably by at least 99.99%, and even more preferably by at least 99.999% compared to cattle not receiving nor having received the immunogenic or vaccine composition.

[0080] Another clinical sign of BVDV-1 and / or BVDV-2 infection is transplacental fetal infection. The term “transplacental fetal infection” or “transplacental BVDV-1 and / or BVDV- 2 fetal infection” or “transplacental infection”, also known as vertical transmitted fetal infection, refers to the transmission of BVDV-1 and / or BVDV-2 virus from a pregnant female bovine to a fetus or embryo trough the placenta. In a particular embodiment of the medical use of the invention the treatment and / or prevention of BVDV-1 and / or BVDV-2 infection is the reduction of transplacental fetal BVDV-1 and / or BVDV-2 infection. In another particular embodiment of the medical use of the invention the treatment and / or prevention of BVDV- 1 and / or BVDV-2 infection is the reduction of transplacental fetal BVDV-1 and / or BVDV-2 infection, wherein the transplacental fetal BVDV-1 and / or BVDV-2 infection is reduced in pregnant cattle receiving or having received the immunogenic or vaccine composition comprising a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain of the present invention by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably by at least 99.9%, more preferably by at least 99.99%, and even more preferably by at least 99.999%, in comparison with pregnant cattle not receiving nor having received the immunogenic or vaccine composition.

[0081] In another particular embodiment of the medical use of the invention, the prevention of BVDV-1 and / or BVDV-2 infection is the prevention of transplacental BVDV-1 and / or BVDV-2 infection. In yet another particular embodiment of the medical use of the invention, the prevention of BVDV-1 and / or BVDV-2 infection is the protection of a fetus or embryo against transplacental BVDV-1 and / or BVDV-2 infection.

[0082] A further consequence of infection by BVDV-1 and / or BVDV-2 is the birth of “persistently infected calves”, due to the vertical transmission of the virus from the mother to the fetus / embryo / calf, either by the placenta or during birth. In a particular embodiment of the medical use of the invention the treatment and / or prevention of BVDV-1 and / or BVDV- 2 infection is the reduction of the birth of persistently BVDV-1 and / or BVDV-2 infected calves, wherein the birth of persistently BVDV-1 and / or BVDV-2 infected calves is reduced in cattle receiving or having received the immunogenic or vaccine composition comprising a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain of the present invention by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably by at least 99.9%, more preferably by at least 99.99%, and even more preferably by at least 99.999%, in comparison with cattle not receiving nor having received the immunogenic or vaccine composition.

[0083] In another particular embodiment of the medical use of the invention, the prevention of BVDV-1 and / or BVDV-2 infection is the prevention of the birth of persistently infected calves. In yet another particular embodiment of the medical use of the invention, the prevention of BVDV-1 and / or BVDV-2 infection is the protection of calves from being persistently infected. In yet another particular embodiment of the medical use of the invention, the prevention of BVDV-1 and / or BVDV-2 infection is the protection of calves from being persistently infected including complete fetal protection against horizontal transfer of BVDV-1 and / or BVDV-2.

[0084] As a consequence of the immunogenic or vaccine composition for use of the invention, newborn calves may obtain protection from BVDV-1 and / or BVDV-2 infection due to the maternally-derived antibodies (MDA) which are passed to the calf via the colostrum. Therefore, in a particular embodiment of the medical use of the invention, the prevention is the prevention of the progeny of a pregnant cow from a BVDV-1 and / or BVDV-2 infection by passive immunization by the colostrum. The term “colostrum” refers to the lactation product known as colostrum milk produced by a female mammal after birth usually for about 5 to 7 days after parturition. In the context of the present invention the term “colostrum” means bovine colostrum, and particularly colostrum produced by a cow after parturition of its progeny which contains high levels of immunoglobulins, antimicrobial peptides, and growth factors among other nutritional and functional components. Colostrum is important for supporting the growth, development, and immunologic defense of newborn mammals. In a particular embodiment of the medical use of the invention, the prevention is the prevention of the progeny of a pregnant cow from a BVDV-1 and / or BVDV-2 infection by passive immunization. In another particular embodiment of the medical use of the invention, the prevention is the prevention of the progeny of a pregnant cow from a BVDV-1 and / or BVDV-2 infection by passive immunization by the colostrum.

[0085] The term “circulating antibodies”, as used herein, refers to antibodies previously present in a subject as a response to an infection with any viral microorganism causing bovine respiratory disease (BRD) by having developed either their own active immune response or by acquired (passive) immune response of maternal origin, namely maternally derived antibodies also known as MDAs. The term “maternally derived antibodies”, or its acronym “MDAs”, as used herein, also refers to antibodies which are inherited by the offspring of the mother and derived from exposure of said mother to natural infections or to previous inoculation with immunogenic or vaccine compositions, including the immunogenic or vaccine compositions of the present invention. 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 is caused by Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or Bovine Viral Diarrhoea Virus type 2 (BVDV-2). In a particular embodiment of the medical use of the invention, the circulating anti-BVDV-1 and / or anti-BVDV-2 antibodies are maternally derived antibodies (MDAs).

[0086] In a particular embodiment of the medical use of the invention, the cattle has circulating maternally-derived antibodies (MDA) anti-Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or maternally-derived antibodies (MDA) anti-Bovine Viral Diarrhoea Virus type 2 (BVDV-2). The immunogenic or vaccine composition of the medical use of the invention comprises the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2).

[0087] 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 may have 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. The “recombinant protein / polypeptide” can be obtained from methods known in the art. Said methods generally include cloning at least one protein / polypeptide into an expression vector, preferably a plasmid, transfecting eukaryotic or prokaryotic cells with said plasmid vector, expressing said protein / polypeptide in said cells and purifying the at least one protein / polypeptide from the cells or from their supernatant.

[0088] The recombinant DNA technology is known from the art and standard recombinant techniques can be carried out as described in well-known manuals to the skilled person in the art such as, for example, J. Sambrook and D. W. Russell, Molecular Cloning: A laboratory manual, 4th edition, Cold Spring Harbor Laboratory Press, New York, 2012). In addition to use in recombinant expression systems, the protein / polypeptides of interest can also be used to transform viruses that transfect host cells in animals. Live attenuated viruses, such as vaccinia or adenovirus, are other alternatives for preparing immunogenic and vaccine compositions comprising recombinant proteins / polypeptides because they are inexpensive to produce and are easily transported and administered. Other expression systems useful to produce recombinant protein / polypeptide of the invention are the use of mammalian cells (such as CHO cells, HEK293 cells, CAP (human aminocyte cells), bacteria (for example Escherichia coli) or yeast cells such as Pichia spp. and Saccharomyces spp.

[0089] In a particular embodiment of the medical use of the invention, the recombinant E2 protein of BVDV-1 and E2 protein of BVDV-2 are produced in a mammalian host cell expression system. In a particular embodiments of the medical use of the invention, the mammalian cell is a CHO cell (Chinese hamster ovarian), a CAP cell (human aminocyte), a PER.C6 cell (human retina), a NSO or Sp2 / 0 cell (mouse myeloma), an EB66 cell (duck), a BHK cell (hamster), a freestyle HEK 293-F, a HEK 293 6E or a HEK 293T cell (human embryonic kidney). In another particular embodiment of the medical use of the invention, the mammalian host cell is a CAP cell.

[0090] Typical transfection and cell growth methods can be used to culture the cells. Expression vectors, e.g., vectors comprising polynucleotides that encode fusion proteins, can be transfected into host cells according to methods well known in the art. For example, introducing nucleic acids into eukaryotic cells can be achieved by calcium phosphate coprecipitation, electroporation, microinjection, lipofection, and transfection employing polyamine transfection reagents. In one particular embodiment of the medical use of the invention, the expression vector is a plasmid vector.

[0091] “An expression vector” as used herein is a plasmid or virus designed for gene expression in cells. The expression vector is used to introduce a specific gene of interest into a target cell, and can commandeer the cell's mechanism to produce the protein encoded by the gene. In a particular embodiment of the medical use of the present invention, the plasmid vector is a mammalian expression vector.

[0092] An expression vector as used herein comprises at least one expression cassette. As used herein an “expression cassette” refers to a component of the expression vector which contains the gene of interest under the control of the regulatory sequences for expression.

[0093] In a particular embodiment of the medical use of the invention, the recombinant E2 protein of BVDV-1 and E2 protein of BVDV-2 once expressed is further collected and purified from the culture supernatant.

[0094] 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 of the medical use of the invention, the purified recombinant E2 protein of BVDV-1 and recombinant E2 protein of BVDV-2 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 of the medical use of the invention, the purified recombinant E2 protein of BVDV-1 and recombinant E2 protein of BVDV-2 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 of the medical use of the invention, purified means that the level of contaminants is below a level acceptable for administration to a subject. In other particular embodiment of the medical use of the invention, the term purified protein means a protein suitable for pharmaceutical use. Therefore, in the context of the present invention the terms “recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 )” and “recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2)” refer to an artificially or synthetically produced recombinant E2 protein of a BVDV-1 or a E2 protein of a BVDV-2 strain, which may be isolated and purified. In a particular embodiment of the medical use of the invention, the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain and the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain are isolated. In a particular embodiment of the medical use of the invention, the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain and the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2) are isolated and purified.

[0095] In the context of the present invention the terms “E2 protein” or “E2 structural protein” or “E2 glycoprotein” or “BVDV E2 protein”, as used herein, refer to a membrane-anchored type I glycoprotein which derives from the proteolytic cleavage of the polyprotein encoded by the viral BVDV genome. E2 is one of the three glycosylated envelope proteins of the BVDV virus, the other proteins being Erns and E1. In the context of the present invention the “E2 protein” refers both to the E2 protein of a BVDV type 1 (BVDV-1 ) and of a BVDV type 2 virus (BVDV-2). In a particular embodiment of the medical use of the invention the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain comprises the sequence according to SEQ ID NO: 1 or a functionally equivalent variant thereof. In another particular embodiment of the medical use of the invention the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain comprises the sequence according to SEQ ID NO: 2 or a functionally equivalent variant thereof. In another particular embodiment of the medical use of the invention, the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain consists of the sequence according to SEQ ID NO: 1. In another particular embodiment of the medical use of the invention the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain consists of the sequence according to SEQ ID NO: 2.

[0096] SEQ ID NO: 1

[0097] DLDCKPEFSYAIARDERIGQLGAEGLTTTWKDYSPEMKLEDTMVIAWCKDGKFTYL PRCTRETRYLAILHTRALPTSWFKKLFDGRKQEDVVEMDDNFEFGLCPCDAKPIVRGKF NTTLLNGSAFQMVCPIGWTGTVSCMSFNMDTLATTVIRTYRRSKPFPHRQGCITQKTLGE DLHNCILGGNWTCVPGDMLLYKGGSIESCKWCGYQFKESEGLPHYPIGKCRLENETGYR LVDDTSCDREGVAIVPQGTLRCKIGKTTIQVIAMDTKLGPMPCRPYEIISSEGPVERTACTF NYTKTLKNKYFEPRDSYFQQYMLKGEYQYWFDLEVTDHHRD

[0098] SEQ ID NO: 2

[0099] FPECKEGFQYAISKDRKMGLLGPESLTTTWHLPTKKIVDSMVHVWCEGKDLKILKT CTKEERYLVAVHERALSTSAEFMQISDGTIGPDVIDMPDDFEFGLCPCDSKPVIKGNFNAS LLNGPAFQMVCPQGWTGTIECTLANQDTLDTTVVRTYRRTTPFQRRKWCTYEKIIGEDIH ECILGGNWTCITGNHSRLKDGPIKKCKWCGYDFVNSEGLPHYPIGKCMLINESGYRYVD DTSCDRGGVAIVPTGTVKCRIGNVTVQVIATNNDLGLMPCSPAEVIASEGPVEKTACTFNY SRTLPNKYYEPRDRYFQQYMLKGEWQYWFDLDSVDHHKD

[0100] In a particular embodiment of the medical use of the invention, the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain comprises the sequence encoded by the sequence according to SEQ ID NO: 3 or a functionally equivalent variant of said sequence encoded by the sequence according to SEQ ID NO: 3. In another particular embodiment of the medical use of the invention the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain comprises the sequence encoded by the sequence according to SEQ ID NO: 4 or a functionally equivalent variant of said sequence encoded by the sequence according to SEQ ID NO: 4. In another particular embodiment of the medical use of the invention, the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain consists of the sequence encoded by the sequence according to SEQ ID NO: 3. In another particular embodiment of the medical use of the invention the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain consists of the sequence encoded by the sequence according to SEQ ID NO: 4.

[0101] SEQ ID NO: 3

[0102] GACCTGGACTGCAAGCCCGAGTTCAGCTACGCCATCGCCAGGGACGAGAGGA

[0103] TCGGCCAGCTGGGCGCCGAGGGCCTGACCACCACCTGGAAGGACTACAGCCCCGA

[0104] GATGAAGCTGGAGGACACCATGGTGATCGCCTGGTGCAAGGACGGCAAGTTCACCT ACCTGCCCAGGTGCACCAGGGAGACCAGGTACCTGGCCATCCTGCACACCAGGGCC CTGCCCACCAGCGTGGTGTTCAAGAAGCTGTTCGACGGCAGGAAGCAGGAGGACGT GGTGGAGATGGACGACAACTTCGAGTTCGGCCTGTGCCCCTGCGACGCCAAGCCCA TCGTGAGGGGCAAGTTCAACACCACCCTGCTGAACGGCAGCGCCTTCCAGATGGTG TG CCCCATCG G CTGGACCG GCACCGTG AG CTG CATGAG CTTCAACATG GACACCCT GGCCACCACCGTGATCAGGACCTACAGGAGGAGCAAGCCCTTCCCCCACAGGCAGG GCTGCATCACCCAGAAGACCCTGGGCGAGGACCTGCACAACTGCATCCTGGGCGGC AACTGGACCTGCGTGCCCGGCGACATGCTGCTGTACAAGGGCGGCAGCATCGAGAG

[0105] CTGCAAGTGGTGCGGCTACCAGTTCAAGGAGAGCGAGGGCCTGCCCCACTACCCCA TCGGCAAGTGCAGGCTGGAGAACGAGACCGGCTACAGGCTGGTGGACGACACCAG

[0106] CTGCGACAGGGAGGGCGTGGCCATCGTGCCCCAGGGCACCCTGAGGTGCAAGATC G GC AAG ACCACCATCCAG GTG ATCG CCATG G ACACCAAG CTGG G CCCCATG CCCTG

[0107] CAGGCCCTACGAGATCATCAGCAGCGAGGGCCCCGTGGAGAGGACCGCCTGCACCT TCAACTACACCAAGACCCTGAAGAACAAGTACTTCGAGCCCAGGGACAGCTACTTCC AGCAGTACATGCTGAAGGGCGAGTACCAGTACTGGTTCGACCTGGAGGTGACCGAC

[0108] CACCACAGGGAC

[0109] SEQ ID NO: 4

[0110] TTCCCCGAGTGCAAGGAGGGCTTCCAGTACGCCATCAGCAAGGACAGGAAGAT

[0111] GGGCCTGCTGGGCCCCGAGAGCCTGACCACCACCTGGCACCTGCCCACCAAGAAG ATCGTGGACAGCATGGTGCACGTGTGGTGCGAGGGCAAGGACCTGAAGATCCTGAA

[0112] GACCTGCACCAAGGAGGAGAGGTACCTGGTGGCCGTGCACGAGAGGGCCCTGAGC ACCAGCGCCGAGTTCATGCAGATCAGCGACGGCACCATCGGCCCCGACGTGATCGA

[0113] CATGCCCGACGACTTCGAGTTCGGCCTGTGCCCCTGCGACAGCAAGCCCGTGATCA AGGGCAACTTCAACGCCAGCCTGCTGAACGGCCCCGCCTTCCAGATGGTGTGCCCC

[0114] CAGGGCTGGACCGGCACCATCGAGTGCACCCTGGCCAACCAGGACACCCTGGACA CCACCGTGGTGAGGACCTACAGGAGGACCACCCCCTTCCAGAGGAGGAAGTGGTGC

[0115] ACCTACG AG AAG ATC ATCG G CG AG G ACATCCACG AGTG CATCCTG G GCG G CAACTG GACCTGCATCACCGGCAACCACAGCAGGCTGAAGGACGGCCCCATCAAGAAGTGCA

[0116] AGTGGTGCGGCTACGACTTCGTGAACAGCGAGGGCCTGCCCCACTACCCCATCGGC AAGTGCATGCTGATCAACGAGAGCGGCTACAGGTACGTGGACGACACCAGCTGCGA CAGGGGCGGCGTGGCCATCGTGCCCACCGGCACCGTGAAGTGCAGGATCGGCAAC

[0117] GTGACCGTGCAGGTGATCGCCACCAACAACGACCTGGGCCTGATGCCCTGCAGCCC CGCCGAGGTGATCGCCAGCGAGGGCCCCGTGGAGAAGACCGCCTGCACCTTCAAC TACAGCAGGACCCTGCCCAACAAGTACTACGAGCCCAGGGACAGGTACTTCCAGCAG TACATGCTGAAGGGCGAGTGGCAGTACTGGTTCGACCTGGACAGCGTGGACCACCA CAAGGAC

[0118] The terms “functional variant” and “functionally equivalent variant” are interchangeable and are herein understood as all those peptides derived from the E2 protein of a Bovine Viral Diarrhoea Virus type 1 or type 2 (BVDV-1 or BVDV-2) strain by means of modification, insertion and / or deletion of one or more amino acids, provided that the function of eliciting an immune response as defined above in the cattle to which is administered is substantially maintained. As used herein, functional variant is also meant to include the full-length sequence of any E2 protein of a BVDV-1 strain or a BVDV-2 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 as defined 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 E2 protein of BVDV-1 or BVDV-2 strains is also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens. See, e.g., Bergmann et al. (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.

[0119] In order to determine if a functional variant or functionally equivalent variant of a E2 protein of a Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) or of a Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain is still capable of eliciting an immune response as defined above, in the subject to which is administered, traditional experiments well known by an expert in the art may be performed, such as vaccination of cattle with the immunogenic or vaccine composition of the medical use of the invention comprising the functional or functionally equivalent E2 protein of a BVDV-1 strain and / or the E2 protein of a BVDV-2 strain and challenging the cattle with an infectious virus strain, as described in the Examples section of the present invention. In a particular embodiment of the medical use of the invention, the functionally equivalent variants of the E2 protein of a BVDV-1 strain and / or of the E2 protein of a BVDV-2 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 E2 protein of a BVDV-1 strain and / or of the E2 protein of a BVDV- 2 strain, respectively. In a particular embodiment of the medical use of the invention, the functionally equivalent variants of the E2 protein of a BVDV-1 strain and / or the E2 protein of a BVDV-2 strain are those that have a degree of identity with respect to SEQ ID NO: 1 and / or SEQ ID NO: 2 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. In a particular embodiment of the medical use of the invention, the degree of identity is calculated along the whole length of SEQ ID NO: 1 or SEQ ID NO: 2.

[0120] In a particular embodiment of the medical use of the invention, the functionally equivalent variants of the E2 protein of a BVDV-1 strain and / or of the E2 protein of a BVDV- 2 strain are those that have a degree of identity with respect to the sequence encoded by SEQ ID NO: 3 and / or SEQ ID NO: 4 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. In a particular embodiment of the medical use of the invention, the degree of identity is calculated along the whole length of the sequence encoded by SEQ ID NO: 3 or SEQ ID NO: 4.

[0121] 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.

[0122] 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.

[0123] The BVDV viruses are some of the infectious agents which cause bovine respiratory disease (BRD) among others, as previously mentioned. Therefore, in a particular embodiment of the medical use of the invention, the immunogenic or vaccine composition further comprises an immunogenic active component derived from a virus selected from the group consisting of: Bovine Respiratory Syncytial Virus (BRSV), Parainfluenza-3 virus (Pl- 3), Bovine Herpesvirus type 1 (BoHV-1 ), bluetongue disease virus (BTV), Vesicular stomatitis virus (VSV), malignant catarrhal fever virus, pseudorabies virus, rabies virus, rinderpest virus, bovine coronavirus, bovine rotavirus, bovine adenovirus, bovine rhinovirus, bovine leukosis virus, foot and mouth disease virus (FMDV), and any combination thereof. In another particular embodiment of the medical use of the invention, the immunogenic or vaccine composition further comprises an immunogenic active component derived from bacteria selected from the group consisting of: Haemophilus somnus, Haemophilus parasuis, Bordetella bronchiseptica, Bacillus anthracis, Actinobacillus pleuropneumonie, Pasteurella multocida, Mannhemia haemolytica, Mycoplasma bovis, Mycobacterium bovis, Mycobacterium paratuberculosis, Clostridial spp., Streptococcus uberis, Staphylococcus aureus, Erysipelothrix rhusopathiae, Chlamydia spp., Brucella spp., Vibrio spp., Salmonella enterica serovars, Leptospira spp., and any combination thereof.

[0124] In another particular embodiment of the medical use of the invention, the immunogenic or vaccine composition further comprises an immunogenic active component derived from a virus selected from the group consisting of: Bovine Respiratory Syncytial Virus (BRSV), Parainfluenza-3 virus (PI-3), Bovine Herpesvirus type 1 (BoHV-1 ), and any combination thereof. In a more particular embodiment of the medical use of the invention the immunogenic or vaccine composition comprises an immunogenic active component derived from a Bovine Respiratory Syncytial Virus (BRSV) strain and a Bovine Herpesvirus type 1 (BoHV-1 ) strain, or a Bovine Respiratory Syncytial Virus (BRSV) strain and a Parainfluenza-3 virus (PI-3) strain, or a Bovine Herpesvirus type 1 (BoHV-1 ) strain and a Parainfluenza-3 virus (PI-3) strain, or a Bovine Respiratory Syncytial Virus (BRSV) strain and a Bovine Herpesvirus type 1 (BoHV-1 ) strain and Parainfluenza-3 virus (PI-3) strain.

[0125] In a further particular embodiment of the medical use of the invention, the immunogenic or vaccine compositions treats and / or prevents the infection by a virus selected from the group consisting of: Bovine Respiratory Syncytial Virus (BRSV), Parainfluenza-3 virus (PI-3), Bovine Herpesvirus type 1 (BoHV-1 ) and any combination thereof.

[0126] The term “immunogenic active component” as used herein refers to a material, such as a killed or inactivated, attenuated virus either live attenuated or genetically modified, a polypeptide or protein, or an immunogenic fragment thereof, an antigenic molecule, or any other component 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 (see previous definitions regarding an immunogenic composition, or an antigen polypeptide or fragment or an antigenic molecule, which are equally valid for the present case). For avoidance of doubt, an “immunogenic fragment” of an antigen according to the present invention is a partial amino acid sequence of the antigen or a functional equivalent of such a fragment that also acts as an antigen, that is detected and bound by antigen-specific antibody or B-cell receptor. An immunogenic fragment of an antigen is shorter than the complete antigen and is preferably between about 10, 50 or 100 and about 1000 amino acids long, more preferably between about 10, 50 or 30 and about 500 amino acids long, even more preferably between about 50 and about 350 amino acids long. A fragment of the E2 antigen includes amino acids having at least 15, 20 or 65 contiguous amino acid residues having at least about 50%, at least about 60%, at least 70%, at least about 80%, at least about 90%, preferably at least about 95%, more preferably at least about 98% sequence identity with at least about 15, 20 or 65 contiguous amino acid residues of SEQ ID NO. 1 or SEQ ID NO. 2, respectively. Depending on the expression system chosen, the protein fragments may or may not be expressed in native glycosylated form. A protein or fragment that "corresponds substantially to" a protein or fragment of the E2 BVDV virus, either BVDV-1 or BVDV-2, is a protein or fragment that has substantially the same amino acid sequence and has substantially the same functionality as the specified protein or fragment of the E2, either BVDV-1 or BVDV-2. A protein or fragment that has "substantially the same amino acid sequence" as a protein or fragment of the E2 BVDV virus, either BVDV-1 or BVDV-2, typically has more than 90% amino acid identity with this protein or fragment. Included in this definition are conservative amino acid substitutions.

[0127] In a particular embodiment of the medical use of the invention, the immunogenic active component is selected from a group consisting of: attenuated viral strain, inactivated viral strain, and recombinant protein derived from a viral strain.

[0128] The term “viral strain” as used herein refers to a genetically distinct lineage separated from another strain by one or more mutations. A strain is a genetic variant (not to be confused with a viral variant) or subtype of a virus.

[0129] The term “attenuated” as used herein refers to a virus with compromised or abolished virulence in the intended subject upon administration. The goal of attenuated virus is to produce a virus that does not produce infection symptoms, or very light infection symptoms, as to when used in an immunogenic or vaccine composition it stills is able to produce an immune response as provide treatment and / or prevention and / or protection when the cattle is infected with a wild type virus. The term “wild-type” indicates that the virus existed (at some point) in the field, and was isolated from a natural host, in the present case the cattle.

[0130] Attenuated virus can be obtained by multiply methods well known in the art by the skilled person, such as genetic modification of the viral genome in order to eliminate or knockdown genes responsible for the virus infection propagation, or by repeated passages of the virus in primary cell cultures, in order to obtain “naturally” modified viruses which have a reduced capacity of infection propagation (see Hajra D., et al, Vaccine Delivery Technology, 2021 , Volume 2183). Therefore, in a particular embodiment, the attenuated viral strain is obtained genetic modification of the viral genome, or by repeated virus passage in primary cell cultures. The particular type of attenuation is not important for the invention as such.

[0131] Many attenuated BRSV strains are known in the art, such as for example BRSV strain Lym-56 from the commercially available vaccines HIPRABOVIS® 4 and NASYM® (Laboratorios HIPRA, S.A.), BRSV strain 375 from the commercially available vaccine Rispoval® 4 (Zoetis UK Limited), BRSV strain Bio 24 / A from the commercially available vaccine Bovalto® Respi Intranasal (Boehringer Ingelheim Animal Health Espana, S.A.U.).

[0132] In a particular embodiment of the medical use of the invention, the attenuated BRSV is attenuated by serial passages in cell cultures. In a further particular embodiment, the serial passages are performed in Vero cell lines. In another particular embodiment of the medical use of the invention, the attenuated BRSV strain is attenuated after 56 passages of a wild-type strain in Vero cells. In a further particular embodiment of the medical use of the invention, the attenuated BRSV is the strain Lym-56, which is available in commercial vaccines such as HIPRABOVIS® 4 and and NASYM® (Laboratorios HIPRA, S.A., Avda. La Selva, 135, 17170 Amer, Girona, Spain).

[0133] Many attenuated BoHV-1 strains are known in the art and available from commercial vaccines, such as for example strain CEDDEL from the commercially available vaccine HIPRABOVIS® IBR MARKER LIVE (Laboratorios HIPRA, S.A.), strain GK / D from the commercially available vaccine Bovilis® IBR-MARKER LIVE (Merck Sharp & Dohme Animal Health, S.L.), strain Bio-27 from the commercially available vaccines Rhinovac IBR-Marker Live (Animal Health Distributors Limited) and BOPROTEC IBR Marker Viva (Vetia Animal Health S.A.U.), and strain Difivac from the commercially available vaccine Rispoval® IBR- Marker Live (Zoetis Spain, S.L.).

[0134] In a particular embodiment of the medical use of the invention, the attenuated BoHV- 1 is attenuated by mutations (insertions, substitutions and / or deletions) in one or more virulence genes. In a particular embodiment of the medical use of the invention, the attenuated BoHV-1 is attenuated by mutations in two virulence genes. In another particular embodiment of the medical use of the invention, the attenuated BoHV-1 is attenuated by two deletions in two virulence genes. In another particular embodiment of the medical use of the invention the attenuated BoHV-1 is attenuated by a deletion in the glycoprotein E (gE) gene and in the enzyme thymidine kinase (tk) gene. In a further particular embodiment of the medical use of the invention, the attenuated BoHV-1 is the attenuated strain CEDDEL, which is present in commercially available vaccines, such as HIPRABOVIS® IBR MARKER LIVE (Laboratorios HIPRA, S.A., Avda. La Selva, 135, 17170 Amer, Girona, Spain).

[0135] In another particular embodiment of the medical use of the invention, the attenuated viral strain is an attenuated Bovine Respiratory Syncytial Virus (BRSV) strain and / or an attenuated Bovine Herpesvirus type 1 (BoHV-1 ) strain. In a further particular embodiment of the medical use of the invention, the attenuated Bovine Respiratory Syncytial Virus (BRSV) strain is the Bovine Respiratory Syncytial Virus (BRSV) Lym-56 strain and / or the attenuated Bovine Herpesvirus type 1 (BoHV-1 ) strain is the CEDDEL strain.

[0136] The level of attenuation of a virus can be measured by the haemadsorption assay (HA). The term “haemadsorption” as used herein refers to a phenomenon whereby cells infected with the virus of interest adsorb erythrocytes (red blood cells) on their surface. The degree of haemadsorption induced by a virus may be measured using a haemadsorption assay. The hemagglutination assay (HA) and hemagglutination inhibition assay (HIA), and its units of hemagglutination units (HU), 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). For example, cells may be transfected with a protein or infected with a virus of interest, then red blood cells added and the degree of haemadsorption detected by imaging.

[0137] In a particular embodiment of the medical use of the invention, the level of attenuation of the attenuated Bovine Respiratory Syncytial Virus (BRSV) strain and / or an attenuated Bovine Herpesvirus type 1 (BoHV-1 ) strain is measured by haemadsorption and the attenuated Bovine Respiratory Syncytial Virus (BRSV) strain and / or an attenuated Bovine Herpesvirus type 1 (BoHV-1 ) strain have reduced haemadsorption when compared to the wild type Bovine Respiratory Syncytial Virus (BRSV) strain and / or an Bovine Herpesvirus type 1 (BoHV-1 ) strain. In a particular embodiment of the medical use of the invention, the attenuated Bovine Respiratory Syncytial Virus (BRSV) strain and / or the attenuated Bovine Herpesvirus type 1 (BoHV-1 ) strain have an haemadsorption capacity reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% when compared the wild type strains.

[0138] The term "killed virus” or “inactivated virus" as used herein refers to a virus that under natural conditions is virulent, but which has been modified so that is no longer capable of replicating anymore. Thus, the inactivated virus cannot cause disease or clinical signs / symptoms associated with the disease but it preserves the capacity of inducing an immune response. The term “inactivated virus” according to the present invention includes whole inactive virus, subunits derived of virus strains, and / or antigens derived of viral strains. Therefore, in a particular embodiment of the medical use of the invention, the inactivated viral strain is a whole killed viral strain, a subunit derived of the viral strain, and / or an antigen derived of the viral strain. The term “inactivated virus” according to the present invention is for use in a method to treat and / or prevent the infection by a virus selected from group consisting of: Bovine Respiratory Syncytial Virus (BRSV), Parainfluenza-3 virus (Pl- 3), Bovine Herpesvirus type 1 (BoHV-1 ), and any combination thereof. Preferably, is an inactivated Parainfluenza-3 virus (PI-3). The particular type of inactivation is not important for the invention as such.

[0139] Many inactivated PI-3 strains are known in the art, such as for example strain SF4 form the commercially available vaccines HIPRABOVIS® 3 and HIPRABOVIS® 4 (Laboratorios HIPRA, S.A.), BOVILIS® BOVIPAST RSP (Merck Sharp & Dohme Animal Health, S.L.), and strain Bio-23 from the commercially available vaccine BOVALTO RESPI 4 (Boehringer Ingelheim Animal Health Espana, S.A.U.).

[0140] In a particular embodiment of the medical use of the invention, the inactivated PI-3 is chemically inactivated. In a further particular embodiment of the medical use of the invention, the inactivated PI-3 is inactivated with binary ethyleneimine (BEI). In a further particular embodiment of the medical use of the invention, the inactivated PI-3 is the strain SF4, which is present in commercially available vaccines, such as HIPRABOVIS® 3 and HIPRABOVIS® 4 (Laboratorios HIPRA, S.A., Avda. La Selva, 135, 17170 Amer, Girona, Spain), and also from the ATCC (American Type Culture Collection), with accession number VR-281.

[0141] The term “subunit”, as used herein, refers to a portion of the virus or viral strain which is itself antigenic, i.e., capable of inducing an immune response in an animal. The term should be construed to include subunits which are obtained by both recombinant and biochemical methods. Subunit vaccines are easy to produce and more stable than other types of vaccines, such as vaccines based on mRNA or containing whole viruses, either attenuated or inactivated.

[0142] The term “antigen” or “immunogen” of the viral strain” as used herein, refers to a molecule that contains one or more epitopes (linear, conformational or both) that upon exposure to a subject induce an immune response in said subject that is specific for that antigen and for that viral strain. The term "antigen" or “immunogen” may comprise a whole organism, killed or inactivated viruses, and live viruses either live attenuated, or modified live viruses. The term "antigen" or “immunogen” can also refer to a subunit or partial fragment of an organism, a recombinant vector containing an insert with immunogenic properties, a fragment of DNA capable of inducing an immune response upon presentation to a subject, a polypeptide, a protein or a fragment thereof, an epitope, or any combination thereof. The term "antigen" can also refer to antibodies, or fragments thereof, and to synthetic peptide mimotopes that can mimic an antigen or antigenic determinant (epitope). The term "antigen" can also refer to an oligonucleotide or polynucleotide that expresses an antigen or antigenic determinant in vivo, such as in DNA immunization applications.

[0143] The term “recombinant protein of the viral strain” as used herein, refers to a recombinant protein (term previously defined and which applies to the present definition) of the viral strain, wherein said recombinant protein will serve as an antigen or immunogen. In a particular embodiment of the medical use of the invention, the recombinant protein is a subunit of the viral strain, an antigen of a viral strain and / or an antigen fragment of a viral strain.

[0144] Typically viruses are inactivated by several methods, including but not limited to, freeze-thawing, chemical treatment (such as thimerosal, binary ethyleneimine (BEI), formalin, beta-propiolactone), sonication, radiation, heat or any other convention means sufficient to prevent replication or growth of the organism while maintaining its immunogenicity. In a particular embodiment of the medical use of the invention the inactivated viral strain is an inactivated Parainfluenza-3 virus (PI-3) strain. In a further particular embodiment, the inactivated Parainfluenza-3 virus (PI-3) strain is the SF4 strain.

[0145] The immunogenic or vaccine composition of the medical use of the invention can comprise not only the immunogenic components but in addition may further comprise excipients, acceptable pharmaceutical adjuvants. In a particular embodiment of the medical use of the invention, the immunogenic or vaccine composition further comprises an excipient and / or an acceptable pharmaceutical adjuvant.

[0146] 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 et al., Handbook of Pharmaceutical Excipients, Pharmaceutical Press, 6thEd. (2009). In a particular embodiment of the medical use of the invention, the immunogenic or vaccine composition further comprises a lyophilization or freeze-drying excipient.

[0147] In an embodiment, the composition of the medical use of the invention may further comprise one or more veterinary acceptable excipient. 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, monoglycerides and diglycerides of fatty acids and any combinations thereof.

[0148] The term "veterinary acceptable” excipient means a component that can be administered to subject along with the immunogenic or vaccine composition of the medical use 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 cattle.

[0149] 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 subject upon exposure to the mixture. As such, in another particular embodiment, the vaccine composition of the medical use of the invention further comprises an acceptable pharmaceutical adjuvant. Illustrative non-limitative examples of acceptable pharmaceutical adjuvants that can be included in the vaccine composition of the medical use of the invention include, mineral oil, ginseng, chitosan, squalane, squalene, dimethylaminoethyl (DEAE), esters of acids or alcohols containing a linear alkyl group, 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), Montanide ISA, Montanide IMS, Montanide GEL 01 , Montanide GEL 02 (Montanide range all from SEPPIC), vegetable oils, water-in-oil emulsion, oil-in-water emulsion, and water-in-oil-in-water emulsion, and any combination thereof. The oil is used in combination with emulsifiers to form an emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide, of glycol, of polyglycerol, of propylene glycol, and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethlyelene copolymer blocks, in particular Pluronic products. In a particular embodiment of the medical use of the invention, the acceptable pharmaceutical adjuvant is selected from a group consisting of: mineral oil, ginseng, chitosan, dimethylaminoethyl (DEAE), aluminum hydroxide, phosphate buffer, Montanide IMS and any combination thereof. In a further particular embodiment of the medical use of the invention, the acceptable pharmaceutical adjuvant is Montanide IMS. In an embodiment of the medical use of the invention, the acceptable pharmaceutical adjuvant is an emulsion.

[0150] In a particular embodiment of the medical use of the invention, the acceptable pharmaceutical adjuvant is present in the immunogenic or vaccine composition at a concentration of between about 25 % volume to volume (v / v) to about 75% v / v, preferably about 30% v / v, about 35% v / v, about 40% v / v, about 45% v / v, about 50% v / v, about 55% v / v, about 60% v / v, about 65% v / v, about 70% v / v.

[0151] The use of the invention relates to the administration of an immunogenic or vaccine composition in a subject. The term “subject” means an individual. In one aspect, a subject is a mammal such as a primate, including humans. In another aspect, the mammal is a nonhuman primate. The term “subject” also includes domesticated animals such as cats, dogs and livestock such as cattle, horses, pigs, etc. As used herein, the subject is preferably cattle. The term “cattle” as used herein refers to an animal that is a member of the biological subfamily Bovinae, including, but not limited to, cows I bulls, bison, African buffalo and Asian buffalo. In preferred embodiments of the medical use of the invention, the animal is a cow. As used in this description, the term "cow" is cattle of any gender or age and is a member of the biological genus Bos, including Bos taurus and Bos indicus species. Thus, the term "cow" includes dairy cattle, beef cattle, bulls, heifers, oxen, calves, dams and gobies. In a particular embodiment of the medical use of the invention, the cattle is a cow, a bull, a calf or a heifer. In another particular embodiment of the medical use of the invention the cattle is a female, preferably a pregnant or lactating female.

[0152] The immunogenic or vaccine composition for the medical use of the invention can be administered by several routes which are well known by the skilled person. In a particular embodiment of the medical use of the invention, the immunogenic or vaccine composition is administered by subcutaneous, intradermal, or intramuscular route, preferably by intramuscular route.

[0153] The immunogenic or vaccine composition of the medical use of the invention can be formulated to be administered in single or multiple doses. The term “dose", is 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. A dose may be administered in a single dosage form, such as a single injection or single shot.

[0154] In a particular embodiment of the medical use of the invention, the immunogenic or vaccine composition is administered in at least two, at least three or more doses, preferably at least two doses.

[0155] In a particular embodiment of the medical use of the invention, the immunogenic or vaccine composition is administered in two doses. In another particular embodiment of the medical use of the invention, the two doses are separated three weeks apart.

[0156] In a particular embodiment of the medical use of the invention the immunogenic or vaccine composition is administered in a subject of at least 10, at least 15, at least 20, or more weeks of age, preferably least 10 weeks of age.

[0157] In another particular embodiment of the medical use of the invention, the first dose is administered in a subject of at least 10, at least 15, at least 20 or more weeks old, preferably at least 10 weeks old. In another particular embodiment of the medical use of the invention, the second dose is administered at least about 21 days, at least about 28 days, at least about two months after the first dose, preferably at least about 3 weeks after the first dose.

[0158] The term “two doses” refers to the fact that the immunogenic or vaccine composition of the medical use of the invention is effective to achieve a therapeutic and / or prophylactic effect against bovine respiratory disease (BRD) caused by all known BRD causing virus as previously defined. This means that two doses are effective for treating, preventing, reducing, controlling and / or ameliorating clinical signs associated to BRD. After the administration of two doses there is no need to administer a third or further doses (three, four, etc.) to maintain the treatment and / or prevention effect. After the administration of the two doses, the therapeutic or prophylactic effect will last at least about 180 days, preferably about 182 days, more preferably about 6 months, yet preferably about 0.5 years. This means that the two doses, as used herein, have a duration of immunity (also referred as DOI) of at least 180 days, preferably 182 days, more preferably 6 months, yet preferably 0.5 year. In a particular embodiment of the medical use of the invention, the treatment and / or prevention of Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or Bovine Viral Diarrhoea Virus type 2 (BVDV-2) infection in cattle is conferred for at least six months after administration of at least two doses.

[0159] After the duration of immunity has elapsed, a further single third dose can be administered. Thus, in a particular embodiment of the medical use of the invention, a further dose is administered no longer than about 1 month, no longer than about 2 months, no longer than about 3 months, no longer than about 4 months, no longer than about 5 months, no longer than about 6 months, no longer than about 7 months, no longer about 8 months, no longer than about 9 months, no longer than about 10 months, no longer than about 11 months, no longer than about 12 months, after the second dose as a booster. More, preferably the further dose is administered no longer about 6 months, after the second dose as a booster.

[0160] In a particular embodiment of the medical use of the invention, the immunogenic or vaccine composition is administered in at least three doses. In another particular embodiment of the medical use of the invention, a further dose (3rddose) is administered no longer than about 6 months after the second dose as a booster.

[0161] After the administration of three doses, the therapeutic or prophylactic effect will last at least about 360 days, preferably about 365 days, more preferably about 12 months, yet preferably about 1 years. This means that the three doses, as used herein, have a duration of immunity (also referred as DOI) of at least 360 days, preferably 365 days, more preferably 12 months, yet preferably 1 years.

[0162] In a particular embodiment of the medical use of the invention, the immunogenic or vaccine composition is administered as an annual booster no longer than 12 months after the third dose.

[0163] In a particular embodiment of the medical use of the invention, the treatment and / or prevention of Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or Bovine Viral Diarrhoea Virus type 2 (BVDV-2) infection in cattle is conferred for at least 12 months after administration of at least three doses. In a particular embodiment of the medical use of the invention a further dose is administered as an annual booster no longer than 12 months after the third dose. The single dose as a six month booster is also capable of providing treatment and / or prevention of BVDV-1 and / or BVDV-2 infection. In this way, the subject is protected over time against bovine respiratory disease (BRD) outbreaks. The term “booster”, as used herein, refers to an extra administration of the immunogenic or vaccine composition of the medical use of the invention which is given after an earlier administration of two doses known as primary vaccination. 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.

[0164] Booster doses are administered when protection provided by the primary dose has begun to decrease over the time.

[0165] The objective of a booster dose is to restore vaccine effectiveness from that deemed no longer sufficient.

[0166] 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.

[0167] The immunogenic or vaccine composition of the medical use of the invention is administered to the cattle comprising an effective amount of the immunogenic active components. By “effective amount” is meant an amount that induces an immunogenic and protective immunological response in the uninfected, infected or unexposed individual to whom the immunogenic or vaccine composition is administered. The “effective amount” also refers to an amount of the immunogenic active component sufficient to achieve a therapeutic and / or prophylactic effect against bovine respiratory disease (BRD) caused by all known BRD causing virus as previously defined. The “effective amount” also refers to an amount of the immunogenic active components sufficient to induce an immune response that reduces at least one symptom or clinical sign which is associated to BVDV-1 and / or BVDV-2 infection or associated disease. The amount of each of the components as defined above can be determined readily by the skilled artisan, for example, by identifying doses effective to elicit a prophylactic or therapeutic immune response, e.g., by measuring the serum titre of vaccine specific immunoglobulins or by measuring the inhibitory ratio of serum samples compared to a control that does not receive the component. Further, the skilled artisan would also be able to adapt the dose of each of the components to the subject. For example, the dose tested in mice models may be extrapolated to cattle by including the same dosage tested in mice models or multiplying by 2, 3, 4, 5, 6, 7, or 8 times the dosage tested in mice models.

[0168] In a particular embodiment of the medical use of the invention, the immunogenic or vaccine composition is administered at a dose of between about 15 pg per dose to about 75 pg per dose of each of the recombinant E2 protein of the BVDV-1 strain and of the recombinant E2 protein of the BVDV-2 strain. In another particular embodiment of the medical use of the invention, the immunogenic or vaccine composition is administered at a dose of about 15 pg , about 20 pg, about 25 pg, about 30 pg, about 35 pg, about 40 pg, about 45 pg, about 50 pg, about 55 pg, about 60 pg, about 65 pg, about 70 pg, or about 75 pg of each of the recombinant E2 protein of the BVDV-1 strain and of the recombinant E2 protein of the BVDV-2 strain, preferably about 30 pg, 40 pg, 50 pg or 60 pg per dose of each of the recombinant E2 protein of the BVDV-1 strain and of the recombinant E2 protein of the BVDV-2 strain. In another particular embodiment of the medical use of the invention, the immunogenic or vaccine composition is administered at a dose of at least about 15 pg, at least about 20 pg, at least about 25 pg, at least about 30 pg, at least about 35 pg, at least about 40 pg, at least about 45 pg, at least about 50 pg, at least about 55 pg, at least about 60 pg, at least about 65 pg, at least about 70 pg, or at least about 75 pg of each of the recombinant E2 protein of the BVDV-1 strain and of the recombinant E2 protein of the BVDV-2 strain, preferably at least about 30 pg, 40 pg, 50 pg or 60 pg per dose of each of the recombinant E2 protein of the BVDV-1 strain and of the recombinant E2 protein of the BVDV-2 strain, more preferably at least about 30 pg and / or 40 pg per dose.

[0169] In a further particular embodiment of the medical use of the invention, the immunogenic or vaccine composition is administered at a dose of at least between about 1046to about 1066cell culture infections dose 50% (CCID50) of attenuated Bovine Respiratory Syncytial Virus (BRSV), and of at least about 1056CCID50 and / or a dose of between about 1057to about 107 7cell culture infections dose 50% (CCID50) of attenuated Bovine herpesvirus type 1 (BoHV-1 ), and of at least about 1O67CCID5o and / or a dose of between about 380 to 580 hemagglutination units (HAU) of inactivated bovine Parainfluenza 3 (PI-3), preferably 480 HAU PI-3. In a further particular embodiment of the medical use of the invention, the immunogenic or vaccine composition is administered at a dose of at least between about 1046to about 1066cell culture infections dose 50% (CCID50) of attenuated Bovine Respiratory Syncytial Virus (BRSV), and of at least about 1056CCID50 and / or a dose of between about 1057to about 107 7cell culture infections dose 50% (CCID50) of attenuated Bovine herpesvirus type 1 (BoHV-1 ), and of at least about 1O67CCID5o and / or a dose of between about 75 to 580 hemagglutination units (HAU) of inactivated bovine Parainfluenza 3 (PI-3), preferably about 100 to 580 HAU PI-3, or about 240 to 580 HAU PI-3, or about 320 to 580 HAU PI-3, or about 380 to 580 HAU PI-3, more preferably 480 HAU PI-3.

[0170] The expression “cell culture infections dose 50%” or its acronym “CCID50” as used herein refers to an amount of virus required to induce cytopathic effects in 50% of wells containing the inoculated cell culture after a defined period of time. CCID50 assays are virus titration experiments which can be used to quantify virus titers by investigating the cytopathic effects of a virus on an inoculated host cell culture. In CCID50 assays varying virus dilutions are added as an endpoint dilution to host cell populations with the same number of cells and incubated until a cytopathic effect can be seen.

[0171] The immunogenic or vaccine composition of the medical use of the invention can be formulated to be administered in a volume which is determined according to the requirements of the composition, the animal, the veterinary, amongst other variables. Therefore, in a particular embodiment of the medical use of the invention the immunogenic or vaccine composition is administered in a volume of about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, preferably of about 2 ml per dose.

[0172] The immunogenic or vaccine composition for the use of the invention comprises the recombinant E2 protein of a Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain and / or the recombinant E2 protein of a Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain. As these are recombinant proteins, their presence in an animal can be detected and allows to determine if the animal has been infected by BVDV-1 and / or BVDV-2 or administered the immunogenic or vaccine composition.

[0173] In a particular embodiment of the medical use of the invention, the immunogenic or vaccine composition allows the differentiation of infected animals from vaccinated animals (DIVA). The term “differentiation of infected animals from vaccinated animals” or its acronym “DIVA” as used herein refers to method for differentiating an animal which has been administered an immunogenic or vaccine composition which comprises antigenic components, from an animal which has been infected by infectious agents (virus, bacteria) which comprise said antigenic agents. This test is supported by the fact that in an animal which has been administered the composition, only the antigenic agents present in the composition will be detected while other agents only present in the infectious natural agent will not be detected.

[0174] In a particular embodiment of the medical use of the invention, the DIVA test is performed by detecting the recombinant E2 protein of a Bovine Viral Diarrhoea Virus type

[0175] 1 (BVDV-1 ) strain and / or the recombinant E2 protein of a Bovine Viral Diarrhoea Virus type

[0176] 2 (BVDV-2) strain and the p80 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain and / or of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain, wherein the detection of the E2 protein and the lack of detection of the p80 protein is indicative of the animal having been vaccinated by the immunogenic or vaccine composition.

[0177] The term “p80 protein”, as used herein, refers to the BVDV-1 and BVDV-2 nonstructural protein p80 which has an enzymatic activity of trypsin-like serine proteinase.

[0178] In another particular embodiment of the medical use of the invention, the DIVA test is done by an immunoassay.

[0179] The term "immunoassay", as used herein, includes any immunoassay technique based on the formation or use of immune complexes, that is, resulting from the conjugation of antibodies and antigens, as quantification references of a determined analyte (substance under examination), which can be the antibody or the antigen, using for the measurement a molecule as a marker which produces a detectable signal in response to a specific binding.

[0180] Immunoassay techniques which can be used in the context of the present invention are Western-blot or Western transfer, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of protein biochips or microarrays which include specific antibodies or assays based on colloidal precipitation in formats such as dipsticks. In a particular embodiment of the DIVA method the immunoassay is performed by enzyme- linked immunosorbent assay (ELISA). The term “enzyme-linked immunosorbent assay” or its acronym “ELISA” as used herein refers to a commonly used analytical biochemistry assay, that uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence of a ligand (commonly a protein) in a liquid sample using antibodies directed against the protein to be measured. Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation. Several types of ELISA exist, namely, without limitation, direct ELISA, sandwich ELISA, competitive ELISA and reverse ELISA. Several enzymatic markers which allow the results of the assay to be measured upon completion of the assay, can be used in ELISA. The most commonly used are without limitation, OPD (o-phenylenediamine dihydrochloride) which turns amber to detect HRP (Horseradish Peroxidase) and is often used to as a conjugated protein; TMB (3,3',5,5'-tetramethylbenzidine) which turns blue when detecting HRP and turns yellow after the addition of sulfuric or phosphoric acid; ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6- sulfonic acid]-diammonium salt) which turns green when detecting HRP; PNPP (p- Nitrophenyl Phosphate, Disodium Salt) which turns yellow when detecting alkaline phosphatase; and ONPG (o-nitrofenil-p-D-galactopirandsido) which turns yellow when detecting beta-galactosidasa (b-Gal).

[0181] The present invention is further described in the following aspects:

[0182] 1. An immunogenic or vaccine composition for use in a method of treatment and / or prevention of Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or Bovine Viral Diarrhoea Virus type 2 (BVDV-2) infection in cattle, where the immunogenic or vaccine composition comprises a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain.

[0183] 2. The immunogenic or vaccine composition for use according to aspect 1 , wherein the treatment and / or prevention of BVDV-1 and / or BVDV-2 infection is the reduction of the clinical signs associated with BVDV-1 and / or BVDV-2 infection.

[0184] 3. The immunogenic or vaccine composition for use according to aspect 2, wherein the clinical signs associated with BVDV-1 and / or BVDV-2 infection are selected from a group consisting of: viremia, virus shedding, leukopenia, lung lesions, hyperthermia and any combination thereof.

[0185] 4. The immunogenic or vaccine composition for use according to any one of aspects 1 to 3, wherein the prevention of BVDV-1 and / or BVDV-2 infection is the prevention of transplacental BVDV-1 and / or BVDV-2 fetal infection.

[0186] 5. The immunogenic or vaccine composition for use according to any one of aspects 1 to 4, wherein the prevention of BVDV-1 and / or BVDV-2 infection is the prevention of the birth of persistently infected calves.

[0187] 6. The immunogenic or vaccine composition for use according to any one of aspects 1 to 5, wherein the prevention is the prevention of the progeny of a pregnant cow from a BVDV-1 and BVDV-2 infection by passive immunization by the colostrum.

[0188] 7. The immunogenic or vaccine composition for use according to any one of aspects 1 to 6, wherein the cattle has circulating maternally-derived antibodies (MDA) anti- Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or maternally-derived antibodies (MDA) anti-Bovine Viral Diarrhoea Virus type 2 (BVDV-2).

[0189] 8. The immunogenic or vaccine composition for use according to any one of aspects 1 to 7, wherein the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain comprises the sequence according to SEQ ID NO: 1 or a functionally equivalent variant thereof.

[0190] 9. The immunogenic or vaccine composition for use according to aspect 8, wherein the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain consists of the sequence according to SEQ ID NO: 1.

[0191] 10. The immunogenic or vaccine composition for use according to any one of aspects 1 to 7, wherein the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain comprises the sequence encoded by the sequence according to SEQ ID NO: 3 or a functionally equivalent variant of said sequence encoded by the sequence according to SEQ ID NO: 3.

[0192] 11. The immunogenic or vaccine composition for use according to aspect 10, wherein the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain consists of the sequence encoded by the sequence according to SEQ ID NO: 3.

[0193] 12. The immunogenic or vaccine composition for use according to any one of aspects

[0194] 1 to 11 , wherein the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain comprises the sequence according to SEQ ID NO: 2 or a functionally equivalent variant thereof.

[0195] 13. The immunogenic or vaccine composition for use according to aspect 12, wherein the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain consists of the sequence according to SEQ ID NO: 2.

[0196] 14. The immunogenic or vaccine composition for use according to any one of aspects

[0197] 1 to 11 , wherein the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type

[0198] 2 (BVDV-2) strain comprises the sequence encoded by the sequence according to SEQ ID NO: 4 or a functionally equivalent variant of said sequence encoded by the sequence according to SEQ ID NO: 4.

[0199] 15. The immunogenic or vaccine composition for use according to aspect 14, wherein the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain consists of the sequence encoded by the sequence according to SEQ ID NO: 4.

[0200] 16. The immunogenic or vaccine composition for use according to any one of aspects 1 to 15, wherein the immunogenic or vaccine composition further comprises an immunogenic active component derived from a virus selected from the group consisting of: Bovine Respiratory Syncytial Virus (BRSV), Parainfluenza-3 virus (Pl- 3), Bovine Herpesvirus type 1 (BoHV-1 ) and any combination thereof.

[0201] 17. The immunogenic or vaccine composition for use according to aspect 16, wherein the immunogenic or vaccine compositions treats and / or prevents the infection by a virus selected from the group consisting of: Bovine Respiratory Syncytial Virus (BRSV), Parainfluenza-3 virus (PI-3), Bovine Herpesvirus type 1 (BoHV-1 ) and any combination thereof.

[0202] 18. The immunogenic or vaccine composition for use according to aspect 16 or 17, wherein the immunogenic or vaccine composition comprises an immunogenic active component derived from a Bovine Respiratory Syncytial Virus (BRSV) strain and a Bovine Herpesvirus type 1 (BoHV-1 ) strain, or a Bovine Respiratory Syncytial Virus (BRSV) strain and a Parainfluenza-3 virus (PI-3) strain, or a Bovine Herpesvirus type 1 (BoHV-1 ) strain and a Parainfluenza-3 virus (PI-3) strain, or a Bovine Respiratory Syncytial Virus (BRSV) strain and a Bovine Herpesvirus type 1 (BoHV- 1 ) strain and Parainfluenza-3 virus (PI-3) strain. The immunogenic or vaccine composition for use according to any one of aspects 1 to 18, wherein the immunogenic or vaccine composition treats and / or prevents the clinical signs associated with Bovine Respiratory Disease (BRD) selected from a group consisting of: viremia, virus shedding, leukopenia, lung lesions, hyperthermia and any combination thereof. The immunogenic or vaccine composition for use according to any one of aspects 1 to 19, wherein the immunogenic or vaccine composition treats and / or prevents the clinical signs associated with Bovine Respiratory Disease (BRD), wherein the clinical signs are selected from a group consisting of: abortions, congenital defects, cerebellar hypoplasia, encephalitis, weak and abnormally small calves, unthrifty, persistently infected (PI) calves, mortality, failure to nurse, early embryonic death, fetal mummification, fetal malformations, fetal transfer, transplacental fetal infection, and transient infertility. The immunogenic or vaccine composition for use according to any one of aspects 16 to 20, wherein the immunogenic active component is selected from a group consisting of: attenuated viral strain, inactivated viral strain, and recombinant protein derived from a viral strain. The immunogenic or vaccine composition for use according to aspect 21 , wherein the attenuated viral strain is obtained by genetic modification of the viral genome, or by repeated virus passage in primary cell cultures. The immunogenic or vaccine composition for use according to aspect 21 or 22, wherein the inactivated viral strain is a whole inactive viral strain, a subunit derived of the viral strain, and / or an antigen derived of the viral strain. The immunogenic or vaccine composition for use according to any one of aspects 21 to 23, wherein the recombinant protein is a subunit of the viral strain, an antigen of a viral strain and / or an antigen fragment of a viral strain. The immunogenic or vaccine composition for use according to aspect 21 or 22, wherein the attenuated viral strain is an attenuated Bovine Respiratory Syncytial Virus (BRSV) strain, preferably is the attenuated Bovine Respiratory Syncytial Virus (BRSV) Lym-56 strain, and / or the attenuated viral strain is an attenuated Bovine Herpesvirus type 1 (BoHV-1 ) strain, preferably is the Bovine Herpesvirus type 1 (BoHV-1 ) CEDDEL strain. 26. The immunogenic or vaccine composition for use according to aspect 21 or 23, wherein the inactivated viral strain is an inactivated Parainfluenza-3 virus (PI-3) strain.

[0203] 27. The immunogenic or vaccine composition for use according to aspect 26, wherein the inactivated Parainfluenza-3 virus (PI-3) strain is the SF4 strain.

[0204] 28. The immunogenic or vaccine composition for use according to any one of aspects 1 to 27, wherein the immunogenic or vaccine composition further comprises an acceptable pharmaceutical adjuvant.

[0205] 29. The immunogenic or vaccine composition for use according to aspect 28, wherein the acceptable pharmaceutical adjuvant is selected from a group consisting of: mineral oil, ginseng, chitosan, dimethylaminoethyl (DEAE), aluminum hydroxide, phosphate buffer, Montanide IMS and any combination thereof.

[0206] 30. The immunogenic or vaccine composition for use according to aspect 28 or 29, wherein the acceptable pharmaceutical adjuvant is an emulsion.

[0207] 31 . The immunogenic or vaccine composition for use according to any one of aspects 28 to 30, wherein the acceptable pharmaceutical adjuvant is at a concentration of between about 25% v / v to about 75% v / v, preferably 50% v / v.

[0208] 32. The immunogenic or vaccine composition for use according to any one of aspects 1 to 31 , wherein the immunogenic or vaccine composition further comprises a freeze-drying excipient.

[0209] 33. The immunogenic or vaccine composition for use according to any one of aspects 1 to 32, wherein the cattle is a female, more preferably a pregnant or lactating female.

[0210] 34. The immunogenic or vaccine composition for use according to any one of aspects 1 to 33, wherein the immunogenic or vaccine composition is administered by subcutaneous, intradermal, or intramuscular route, preferably by intramuscular route.

[0211] 35. The immunogenic or vaccine composition for use according to any one of aspects 1 to 34, wherein the immunogenic or vaccine composition is administered in at least two doses.

[0212] 36. The immunogenic or vaccine composition for use according to aspect 35, wherein the first dose is administered in a subject of at least 10 weeks old. 37. The immunogenic or vaccine composition for use according to aspect 35 or 36, wherein the second dose is administered at least about three weeks after the first dose.

[0213] 38. The immunogenic or vaccine composition for use according to any one of aspects 35 to 37, wherein a third dose is administered no longer than about 6 months after the second dose as a booster.

[0214] 39. The immunogenic or vaccine composition for use according to any one of aspects 35 to 38, wherein a further dose is administered as an annual booster no longer than 12 months after the third dose.

[0215] 40. The immunogenic or vaccine composition for use according to any one of aspects 1 to 39, wherein the immunogenic or vaccine composition is administered at a dose of between about 15 pg per dose to about 75 pg per dose of each of the recombinant E2 protein of the BVDV-1 strain and of the recombinant E2 protein of the BVDV-2 strain.

[0216] 41. The immunogenic or vaccine composition for use according to aspect 40, wherein the immunogenic or vaccine composition is administered at a dose of about 30 pg, 40 pg, 50 pg or 60 pg per dose of each of the recombinant E2 protein of the BVDV- 1 strain and of the recombinant E2 protein of the BVDV-2 strain

[0217] 42. The immunogenic or vaccine composition for use according to any one of aspects 17 to 27 or any one of aspects 28 to 39 whenever in the context of aspects 17 to 27, wherein the immunogenic or vaccine composition is administered at a dose of between about 1046to about 1066cell culture infections dose 50% (CCID50) of attenuated Bovine Respiratory Syncytial Virus (BRSV), preferably about 1056CCID5o and / or a dose of between about 1057to about 107 7cell culture infections dose 50% (CCID50) of attenuated Bovine herpesvirus type 1 (BoHV-1 ), preferably about 1 O67CCID5O and / or a dose of between about 75 to 580 hemagglutination units (HAU) of inactivated bovine Parainfluenza 3 (PI-3), preferably 480 HAU PI-3.

[0218] 43. The immunogenic or vaccine composition for use according to any one of aspects 1 to 42, wherein the immunogenic or vaccine composition is administered in a volume of about 2 ml per dose.

[0219] 44. The immunogenic or vaccine composition for use according to any one of aspects 1 to 43, wherein the treatment and / or prevention of Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or Bovine Viral Diarrhoea Virus type 2 (BVDV-2) infection in cattle is conferred for at least six months after administration of at least two doses.

[0220] 45. The immunogenic or vaccine composition for use according to any one of aspects 1 to 44, wherein the immunogenic or vaccine composition allows the differentiation of infected animals from vaccinated animals (DIVA).

[0221] 46. The immunogenic or vaccine composition for use according to aspect 45, wherein the DIVA test is performed by detecting the recombinant E2 protein of a Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain and / or the recombinant E2 protein of a Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain and the p80 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain and / or of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain, wherein the detection of the E2 protein and the lack of detection of the p80 protein is indicative of the animal having been vaccinated by the immunogenic or vaccine composition.

[0222] ***

[0223] 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.

[0224] EXAMPLES

[0225] EXAMPLE 1 : Immunoqenicitv study of recombinant E2 proteins of Bovine Viral Diarrhea Virus Type 1 (BVDV-1) and Type 2 (BVDV-2) in calves

[0226] The aim of this study was to determine the ability of vaccine compositions comprising recombinant E2 proteins from BVDV-1 and BVDV-2 to induce both a humoral and cellular immune response in calves. The study also compared the immune response generated with said vaccine compositions with the immune response obtained by inactivated BVDV vaccine compositions.

[0227] A total of 35 calves (Bos taurus) were enrolled in the study. Animals persistently infected with BVDV-1 or BVDV-2 were excluded. The animals selected in the study were between 4 and 6 months of age and seronegative for BVDV-1 and BVDV-2. Animals that completed the enrolment were randomly allocated into 5 different groups of 7 animals each (Groups 1 to 5: Table 1 ), which received a different vaccine composition according to their group assignment. The vaccine compositions were administered following a two-dose protocol by intramuscular (IM) route, one administration on day 0 (DO) and a second administration on day 14 (D14).

[0228] Group 1 : 7 calves received two doses of a 2 ml_ vaccine composition comprising 50 pg / dose of the recombinant E2 protein (SEQ ID NO: 1 ) of BVDV-1 formulated in 50% v / v emulsion with the commercial adjuvant Montanide™ IMS (SEPPIC).

[0229] Group 2: 7 calves received two doses of a 2 ml_ vaccine composition comprising 50 pg / dose of the recombinant E2 protein (SEQ ID NO: 2) of BVDV-2 formulated in 50% v / v emulsion with the commercial adjuvant Montanide™ IMS (SEPPIC).

[0230] Group 3: 7 calves received two doses of a 2 ml_ vaccine composition comprising 106CCID5o / dose (Cell Culture Infectious Dose 50%) of BVDV-1 strain Singer inactivated with binary ethyleneimine (BEI) and formulated in 50% v / v emulsion with the commercial adjuvant Montanide™ IMS (SEPPIC).

[0231] Group 4: 7 calves received two doses of a 2 ml_ vaccine composition comprising 106CCID5o / dose of BVDV-2 V-1054 strain (Ghent) inactivated with binary ethyleneimine (BEI) and formulated in 50% v / v emulsion with the commercial adjuvant Montanide™ IMS (SEPPIC).

[0232] Group 5 (negative control group): 7 animals were left as a negative control group and they did not receive any vaccine composition.

[0233] Table 1 : Design of the study. rE2: recombinant produced E2 recombinant proteins from BVDV-1 and BVDV-2 were obtained by recombinant DNA technology (either in baculovirus or in mammalian cells using standard procedures known in the art such as, for example, J. Sambrook and D. W. Russell, Molecular Cloning: A laboratory manual, 4th edition, Cold Spring Harbor Laboratory Press, New York, 2012). The DNA coding sequence (SEQ ID NO: 3 and 4) for these E2 proteins were introduced, by means of a plasmid, into the chromosome of a cell line of mammalian origin, CAP (CEVEC's Amniocyte Production) cell line as a first study. The DNA was then expressed as the recombinant E2 protein for BVDV-1 and BVDV-2. The E2 proteins were then purified from the supernatant using standard techniques.

[0234] Blood samples were collected from all animals on days 0, 14 and 35 of the study.

[0235] Cellular immune response was assessed by measuring the production of IFN-y in the supernatant of whole blood cultures with the commercial BOVIGAM ELISA kit (Ingenasa). Particularly, the capacity of peripheral lymphocytes to produce IFN-y was measured on days 0, 14 and 35 of the study.

[0236] Humoral immune response (total antibodies production and seroneutralization) was assessed by analyzing the sera of the blood samples taken from all animals on days 0, 14 and 35 of the study by ELISA (enzyme-linked immunosorbent assay) to determine the total IgG specific antibodies against BVDV (I DEXX BVDV Total Ab X3 Test) and also to determine the titre of seroneutralizing antibodies against both BVDV-1 (Groups 1 and 3) and BVDV-2 (Groups 2 and 4) by a seroneutralization assay.

[0237] Safety of the vaccine compositions was also determined during all the study by monitoring and measuring different parameters such as rectal temperature, as well as local clinical signs at the inoculation sites (nodule(s); local inflammation) and general clinical signs (corporal condition; depression).

[0238] The results demonstrated that the vaccine compositions in all groups were safe, with slightly increase of rectal temperature after the administration of the first dose (D1 ) which did not exceed 2 °C increase at individual level or 1.5 °C increase at group level. Moreover, no further increase above these values were observed after administering the second dose (D15). Thus, observed temperature increases were acceptable.

[0239] No nodules or significant local inflammation at the injection site were observed. No general clinical signs such as depression or bad conditions were observed at all during the whole study. Therefore, the vaccine compositions administered in this study were proven to be safe since no local or general clinical signs were observed.

[0240] Humoral response measured as total antibodies against BVDV (Figure 1 ) and the seroneutralizing antibodies against BVDV-1 and BVDV-2 (Figure 2).

[0241] With regards to total antibodies against BVDV, it was clearly observed a higher titer result within the recombinant E2 protein groups (Group 1 and Group 2) when compared with the immune response of animals in groups that received the inactivated vaccine compositions (Group 3 and Group 4). All animals in the recombinant E2 protein groups (Groups 1 and 2) showed titers higher than the animals receiving a composition comprising inactivated BVDV, either BVDV-1 or BVDV-2.

[0242] This was also observed when coming to the seroneutralizing antibodies generated in the recombinant E2 groups. Accordingly, seroneutralizing antibodies, meaning those antibodies with capacity to seroneutralize the virus, were higher in the animals vaccinated with recombinant E2 proteins (Group 1 and Group 2) when compared to animals receiving the inactivated vaccine compositions. Furthermore, it was also observed that the immunological response of the animals that received the recombinant E2 protein was faster than that observed with the inactivated vaccine compositions, since seroconversion was already observed in some animals in Group 1 and Group 2 on day 14, while seroconversion in animals receiving an inactivated vaccine compositions was observed after receiving the 2 doses (after D35 of the study).

[0243] Therefore, the study concluded that vaccine compositions comprising recombinant E2 proteins, either from BVDV-1 or BVDV-2, were immunogenic and they had the capacity to provide higher seroconversion than the immunization with vaccine compositions comprising inactivated BVDV-1 or BVDV-2 virus. The results also showed that for some animals that received the recombinant E2 vaccine composition the seroconversion developed faster, after a single dose than the inactivated counterparts.

[0244] EXAMPLE 2: Efficacy of vaccine compositions comprising recombinant E2 protein of BVDV-1 and BVDV-2: onset of immunity and influence of maternally derived antibodies against Bovine Viral Diarrhea Virus Type 1 (BVDV-1) in calves The aim of this study was to assess the onset of immunity and the influence of maternally derived antibodies (MDA) on the efficacy of vaccine compositions comprising recombinant E2 proteins of BVDV-1 and BVDV-2 after a challenge with a BVDV-1 strain.

[0245] Because maternally acquired immunity could still be present and could interfere with immunity development at the age of 10 weeks in calves, a study to determine whether such interference occurs was performed.

[0246] A total of twenty-four young calves (Bos taurus) of approximately 10 weeks of age were selected. The animals were randomly allocated to three treatment groups (A to C: Table 2).

[0247] Table 2: Design of study killed inactivated virus

[0248] The MDA titers of groups B and C were representative of the titres found in animals of 10 weeks of age under field conditions.

[0249] Groups A and B were vaccinated intramuscularly (IM) with two doses, separated three weeks apart, at Day 0 (DO) and Day 21 (D21 ) of the study, with the vaccine compositions of the invention. Control group C received a mock-up vaccine consisting of sterile PBS through the same route and with the same vaccination scheme.

[0250] Group A: 8 calves without MDA antibodies against BVDV-1 (seronegative for BVDV- 1 ) were vaccinated intramuscularly in the neck muscles with two doses, on Day 0 (DO) and Day 21 (D21 ) of the study. The animals received a 2 ml_ of a vaccine composition comprising: o 40 pg / dose of recombinant E2 protein from BVDV-1 and 40 pg / dose of recombinant E2 protein from BVDV-2; o 1059CCI D5o / dose (cell culture infectious dose 50%) of live attenuated Bovine Respiratory Syncytial Virus (BRSV) strain Lym-56; o 107 0CCID5o / dose of live modified Bovine herpesvirus type 1 (BoHV-1 ), also called Infectious Bovine Rhinotracheitis Virus (IBR), strain CEDDEL; o 320 HAU (hemagglutination units) inactivated bovine Parainfluenza 3 (PI-3) strain SF4.

[0251] Group B: 8 calves having MDA antibodies against BVDV-1 (seropositive to BVDV- 1 ) were vaccinated intramuscularly in the neck muscles with two doses, on Day 0 (DO) and Day 21 (D21 ) of the study. The animals received a 2 ml_ of the same vaccine composition as Group A.

[0252] Group C: 8 calves having MDA antibodies against BVDV-1 (seropositive to BVDV- 1 ) were vaccinated intramuscularly in the neck muscles with two doses, on Day 0 (DO) and Day 21 (D21 ) of the study with a 2 ml_ of a mock-vaccine composition with sterile PBS (phosphate buffer saline).

[0253] The vaccine composition of groups A and B was further formulated in 50% v / v with the commercial adjuvant Montanide™ IMS (SEPPIC).

[0254] The attenuated strain of BRSV and the inactivated strain of PI-3 were obtained from the commercial vaccine HIPRABOVIS® 4 (Laboratorios HIPRA, S.A., Avda. La Selva, 135, 17170 Amer, Girona, Spain). The attenuated strain of IBR was obtained from the commercial vaccine HIPRABOVIS® IBR Marker Live (Laboratorios HIPRA, S.A., Avda. La Selva, 135, 17170 Amer, Girona, Spain). Recombinant E2 proteins from BVDV-1 and BVDV- 2 were obtained by recombinant DNA technology and expressed in mammalian cells (CAP cells), as described in Example 1.

[0255] Animals in Group C (MDA positive, mock-up vaccinated) were weekly monitored to assess the decay of MDAs against BVDV-1 . At the time when the MDAs disappeared in this control group, and after completing the vaccine protocol (i.e., 28 days after second administration of PBS, day 49 (D49) of the study), all animals were experimentally infected (challenged) with a virulent BVDV-1 strain by intranasal route. Accordingly, the experimental infection was performed on Day 49 (D49) of the study, which corresponded to 28 days after the second dose. The animals were inoculated intranasally with a 10 ml_ suspension comprising 107 1CCID50 BVDV-1 virus (half of the dose volume was administered into each nostril through a disposable nasal applicator aerosol generator coupled to a syringe).

[0256] General clinical signs, serology, rectal temperatures, viremia, leukopenia and virus shedding (by nasal swabs) were monitored on a daily basis. Overall, the following clinical parameters were evaluated:

[0257] - General clinical signs and rectal temperature.

[0258] - Serology: Analysis of BVDV antibodies (ELISA) and neutralizing anti-BVDV-1 antibodies (SN).

[0259] - Virus detection and titration from nasal swabs (RT-qPCR).

[0260] - Haematology: White blood cells counts (leukocyte counts).

[0261] - Viremia: Virus detection and titration from the buffy coats (RT-qPCR).

[0262] After challenge, a mild peak of rectal temperatures was observed from day 3 to 5 post-challenge, where the average of rectal temperatures in the control group (Group C) was greater than in the vaccinated groups (Groups A and B). A second peak of rectal temperatures was observed from day 7 to 9 post-challenge, again the average temperature in the control group (Group C) was greater compared to the vaccinated groups, either MDA negative or MDA positive (Group A and B). These differences were also observed on the percentage of animals with hyperthermia (temperature over 39.5°C), in which control animals had significantly greater number of days with hyperthermia compared to vaccinated animals (Groups A and B). No differences on rectal temperatures were observed between vaccinated groups, indicating that the presence of antibodies against BVDV-1 did not interfere on the efficacy of the vaccine on reducing hyperthermia.

[0263] These results of the study demonstrated that the vaccine composition of the invention reduced hyperthermia caused by BVDV-1 infection both in seronegative and also seropositive animals indicating that vaccination with the vaccine compositions of the invention is not interfered by circulating antibodies, in particularly maternally derived antibodies (MDAs) against BVDV-1.

[0264] Total anti-BVDV-1 antibodies and Seroneutralization results:

[0265] All animals were sampled before and after vaccination in order to determine the immune response against BVDV-1 and seroneutralization. After the first dose of vaccine, almost all the seronegative-vaccinated animals presented an increase on the antibody levels (ELISA). This increase was evident after the second dose of vaccine, when all of the vaccinated animals became seropositive for total antibodies (ELISA) and neutralizing antibodies (SN assay) (Figure 3 and Figure 4). In contrast, all control animals presented a marked decrease of the antibody levels from day 0 (DO) to day 49 (D49) and the total anti-BVDV-1 titers were lower than in the vaccinated groups, either MDA seronegatives and MDA seropositives (Groups A and B). No differences were observed between both vaccinated groups (A and B), thus confirming that the vaccine compositions of the invention were effective both in presence and in absence of circulating antibodies against BVDV-1.

[0266] After challenge (D70) it was observed that all groups increased the antibody titres against BVDV-1 indicating that the challenge was correctly conducted in all groups. All animals of the study were seropositive to anti-BVDV-1 antibodies by ELISA and neutralizing anti-BVDV-1 antibodies. The titre of seroneutralizing antibodies was significantly grater in both vaccinated groups (Groups A and B) compared to the control group (Group C). No significant differences were found between vaccinated groups.

[0267] Virus shedding and viremia:

[0268] Regarding virus shedding, it was observed that after challenge, the virus shedding and the percentage of samples where BVDV-1 was excreted and detected by PCR was greater in the control group (Group C) compared to vaccinated groups (Groups A and B). Nine days after challenge, day 58 of the study (D58), BVDV-1 was detected in 75% of the nasal samples in the control group (Group C), which was significantly greater than the nasal samples that excreted BVDV-1 of vaccinated groups (Groups A and B), which were 25% of samples in both vaccinated groups. In terms of the number of days of virus shedding, control animals excreted during more time than both vaccinated groups (Groups A and B).

[0269] Considering the entire post-challenge period, the average viremia titer of control animals (Group C) was significantly higher compared to the vaccinated animals (Groups A and B) (Figure 5). Furthermore, the control group had also significantly higher average viremic days (6.9 days for Group C) in comparison to vaccinated groups (2 days for both Group A and B).

[0270] These results demonstrated that vaccinated animals that received the vaccine composition of the invention in both groups (A: MDA-seronegative and B: M DA seropositive) reduced virus excretion and viremia indicating that said vaccine compositions are efficacious reducing the BVDV-1 excretion and titers of virus in both BVDV-1 seropositive and seronegative animals.

[0271] Overall, the vaccine compositions of the invention when administered to calves are able to reduce clinical signs, hyperthermia, leukopenia, virus shedding, and viremia caused by a BVDV-1 infection in both MDA-negative and MDA-positive animals.

[0272] Given the results of this study the onset of immunity of the vaccine compositions of the invention against infections caused by BVDV-1 was demonstrated at 21 days after vaccination.

[0273] EXAMPLE 3: Efficacy of vaccine compositions comprising recombinant E2 protein of BVDV-1 and BVDV-2: onset of immunity and influence of maternally derived antibodies against Bovine Viral Diarrhea Virus Type 2 (BVDV-2) in calves

[0274] The aim of this study was to assess the onset of immunity and the influence of maternally derived antibodies (MDA) on the efficacy of vaccine compositions comprising recombinant E2 proteins of BVDV-1 and BVDV-2 after a challenge with a BVDV-2 strain.

[0275] Because maternally acquired immunity could still be present and could interfere with immunity development at the age of 10 weeks in calves, a study to determine whether such interference occurs was performed.

[0276] Thus, the goal of this trial was to demonstrate to which extent MDAs could have an impact on the efficacy of the vaccine compositions of the invention in 10-week-old calves by means of challenge.

[0277] A total of thirty-eight young calves (Bos taurus) of approximately 10-weeks of age were selected. The animals were randomly allocated to four treatment groups (A to D: Table 3).

[0278] Table 3: Groups and corresponding treatments. killed inactivated virus

[0279] The MDA titres of groups C or D were representative of the titres found in animals of 10 weeks of age under field conditions.

[0280] Groups A and C were vaccinated intramuscularly (IM) with two doses separated three weeks apart, at Day 0 (DO) and Day 21 (D21 ) of the study, with the vaccine compositions of the invention. Control groups B and D received a mock-up vaccine consisting of sterile PBS through the same route and with the same vaccination scheme.

[0281] Group A: 9 calves without MDA antibodies against BVDV-2 (seronegative for BVDV- 2) were vaccinated intramuscularly in the neck muscles with two doses, on Day 0 (DO) and Day 21 (D21 ) of the study. The animals received a 2 ml_ of a vaccine composition comprising: o 40 pg / dose of recombinant E2 protein from BVDV-1 and 40 pg / dose of recombinant E2 protein from BVDV-2; o 1059CCI D5o / dose (cell culture infectious dose 50%) of live attenuated Bovine Respiratory Syncytial Virus (BRSV) strain Lym-56; o 107 0CCID5o / dose of live modified Bovine herpesvirus type 1 (BoHV-1 ), also called Infectious Bovine Rhinotracheitis Virus (IBR), strain CEDDEL; o 320 HAU (hemagglutination units) inactivated bovine Parainfluenza 3 (PI-3) strain SF4.

[0282] Group B: 9 calves without MDA antibodies against BVDV-2 (seronegative for BVDV- 2) were vaccinated intramuscularly in the neck muscles with two doses, on Day 0 (DO) and Day 21 (D21 ) of the study with a 2 ml_ of a mock vaccine composition with sterile PBS (phosphate buffer saline).

[0283] Group C: 10 calves having MDA antibodies against BVDV-2 (seropositive to BVDV- 2) were vaccinated intramuscularly in the neck muscles with two doses, on Day 0 (DO) and Day 21 (D21 ) of the study with a 2 ml_ of a vaccine composition comprising: o 40 pg / dose of recombinant E2 protein from BVDV-1 and 40 pg / dose of recombinant E2 protein from BVDV-2; o 1059CCI D5o / dose (cell culture infectious dose 50%) of live attenuated Bovine Respiratory Syncytial Virus (BRSV) strain Lym-56; o 107 0CCID5o / dose of live modified Bovine herpesvirus type 1 (BoHV-1 ), also called Infectious Bovine Rhinotracheitis Virus (IBR), strain CEDDEL; o 320 HAU (hemagglutination units) inactivated bovine Parainfluenza 3 (PI-3) strain SF4,

[0284] Group D: 10 calves having MDA antibodies against BVDV-2 (seropositive to BVDV- 2) were vaccinated intramuscularly in the neck muscles with two doses, on Day 0 (DO) and Day 21 (D21 ) of the study with a 2 ml_ of a mock-vaccine composition with sterile PBS (phosphate buffer saline).

[0285] The vaccine composition of groups A and C was further formulated in 50% v / v with the commercial adjuvant Montanide™ IMS (SEPPIC).

[0286] The attenuated strain of BRSV and the inactivated strain of PI-3 were obtained from the commercial vaccine HIPRABOVIS® 4 (Laboratorios HIPRA, S.A., Avda. La Selva, 135, 17170 Amer, Girona, Spain). The attenuated strain of IBR was obtained from the commercial vaccine HIPRABOVIS® IBR Marker Live (Laboratorios HIPRA, S.A., Avda. La Selva, 135, 17170 Amer, Girona, Spain). Recombinant E2 proteins from BVDV-1 and BVDV- 2 were obtained by recombinant DNA technology and expressed in mammalian cells (CAP cells), as described in Example 1.

[0287] Animals in Group D (MDA positive, mock-up vaccinated) were weekly monitored to assess the decay of MDAs against BVDV-2. At the time when the MDAs disappeared in this control group, and after completing the vaccine protocol (i.e., after D21 ), all animals were experimentally infected (challenged) with a virulent BVDV-2 strain by intranasal route.

[0288] Accordingly, the experimental infection was performed on Day 42 (D42) of the study, which corresponded to 21 days after the second dose. The animals were inoculated intranasally with a 10 mL suspension comprising 105CCID50 BVDV-2 virulent strain (half of the dose volume was administered into each nostril through a disposable nasal applicator aerosol generator coupled to a syringe).

[0289] General clinical signs, respiratory clinical signs, and rectal temperatures were monitored on a daily basis for 21 days after the experimental challenge. Blood samples were collected daily up to 21 days after the experimental infection to assess leukopenia, viremia and serological response. Nasal swabs samples were also collected daily and up to 21 days after the experimental infection to assess virus shedding. Overall, the following clinical parameters were evaluated:

[0290] - General clinical signs and rectal temperatures.

[0291] - Serology: Analysis of BVDV antibodies (ELISA) and seroneutralizing anti-BVDV-2 antibodies (SN).

[0292] - Virus detection and titration from nasal swabs (RT-qPCR).

[0293] - Haematology: White blood cells counts (leucocyte counts).

[0294] - Viremia: Virus detection and titration from the buffy coats (RT-qPCR).

[0295] During the entire post-challenge period, vaccinated groups with and without MDAs had similar values of rectal temperature at all monitored times

[0296] After challenge, a peak of rectal temperatures was observed from day 3 to day 9 postchallenge, where the average of rectal temperatures in the control groups (B and D) was significantly higher than in the vaccinated groups (A and C). These significant differences were also observed on the percentage of animals with hyperthermia (over 39.5°C) where control groups, either MDA positive or MDA negative, showed significantly higher number of days with hyperthermia compared with vaccinated groups A (MDA negative) and C (MDA positive).

[0297] These results of the study demonstrated that the vaccine composition of the invention reduced hyperthermia caused by BVDV-2 infection both in seronegative and also seropositive animals indicating that vaccination with the vaccine compositions of the invention is not interfered by circulating antibodies, in particular maternally derived antibodies (MDAs) against BVDV-2.

[0298] Total anti-BVDV-2 antibodies and Seroneutralization results:

[0299] All animals were sampled before and after vaccination in order to determine the immune response against BVDV-2 and seroneutralization.

[0300] After the vaccination scheme was completed (D42 of the study), a clear increase of antibody levels (ELISA) and neutralizing antibody titres (SN assay) were observed in both vaccinated groups, either MDA seronegatives and MDA seropositives (Group A and C, respectively) (Figure 6). All animals became seropositive by total antibodies and neutralizing antibodies against BVDV-2 (Figure 7) and vaccinated groups had significantly greater titres and percentage of seroneutralization compared to control groups (B and D). No differences were observed between both vaccinated groups (A and C), thus confirming that the vaccine compositions of the invention were effective both in presence and in absence of circulating antibodies against BVDV-2.

[0301] Furthermore, it was observed that all groups increased the antibody titre against BVDV-2 after challenge (D63) indicating that the challenge was correctly conducted in all groups. All the animals of the study were seropositive by total antibodies (ELISA) and neutralizing antibodies (SN assay). The titre of seroneutralizing antibodies was significantly higher in vaccinated animals (Groups A and C) compared to control groups (Groups B and D). No significant differences were found between vaccinated groups.

[0302] Virus shedding and viremia:

[0303] Regarding virus shedding, it was observed that after challenge, control animals B and D had a higher average titre than vaccinated groups A and C, for example nasal excretion of BVDV-2 in control groups (Groups B and D) showed a higher excretion than vaccinated groups (Groups A and C) from day 6 to day 9 after the experimental infection (Figure 8).

[0304] Furthermore, considering the entire post-challenge period (until D14 post-challenge), the average of BVDV-2 virus shedding of control animals (Groups B and D) was significantly higher compared to the vaccinated animals (Groups A and C). No significant differences were found between vaccinated groups (Groups A and C), either MDA-negative or MDA- positive. In terms of the number of days of virus shedding, control animals (Groups B and D), either seronegative or seropositive, excreted BVDV-2 during more time than vaccinated groups (A and C). No significant differences were found between vaccinated groups regarding the number of days of virus shedding.

[0305] Considering the entire post-challenge period, the average of viremia titre of control animals, MDA-seronegative and MDA seropositive, was significantly higher compared to vaccinated groups (A: MDA-seronegative and C: MDA seropositive) (Figure 9). No significant differences were found between vaccinated groups A and C. In terms of days with viremia, again control animals in groups B and D (MDA- and MDA+) had significantly higher average of viremic days in comparison to vaccinated groups A and C (MDA- and MDA+). These results demonstrated that vaccinated animals that received the vaccine composition of the invention in both groups (A: MDA-seronegative and C: M DA seropositive) reduced virus excretion and viremia indicating that said vaccine compositions are efficacious reducing the BVDV-2 excretion and titres of virus in both BVDV-2 seropositive and seronegative animals.

[0306] Overall, the vaccine compositions of the invention when administered to calves are able to reduce clinical signs, hyperthermia, leukopenia, virus shedding, and viremia caused by a BVDV-2 infection in both MDA-negative and MDA-positive animals.

[0307] Finally, given the results of this study the onset of immunity of the vaccine compositions of the invention against infections caused by BVDV-2 was demonstrated at 21 days after vaccination.

[0308] EXAMPLE 4: Efficacy of vaccination against Bovine Respiratory Disease caused by Bovine Respiratory Syncytial Virus (BRSV), Parainfluenza Type 3 (PI-3) Virus, and Bovine Herpesvirus type-1 Virus (BoHV)

[0309] Because the efficacy of the vaccine compositions of the invention was surprisingly observed against respiratory and reproductive parameters caused by both Bovine Viral Diarrhea Virus Type 1 (BVDV-1 ) and Bovine Viral Diarrhea Virus Type 2 (BVDV-2) in calves, even in presence of maternally derived antibodies (MDA), the aim of this further study was to assess the efficacy against different viral respiratory pathogens that are involved in the Bovine Respiratory Disease (BRD) complex including Bovine Respiratory Syncytial Virus (BRSV), Parainfluenza Type 3 (PI-3) Virus, and Bovine Herpesvirus type-1 Virus (BoHV-1 ). The efficacy was tested in both seronegative and seropositive animals in order to assess the influence of MDAs on the efficacy of the vaccine compositions of the invention. The onset of immunity (OOI) of the vaccine composition for each viral respiratory pathogen was also assayed.

[0310] With these aims, different efficacy sub-studies were performed, one for each viral respiratory pathogen to be assessed. In Sub-study 1 the efficacy of the vaccine compositions of the invention against BRSV infection and clinical signs thereof were assessed. In Sub-study 2 the efficacy of the vaccine compositions of the invention against PI-3 infection and associated clinical signs were studied. Finally, in Sub-study 3 the efficacy of the vaccine compositions of the invention against BoHV-1 infection and associated clinical signs were assessed.

[0311] For each efficacy sub-study, female and male calves (Bos taurus) of approximately 10-weeks of age were selected and randomly assigned to a different Sub-study group according to their immunological status. All calves enrolled in each sub-study were sampled before vaccination to determine their serological status and confirm that animals allocated in the seronegative groups had no antibodies against each viral respiratory pathogen to be tested and those in the seropositive group had MDA titers representative of the MDA titers usually found in field conditions in commercial feedlots for each viral respiratory pathogen to be tested at the age of 10-weeks.

[0312] All calves enrolled in the study were vaccinated intramuscularly (IM) with two doses of a vaccine composition comprising 5 immunogenic components: a live genetically modified BoHV-1 virus, a live attenuated BRSV, an inactivated PI-3 virus, a BVDV-1 recombinant E2 protein and a BVDV-2 recombinant E2 protein, administered three weeks apart, on Day 0 (DO) and Day 21 (D21 ) of the study according to their group assignment. The different Sub-study designs are described in the following tables (Tables 4 to 6).

[0313] Table 4: Design of Sub-study 1. Efficacy on BRSV r: recombinant-produced protein, LAV: live attenuated virus, LMV: live modified virus,

[0314] K: killed inactivated virus.

[0315] Table 6: Design of Sub-study 3. Efficacy on BoHV-1 (IBR) r: recombinant-produced protein, LAV: live attenuated virus, LMV: live modified virus, K: killed inactivated virus

[0316] Calves in Groups B and D of Sub-study 1 , and calves in Group C of Sub-study 2 and Sub-study 3 were treated as a control groups and all of them received a mock-up vaccine consisting of 2 mL sterile phosphate buffer saline (PBS) composition. These animals were vaccinated following the same route (IM) and the same vaccination scheme of two doses on DO and D21 of the study.

[0317] On the other hand, calves assigned to Groups A and C of Sub-study 1 , and calves assigned to Groups A and B of both, Sub-study 2 and Sub-study 3, received a 2 ml of a vaccine composition of the invention comprising:

[0318] • Between 16.3 to 30 pg / dose of recombinant E2 protein from BVDV-1 and between 16.3 to 30 pg / dose of recombinant E2 protein from BVDV-2; • Between 1052and 1065CCID5o / dose (cell culture infectious dose 50%) of live attenuated Bovine Respiratory Syncytial Virus (BRSV) strain Lym-56;

[0319] • Between 1063and 107 6CCID5o / dose of live modified Bovine herpesvirus type 1 (BoHV-1 ), also called Infectious Bovine Rhinotracheitis Virus (IBR), strain CEDDEL;

[0320] • Between 130.8 and 240 HAU / dose (hemagglutination units) inactivated bovine Parainfluenza 3 (PI-3) strain SF4.

[0321] The attenuated strain of BRSV and the inactivated strain of PI-3 were obtained from the commercial vaccine HIPRABOVIS® 4 (Laboratorios HIPRA, S.A., Avda. La Selva, 135, 17170 Amer, Girona, Spain). The attenuated strain of BoHV-1 was obtained from the commercial vaccine HIPRABOVIS® IBR Marker Live (Laboratorios HIPRA, S.A., Avda. La Selva, 135, 17170 Amer, Girona, Spain). Recombinant E2 proteins from BVDV-1 and BVDV- 2 were obtained by recombinant DNA technology and expressed in mammalian cells (CAP cells), as described in Example 1.

[0322] In particular, in Sub-study 1 the final vaccine composition comprised 16.4 pg / dose of recombinant E2 protein from BVDV-1 , 16.4 pg / dose of recombinant E2 protein from BVDV- 2, 1052CCID5o / dose of the live attenuated BRSV strain, 1066CCID5o / dose of the live modified BoHV-1 strain, and 130.8 HAU / dose of the inactivated PI-3 strain.

[0323] In Sub-study 2 the final vaccine composition comprised 30 pg / dose of recombinant E2 protein from BVDV-1 , 30 pg / dose of recombinant E2 protein from BVDV-2, 1057CCID5o / dose of the live attenuated BRSV strain, 107° CCID5o / dose of the live modified BoHV-1 strain, and 240 HAU / dose of the inactivated PI-3 strain.

[0324] In Sub-study 3 the final vaccine composition comprised 16.5 pg / dose of recombinant E2 protein from BVDV-1 , 16.5 pg / dose of recombinant E2 protein from BVDV-2, 1055CCID5o / dose of the live attenuated BRSV strain, 1063CCID5o / dose of the live modified BoHV-1 strain, and 132.2 HAU / dose of the inactivated PI-3 strain.

[0325] All the vaccine compositions of the invention were further formulated in 50% v / v with the commercial adjuvant Montanide™ IMS (SEPPIC).

[0326] MDA titers in all calves assigned to a seropositive control group and mock-up vaccinated, namely those in Sub-study 1 Group D, Sub-study 2 Group C, Sub-study 3 Group C, were monitored weekly to assess the decay of MDAs. At the time when the MDAs disappeared in said control groups all animals in all groups and sub-studies were experimentally infected (challenged) with a different wild-type respiratory virus according to the Sub-study which they belonged. Experimental infection (challenge) was always performed after completing the whole vaccine schedule, i.e. after day 21 of the study.

[0327] • Sub-Study 1 : animals were experimentally infected on day 42 (D42) of the study, i.e., 21 days after receiving the second dose, with a virulent BRSV strain by aerosol inhalation. The challenge was conducted by preparing a 100 ml of a suspension comprising 10346CCID5o / ml of the virulent BRSV, followed by nebulizing the 100 ml suspension onto the 28 animals of the Sub-study 1. The animals were exposed to the nebulized suspension for a duration of 45 minutes.

[0328] • Sub-Study 2: animals were experimentally infected on day 42 (D42) of the study, i.e., 21 days after receiving the second dose, with a virulent PI-3 strain by intranasal route. The challenge was performed by aerosol inhalation of a 10 ml suspension comprising 107° CCID5o / ml, 5 ml of the suspension were administered to each nostril.

[0329] • Sub-Study 3: animals were experimentally infected on day 66 (D66) of the study, i.e., 45 days after receiving the second dose, with a virulent BoHV-1 strain by intranasal route. The challenge was performed by aerosol inhalation of a 2 ml suspension comprising 1067CCID5o / ml of the virulent IBR strain, 1 ml of the suspension was administered into each nostril.

[0330] General and respiratory clinical signs were monitored on a daily basis over the duration of the study. The response of all animals in each sub-study against the corresponding respiratory virus challenge was also evaluated through the study. Blood samples were collected from all calves at different sampling points. Serological response was monitored from day 0 (DO) of the study until 14 days post-challenge (D56 of the study) for Sub-studies 1 (BRSV) and 2 (PI-3), and for Sub-study 3 (BoHV-1 ) it was monitored until 21 days post-challenge (D87 of the study). Seroneutralization and ELISA techniques were used to determine specific total antibody titres in serum of calves for each viral respiratory pathogen to be tested (BRSV, PI-3 and BoHV-1 ).

[0331] After challenge, all animals were monitored daily for virus excretion (nasal swabs) and clinical signs. Lung lesions were assessed in necropsied samples from humanely euthanized calves. Virus isolation from samples was conducted by PCR, cell culture and immunohistochemistry (IHC), depending on the virus to be isolated. Particularly, molecular techniques (PCR) were used to detect the BRSV virus in the nasal swabs in order to check the presence of the virus in said samples and thus calculating the virus shedding both in control and vaccinated groups. Virus isolation for PI-3 and BoHV- 1 were done in cell culture and by immunohistochemistry to detect the presence of the virus in nasal swabs and calculating virus shedding also in both control and vaccinated groups.

[0332] Clinical signs were assessed following the scoring system described in Table 7.

[0333] At the end of the study, animals were humanely euthanized, and lung lesions were assessed for BRSV and PI-3 during necropsies. Euthanasia was performed 14 days after challenge (D56 of the study) for Sub-studies 1 (BRSV) and 2 (PI-3), and 21 days post- challenge (D87 of the study) for Sub-study 3 (BoHV-1 ).

[0334] Lung lesions (percentage of affection) were assessed by recoding the percent of pneumonic area per lung lobe, especially for consolidation or acute bronchopneumonia lesions. The percentage of pneumonic lung for each calf necropsied was assessed according to the method described in Ellis J. et al., 2018. Table 7: General clinical signs evaluation 1lf mucous or blood was observed, it was recorded as observation, n.a.: not applicable.

[0335] Results of Sub-study 1 (Efficacy against BRSV)

[0336] Virus shedding:

[0337] Previous to challenge, BRSV was not detected in any of the samples collected.

[0338] Individual quantitative value (Iog2total virus / ml) was obtained for each animal and time point before and after challenge. Using them, the individual virus shedding from day 1 to 14 post-challenge was calculated to assess the total shedding during the post-challenge period. Furthermore, the number of days excreting BRSV were also calculated during the post-challenge phase to assess differences in days of shedding. Daily shedding and days of shedding data per group were compared. Total virus shedding was also compared to assess differences between groups.

[0339] Conversely, after challenge control Groups B (MDA seronegative) and D (MDA seropositive) had significantly higher average virus excretion during the whole postchallenge period, from day 1 to day 14 after challenge (Table 8, Figure 10). However, vaccinated animals showed significantly lower excretion. Moreover, no significant differences were observed among vaccinated groups A (MDA seronegative) and C (MDA seropositive) at any time point after challenge. The differences observed in virus shedding between vaccinated and control groups were also seen when assessing the average of total virus excretion per group during the whole post-challenge period. Vaccinated animals, both MDA-seronegative and MDA-seropositive, had significantly lower total virus excretion compared to control animals post-challenge.

[0340] Furthermore, significant differences were also observed regarding the days that vaccinated or control groups excreted BRSV. Vaccinated groups, either MDA-seronegative or MDA-seropositive, had significantly lower average days of BRSV excretion (2.14 and 4.42 days in Groups A and C, respectively) compared to control groups (9.71 and 8.85 days in Groups B and D, respectively). No significant differences were observed between virus shedding for vaccinated groups. These results demonstrated that vaccine compositions of the invention administered in two doses, significantly reduced the virus shedding (excretion) in cattle caused by an infection of BRSV both in terms of mean titers and mean duration of excretion regardless their immunological status (MDA negative, MDA positive). Table 8: Mean titer of virus excretion of BRSV (log? total virus / ml) and mean days of virus shedding per group from the day of challenge to day 14 post-challenge.

[0341] General clinical signs were monitored during the entire study and no alterations due to the vaccine composition were observed during the vaccination phase.

[0342] After challenge, all clinical signs recorded including any respiratory distress were considered and reported as average of clinical signs per day. Control groups, either MDA- seronegative or MDA-seropositive, (Groups B and D respectively) showed an increase on the average score, especially from day 5 to day 12, with a maximum mean score of 8.6 and 10.0, respectively, whereas vaccinated groups reached a maximum mean score of 5.4 and 5.6 for MDA-negative and MDA-positive (Groups A and C), respectively. From day 6 to day 11 , vaccinated groups showed lower average score respect to the control groups. No significant differences were observed between vaccinated groups A and C. Hence, a notable reduction in clinical signs was observed after the BRSV infection in animals vaccinated with the vaccine compositions administered in two doses, regardless their immunological status (MDA negative, MDA positive).

[0343] Hyperthermia:

[0344] Overall rectal temperature after challenge were also recorded and reported. The results shown that an increase in rectal temperatures was observed after the virus excretion peak the first days after challenge (from day 5 to day 8 post-challenge). Both vaccinated groups, A and C, had significantly lower rectal temperatures compared to control groups B and D (Figure 11 ). The study also demonstrated that the overall recorded rectal temperatures were significantly greater in both control groups, either MDA-seronegative and MDA-seropositive (Groups B and D), with an average rectal temperature of 39.29°C and 39.30°C, respectively, compared to vaccinated groups A and C (MDA-seronegative and MDA-positive), with an average rectal temperature of 38.83°C and 38.85°C, respectively. Therefore, the vaccine compositions of the invention also reduced hyperthermia (increase of rectal temperatures) caused by a BRSV infection, when it is administered in two doses, regardless the immunological status (MDA negative, MDA positive) of the calves at the time of vaccination.

[0345] Lung lesions:

[0346] Lung lesions were assessed at the end of the study in necropsied animals. It was clearly seen that the experimental infection by BRSV caused notable respiratory lesions, mainly consisting of pneumonic consolidation of the lung, in samples obtained in both control groups B and D. Remarkably, control groups B and D had significantly greater percentage of lobe lesion compared to both vaccinated groups A and C in different pulmonary lobes. The average of total lung affection in both vaccinated groups, either MDA- negative and MDA-positive (Groups Aand C, respectively) was less than 2% in both groups, particularly 0.87% and 1.61 % (Groups A and C, respectively), which was significantly lower than the percentage observed in the control groups, 24.8% in MDA-seronegative control group (Group B) and 31.3% in MDA-seropositive control group (Group D). No significant differences were observed between both vaccinated groups A and C. The results indicated that lung affections of control groups B and D were clearly evident, whereas lung lesions observed in both vaccinated groups A and C were practically null. Accordingly, the results obtained in this study also demonstrated that vaccination with vaccine compositions of the invention reduced lung lesions caused by experimental infection of BRSV, when it is administered in two doses, regardless of the immunological status of the animals, either seropositive or seronegative.

[0347] Anti-BRSV antibodies and Seroneutralization:

[0348] All animals were sampled before vaccination, as expected all animals in the vaccinated Groups C (MDA-positive) and in the control Group D (MDA-seropositive) had specific antibodies against BRSV, whereas all animals in the vaccinated group A (MDA- seronegative) and Group B (MDA-seronegative) had no specific antibodies against BRSV. Consequently, the results of serology confirmed that all animals included in seropositive groups had antibodies against BRSV on DO of the study, while all seronegative animals, either in the vaccination or control groups, were negative at vaccination (DO). The day of challenge (D42 of the study) all animals in the control group, either MDA-seronegative and MDA-seropositive, developed specific antibodies against BRSV indicating that the experimental challenge was correctly conducted. No significant differences were observed between both vaccinated groups A and C from day 7 (D7) to day 56 (D56) of the study (Figure 12). Although vaccinated seronegative animals (Group A) had a higher percentage of positive animals that seroconverted to BRSV, the protection was achieved at the same level in both vaccinated groups A and C, as detailed in the previous efficacy parameters.

[0349] Parameters to demonstrate protection from a BRSV challenge showed that the efficacy of the vaccine composition of the invention when administered in two doses is similar in both MDA-negative and MDA-positive animals.

[0350] To sum up, results obtained in efficacy sub-study 1 against BRSV, demonstrated that vaccine compositions of the invention reduces virus shedding (excretion), lung lesions, clinical sings and hyperthermia caused by BRSV after an experimental infection in either seropositive or seronegative animals, when it is administered by intramuscular route in two doses.

[0351] Results of Sub-study 2 (Efficacy against PI-3)

[0352] Virus shedding:

[0353] Previous to challenge, nasal swabs were obtained from all animals at different time points to assess PI-3 presence before challenge. The analysis confirmed that all samples were free from PI-3 and the virus was not detected in any of the samples.

[0354] The differences in virus shedding between vaccinated groups A and B (MDA- seronegative and MDA-seropositive, respectively) and control group C (MDA-positive) were measured analyzing the average of total virus shedding per group on the excreting days from day 43 to day 49 of study, corresponding to day 1 and day 7 after challenge. Individual quantitative value (CCID5o / ml) was obtained for each animal and time point. These values were log transformed. Using them, the individual virus shedding from day 1 to 14 postchallenge was calculated to assess the total shedding during the post-challenge period. Furthermore, the number of days shedding PI-3 virus were also calculated during the postchallenge period to assess differences in days of virus shedding. Daily shedding and days of shedding data per group were compared. Total virus shedding was also compared to assess differences between groups.

[0355] Vaccinated animals in groups A and B excreted significantly lower PI-3 virus (total virus shedding) compared to control animals in group C between days 1 and 7 after challenge (Table 9, Figure 13). This difference was also observed with regards to the number of days that the animals excreted PI-3 virus, i.e. virus shedding. It was observed that vaccinated animals, either MDA-seronegative and MDA-seropositive (Groups A and B, respectively) excreted significantly lower days PI-3 virus, practically null virus shedding was observed, i.e. 0.2 days and 0.0 days (Group A and Group B, respectively). Thus, no differences were observed between both vaccinated groups. In contrast, control group C, excreted PI-3 virus during a mean of 2.3 days. These results indicated that said compositions are efficacious reducing the PI-3 virus excretion and titres of virus in both PI-3 seropositive and seronegative animals.

[0356] Table 9: Titer of PI-3 virus shedding (log CCID5o / ml) and mean virus shedding days per group from days 1 to 7 post challenge, day 43 (D43) to day 49 (D49) of the study.

[0357] Hyperthermia:

[0358] Overall rectal temperature was monitored before and after challenge. All animals in any of the groups had normal rectal temperatures below 39.5 °C before challenge. After, challenge, vaccinated groups, either MDA-negative or MDA-positive, showed similar values during the whole period, thus no differences between both vaccinated groups (A and B) were observed (Figure 14).

[0359] A peak of rectal temperatures was noticed between days 4 and 9 after challenge. Control group C had an average of rectal temperatures, 39.53°C, significantly higher than vaccinated groups, 38.95°C and 38.89°C (Group A and B respectively).

[0360] Hyperthermia was considered on animals with rectal temperature above 39.5°C. Vaccinated groups A and B had the same average of days with hyperthermia (0.8 days in either MDA-seronegative or MDA-seropositive groups). Furthermore, control group C had an average of 3.3 days with hyperthermia, which was significantly higher than the average observed in the vaccinated groups. The results demonstrated that the vaccine compositions of the invention reduced hyperthermia (increase of rectal temperature) caused by a PI-3 infection, when it is administered in two doses, regardless the immunological status of the calves at the time of vaccination (MDA-negative, MDA-positive).

[0361] General clinical signs:

[0362] General clinical signs were monitored during the entire study and no alterations due to the vaccine composition were observed during the vaccination phase. After challenge, all clinical signs recorded including any respiratory distress were considered and reported as average of clinical signs per day.

[0363] During the whole post-challenge period, no differences between vaccinated groups A and B were observed. In addition, control group C had a higher average score (5.5) compared to vaccinated groups, either MDA-negative (4.27) or MDA-positive (4.26) (Groups A and B, respectively) from the day of challenge until the end of the study (D56, 14 days post-challenge). These differences between control group and vaccinated groups were statistically significant. Accordingly, the results demonstrated that clinical signs caused by a PI-3 infection are significantly reduced in animals vaccinated with the vaccine compositions of the invention, when administered in two doses, either in presence or absence of anti-PI-3 antibodies, particularly maternally derived antibodies (MDAs).

[0364] Lung lesions:

[0365] Lung lesions were assessed at the end of the study in necropsied animals (day 14 post-challenge). Particularly, it was observed that the average of total lung affection in control group C was higher (3.62%) than in the vaccinated groups. Both vaccinated groups, either MDA-seronegative or MDA-seropositive animals, had a similar percentage of lung affectation 1.58% and 1.51 % (Groups A and B, respectively).

[0366] Accordingly, the results obtained in this study also demonstrated that vaccination with vaccine compositions of the invention reduced lung lesions caused by experimental infection of PI-3, when it is administered in two doses, regardless of the immunological status of the animals, either seropositive or seronegative to PI-3.

[0367] Anti-PI-3 antibodies and Seroneutralization:

[0368] All animals were sampled before vaccination, as expected all animals in the vaccinated Group B (MDA-positive) and in the control Group C (MDA-seropositive) had specific antibodies against PI-3. In contrast, animals vaccinated in Group A (MDA- seronegative) were free from anti-PI-3 antibodies. The average titres of PI-3 by ELISA and seroneutralization assay demonstrated that all animals included in the seropositive groups had antibodies against PI-3 the day 0 of the study (DO). After the second dose of the vaccine (D21 ), an increase of the anti-PI-3 antibody response was observed in both vaccinated groups, A and B, no significant differences were observed between these two groups from until the end of the study (D56).

[0369] Moreover, both vaccinated groups (Group A and Group B) had significantly higher neutralizing antibody titres against PI-3 compared to control group (Group C) (Figure 15). These results were similar in terms of seroneutralization antibodies, and both vaccinated groups A and B performed in the same way, no differences between them were observed in contrast to control group C.

[0370] Parameters to demonstrate protection from a PI-3 challenge showed that the efficacy of the vaccine composition of the invention when administered in two doses is similar in both MDA-negative and MDA-positive animals.

[0371] In summary, results obtained in efficacy sub-study 2 performed against PI-3, demonstrated that vaccine compositions of the invention significantly reduce virus shedding (excretion), lung lesions, clinical sings and hyperthermia caused by PI-3 after an experimental infection in either seropositive or seronegative animals, when the vaccine composition is administered by intramuscular route in two doses.

[0372] Results of Sub-study 3 (Efficacy against BoHV-1)

[0373] Virus shedding:

[0374] Previous to challenge, nasal swabs were obtained from all animals at different time points to assess BoHV-1 presence before challenge. The analysis confirmed that all samples were free from BoHV-1 and the virus was not detected in any of the samples.

[0375] The differences in virus shedding between vaccinated groups A and B (MDA- seronegative and MDA-seropositive, respectively) and control group C (MDA-positive) were measured analyzing the average of total virus shedding per group on the excreting days from day 1 to day 21 post-challenge (days 66 to 87 of the study). However, because virus excretion was not observed beyond day 15 post-challenge (day 81 of the study), subsequent monitoring was not necessary. Individual quantitative value (CCID50 / ml) was obtained for each animal and time point. These values were log transformed. Using them, the individual virus shedding from day 1 to 21 post-challenge was calculated to assess the total shedding during the post-challenge period. Furthermore, the number of days shedding BoHV-1 were also calculated during the post-challenge phase to assess differences in days of shedding. Daily shedding and days of shedding data per group were compared. Total virus shedding was also compared to assess differences between groups.

[0376] Results of this sub-study demonstrated that BoHV-1 virus shedding was significantly reduced in both vaccinated groups, either MDA-seronegative and MDA-seropositive (Group A and Group B, respectively) when compared to control group C (Figure 16). The control group had significantly higher average of days shedding BoHV-1 virus in comparison to vaccinated groups (8.2 days in control group C vs 3.6 days and 5.0 days in vaccinated animals, either MDA-seronegative or MDA-seropositive (Group A and B respectively). Vaccinated groups A and B showed a reduction of more than 3 days of shedding compared to control group. Furthermore, the average shedding titer (CCID5o / ml) for control group C was significantly higher compared to both vaccinated groups over the entire post-challenge period, from day 67 to day 81 of the study (Table 10).

[0377] Table 10: Titer of BoHV-1 virus shedding (log CCID5o / ml) and mean shedding days per group from days 1 to 15 post challenge, day 67 (D67) to day 81 (D81 ) of the study.

[0378] Hyperthermia:

[0379] Overall rectal temperature was monitored before and after challenge. All animals in any of the groups had normal rectal temperatures below 39.5 °C before challenge. After, challenge, vaccinated groups, either MDA-negative or MDA-positive, showed similar values during the whole period, no differences between both vaccinated groups (A and B) were observed. In contrast, an increase on rectal temperatures was observed in animals in the control group (Group C) between 6 and 9 days after challenge (Figure 17). Considering the number of days with hyperthermia (rectal temperature above 39.5°C), control group (Group C) had an average of 3 days of hyperthermia, which was significantly higher than the observed average in both vaccinated groups A and B, with an average of 1.0 days and 1.2 day in MDA-seronegative and MDA-seropositive groups, respectively.

[0380] The results demonstrated that the vaccine compositions of the invention reduced hyperthermia (increase of rectal temperature) caused by a BoHV-1 infection, when it is administered in two doses, regardless the immunological status of the calves at the time of vaccination (MDA-negative, MDA-positive).

[0381] General clinical signs:

[0382] General clinical signs were monitored during the entire study and no alterations due to the vaccine composition were observed during the vaccination period. After challenge, all clinical signs recorded including any respiratory distress were considered and reported as average of clinical signs per day. In particular, the evaluation of clinical signs associated to BoHV-1 infection was carried out recoding the clinical score for dyspnea, depression, nasal discharge, ocular discharge and cough for 21 days after challenge.

[0383] The results of this efficacy sub-study demonstrated that the average clinical signs from day 1 to day 21 after challenge per group was significantly greater in control group C with a clinical score of 1.50, compared to vaccinated groups A and B, with a clinical score of 0.62 for Group A (MDA-seronegative) and 0.52 for Group B (MDA-seropositive). No significant differences were observed between vaccinated groups A and B. Vaccinated animals only presented mild clinical signs after the BoHV-1 infection, mainly nasal discharge, ocular discharge and sporadically some coughing. In contrast, control animals presented a more severe respiratory affection concurring with nasal discharge, ocular discharge, depression, dyspnea and cough with higher scores compared to vaccinated groups.

[0384] Accordingly, the results demonstrated that clinical signs caused by a BoHV-1 infection are significantly reduced in animals vaccinated with the vaccine compositions of the invention, when administered in two doses, either in presence or absence of anti-BoHV-1 antibodies, particularly maternally derived antibodies (MDAs).

[0385] Anti-BoHV-1 antibodies and Seroneutralization: All animals were sampled before vaccination, as expected all animals in the vaccinated Group B (MDA-positive) and in the control Group C (MDA-seropositive) had specific antibodies against BoHV-1. In contrast, animals vaccinated in Group A (MDA- seronegative) were free from anti-BoHV-1 antibodies. Consequently, the average titres (ELISA and SN antibodies) and percentage of seropositive animals of MDA-seropositive groups (control and vaccinated) were significantly higher compared to MDA-seronegative vaccinated group on day 0.

[0386] After the two-dose vaccination protocol, both vaccinated groups (Group A and Group B) had similar values of anti-BoHV-1 antibody titres (both ELISA and neutralizing antibodies) and percentage of positive animals until the end of the study, demonstrating that MDAs had no interference in the antibody response of the vaccine composition against BoHV-1 infections. In contrast, all control animals, had a marked anti-BoHV-1 decrease from day 9 to day 66 of the study and after challenge, both vaccinated groups Aand B had a statistically higher anti-BoHV-1 antibody titers compared to control group C.

[0387] Regarding seroneutralization antibodies against BoHV-1 , both vaccinated groups A and B had similar values the day of challenge, whereas control group had significantly lower seroneutralization antibodies against BoHV-1 compared to vaccinated groups (Figure 18).

[0388] Parameters to demonstrate protection from a BoHV-1 experimental infection showed that the efficacy of the vaccine composition of the invention when administered in two doses is similar in both MDA-negative and MDA-positive animals.

[0389] Overall, results obtained in efficacy sub-study 3 performed against BoHV-1 , demonstrated that vaccine compositions of the invention significantly reduce virus shedding (excretion), lung lesions, clinical sings and hyperthermia caused by BoHV-1 after an experimental infection in either seropositive or seronegative animals, when the vaccine composition is administered by intramuscular route in two doses.

[0390] Taken as a whole, Example 4 supports the efficacy of the vaccine compositions of the invention for active immunization of cattle to reduce virus shedding, hyperthermia, leukopenia, lung lesions and other clinical sings against different pathogens causing the bovine respiratory disease complex (BRDC) such as Bovine Respiratory Syncytial Virus (BRSV), Parainfluenza Type 3 (PI-3) Virus, Bovine Herpesvirus type-1 Virus (BoHV-1 ), Bovine Viral Diarrhea Virus Type 1 (BVDV-1 ) and Bovine Viral Diarrhea Virus Type 2 (BVDV- 2) either in presence or absence of maternally derived antibodies. EXAMPLE 5: Efficacy of vaccine compositions comprising recombinant E2 protein of BVDV-1 and BVDV-2 against transplacental infection after an experimental Bovine Diarrhea Virus Type 1 infection in pregnant heifers

[0391] The aim of this study was to assess the efficacy of vaccine compositions comprising recombinant E2 proteins of BVDV-1 and BVDV-2 administered to pregnant heifers against transplacental infection after an experimental challenge with BVDV-1 .

[0392] A total of thirty female calves (Bos taurus) of approximately 10-weeks of age were enrolled in this study. The calves were free from bovine diarrhea virus and without antibodies against bovine diarrhea virus. The animals were randomly allocated to two treatment groups (Table 11 ).

[0393] Table 11 : Treatment groups r: recombinant-produced protein, LAV: live attenuated virus, LMV: live modified virus,

[0394] K: killed inactivated virus

[0395] Animals in Group A were vaccinated intramuscularly (IM) with two doses, separated three weeks apart, on Day 0 (DO) and Day 21 (D21 ) of the study, and revaccinated 6 months later, on Day 203 (D203) of the study with the vaccine composition of the invention. Control group B received a mock-up vaccine consisting of sterile phosphate buffer saline (PBS) through the same route and the same vaccination scheme.

[0396] Group A: 20 calves were vaccinated intramuscularly in the neck muscle with two doses, separated three weeks apart, on Day 0 (DO) and Day 21 (D21 ), and then, revaccinated 6 months later, on Day 203 (D203) of the study with a 2 mL of a vaccine composition comprising: o 40 pg / dose of recombinant E2 protein from BVDV-1 and 40 pg / dose of recombinant E2 protein from BVDV-2; o 1054CCID5o / dose (cell culture infectious dose 50%) of live attenuated Bovine Respiratory Syncytial Virus (BRSV) strain Lym-56; o 1066CCID5o / dose of live modified Bovine herpesvirus type 1 (BoHV-1 ), also called Infectious Bovine Rhinotracheitis Virus (IBR), strain CEDDEL; o 480 HAU (hemagglutination units) inactivated bovine Parainfluenza 3 (PI-3) strain SF4.

[0397] Group B: 10 calves were vaccinated intramuscularly in the neck muscle with two doses, separated three weeks apart, on Day 0 (DO) and Day 21 (D21 ) and then, revaccinated 6 months later, on Day 203 (D203) of the study with a 2 ml_ mockvaccine composition comprising sterile PBS (phosphate buffer saline).

[0398] The vaccine composition of Group A was further formulated 50% v / v with the commercial adjuvant Montanide™ IMS (SEPPIC).

[0399] The attenuated strain of BRSV and the inactivated strain of PI-3 were obtained from the commercial vaccine HIPRABOVIS® 4 (Laboratorios HIPRA, S.A., Avda. La Selva, 135, 17170 Amer, Girona, Spain). The attenuated strain of IBR was obtained from the commercial vaccine HIPRABOVIS® IBR Marker Live (Laboratorios HIPRA, S.A., Avda. La Selva, 135, 17170 Amer, Girona, Spain). Recombinant E2 proteins from BVDV-1 and BVDV- 2 were obtained by recombinant DNA technology and expressed in mammalian cells (CAP cells), as described in Example 1.

[0400] To assess the efficacy of the vaccine composition against BVDV-1 transplacental infection, all animals were synchronized and bred by artificial insemination after revaccination. Artificial insemination was conducted on day 260 of the study and pregnancy check on day 292 and at different time points after challenge. At the 84thday of gestation, corresponding to Day 344 (D344) of the study, pregnant animals in both Groups, A and B, were experimentally challenged with BVDV-1 by the intranasal route. The animals were inoculated with a 10 mL suspension comprising 103CCID5o / ml of BVDV-1 non-cytopathic strain V-1146 (half of the dose volume was administered into each nostril through a disposable nasal applicator aerosol generator coupled to a syringe).

[0401] All animals were monitored on daily basis for general clinical signs during the entire study (from day -1 (D-1 ) until the end of the study (D410)). Blood samples were collected from heifers at different sampling points from D-1 to D407 to assess anti-BVDV-1 antibodies and seroneutralizing response against BVDV-1 as well as for BVDV-1 detection and titration by PCR and by cell culture from nasal swabs, buffy coats and fetal tissues. Heparinized blood samples were also collected to obtain peripheral blood mononuclear cells (PBMCs) to determine BVDV-specific IFN-y. Blood samples for white blood cell (WBC) counts were collected in EDTA tubes one day prior to challenge and different days post-challenge. Viremia was monitored from the day of challenge (D344) up to day 21 after challenge. Pregnancy monitoring and checks were performed at different time points before and after challenge. If abortions occurred during the study, any fetal tissue, placenta or liquid available from the aborted fetus was collected in order to check the presence of BVDV-1 in said samples. Heifers were humanely euthanized at the end of the study on Day 410 (D410) and each fetus was recovered to obtain fetal serum and fetal tissues (brain, Peyer’s patches, liver, spleen and thymus gland) for virus isolation.

[0402] Overall, the following parameters were evaluated after BVDV-1 challenge:

[0403] - General clinical signs (monitored all over the study).

[0404] - Serology: Analysis of BVDV-1 antibodies (BVDV ELISA kit, IDEXX), BVDV-1 seroneutralization antibodies (SN test) and interferon gamma (IFNg) (Bovigam ELISA Kit).

[0405] - White blood cell (WBC) counts analyzed using a semi-automated electronic cell counting device (XN-1000 Sysmex).

[0406] - BVDV-1 virus detection and titration from nasal swabs and buffy coats on heifers (RT-qPCR).

[0407] - BVDV-1 virus detection on fetal tissue (Virus isolation in cell culture and IHC).

[0408] Total anti-BVDV-1 antibodies and Seroneutralization results in heifers:

[0409] From D21 until the day of challenge (D344) vaccinated animals (Group A) had a significantly higher average titre of ELISA anti-BVDV-1 antibodies (Figure 19). Similarly, from D41 until challenge (D344), vaccinated animals also had significantly higher average titre of seroneutralizing anti-BVDV-1 antibodies compared to the control group (Group B) (Figure 20). Furthermore, the percentage of seropositive animals, both total anti-BVDV-1 antibody titres (ELISA) and neutralizing antibodies (SN assay), was significantly higher in the vaccinated group (Group A) compared to the control group (Group B) from D41 to D344 (either ELISA or SN assay). A peak mean antibody log? titer of 8.8 (GMT, 438.5) was reached after the third dose of vaccine on Day 226 in vaccinated group (Group A), whereas control group had no neutralizing antibodies prior to challenge. All vaccinated animals became seropositive after the second dose, and after the third dose, anti-BVDV-1 antibodies measured by ELISA or SN assay increased in the vaccinated group A, indicating a booster immune response on these animals.

[0410] After challenge, the total anti-BVDV-1 antibody titers (ELISA) and seroneutralizing anti-BVDV-1 antibody titres (SN assay) of vaccinated group (Group A) remained significantly higher compared to control group (Group B) (Figures 19 and 20). All control animals (Group B) had anti-BVDV-1 antibodies 21 days after challenge, indicating that the challenge was correctly conducted.

[0411] In terms of immune cellular response, the study measured by interferon-gamma analysis, that vaccinated animals (Group A) had significantly higher values on the day of challenge (D344) compared to control group (Group B) (Figure 21 ). This difference was also observed 7 days after challenge (D351 ), when vaccinated animals had a higher average on interferon-gamma compared to the values obtained in the challenge day, whereas control animal had no interferon-gamma expression at all.

[0412] Viremia:

[0413] The detection of BVDV-1 on blood samples by PCR was performed in order to assess the viremia on the animals. The differences in viremia between control animals (Group B) are shown in Figure 22, where the average of virus titre in the control group (Group B) was significantly higher from day 7 to 10 after challenge compared to vaccinated animals (Group A). The percentage of viremic animals was also significantly higher in the control group (Group B) in comparison with the vaccinated group (Group A) on days 7, 8 and 10 after challenge.

[0414] Regarding the number of days with viremia, the control group (Group B) had a significantly higher average of viremic days (2.62 days) in comparison with the vaccinated group (A), for which viremia was practically zero days (0.2 days).

[0415] White Blood Cell (WBC) count:

[0416] After the BVDV-1 challenge, control animals (Group B) showed a clear decrease in white blood cells on days 5 to 7 after challenge. In contrast, vaccinated animals did not show a significant decrease in white blood cells. Thus, the vaccine composition of the invention prevents the immunosuppression caused by BVDV-1 infection.

[0417] BVDV-1 virus detection on fetal tissues by virus isolation in cell culture and IHC:

[0418] All fetal samples from control animals (Group B) were positive for presence of BVDV- 1 in the brain and Peyer’s patches. The BVDV-1 virus was also detected in the thymus gland, liver and spleen samples in most of the control animal fetal samples, and the average BVDV-1 virus titration was significantly higher in the control group (Group B) (Figure 23). The BVDV-1 virus was detected in 100% of the fetal samples from the control group (Group B); whereas it was only detected in 6.7% of the fetal samples from the vaccinated group (Group A). Hence, the control group (Group B) showed a significantly higher percentage of animals with presence of BVDV-1 virus in all the collected fetal samples compared to the vaccinated group (Group A) (Figure 24).

[0419] Overall, the results demonstrate that the vaccine composition of the invention generates an immune response against BVDV-1 that is protective for reproductive parameters. The vaccine compositions clearly reduced the number of BVDV-1 viremic animals and the duration of BVDV-1 viremia caused by experimental infection with BVDV- 1. Moreover, the reduction in the number of days of viremia and in the total viral titer observed in vaccinated animals prevented transplacental infection and birth of persistently infected offspring. Additionally, the results also demonstrated that the vaccine compositions of the invention administered to heifers in a two-dose vaccination protocol followed by a revaccination with a third dose is effective to protect from births of persistently infected animals and from transplacental infection against BVDV-1.

[0420] When a two-dose vaccination regime is followed, the results obtained also demonstrated the efficacy of the vaccine compositions of the invention to protect from births of persistently infected animals and from transplacental infection against BVDV-1 , although a superior response was obtained with the basic two-dose protocol plus a revaccination booster (third dose) after 6 months.

[0421] EXAMPLE 6: Efficacy of vaccine compositions comprising recombinant E2 protein of BVDV-1 and BVDV-2 against transplacental infection after an experimental Bovine Diarrhea Virus Type 2 infection in pregnant heifers

[0422] The aim of this study was to assess the efficacy of vaccine compositions comprising recombinant E2 proteins of BVDV-1 and BVDV-2 administered to pregnant heifers against transplacental infection after an experimental challenge with BVDV-2.

[0423] A total of thirty female calves (Bos taurus) of approximately 10-weeks of age were enrolled in this study. The calves were free from bovine diarrhea virus and without antibodies against bovine diarrhea virus. The animals were randomly allocated to two treatment groups (Table 12). Table 12: Treatment groups. r: recombinant-produced protein, LAV: live attenuated virus, LMV: live modified virus,

[0424] K: killed inactivated virus

[0425] Animals in Group A were vaccinated intramuscularly (IM) with two doses, separated three weeks apart, on Day 0 (DO) and Day 21 (D21 ) of the study, and revaccinated 6 months later, on Day 203 (D203) of the study with the vaccine composition of the invention. Control group B received a mock-up vaccine consisting of sterile phosphate buffer saline (PBS) through the same route and the same vaccination scheme.

[0426] Group A: 20 calves were vaccinated intramuscularly in the neck muscle with two doses, separated three weeks apart, on Day 0 (DO) and Day 21 (D21 ), and then, revaccinated 6 months later, on Day 203 (D203) of the study with a 2 mL of a vaccine composition comprising: o 40 pg / dose of recombinant E2 protein from BVDV-1 and 40 pg / dose of recombinant E2 protein from BVDV-2; o 1056CCID5o / dose (cell culture infectious dose 50%) of live attenuated Bovine Respiratory Syncytial Virus (BRSV) strain Lym-56; o 1067CCID5o / dose of live modified Bovine herpesvirus type 1 (BoHV-1 ), also called Infectious Bovine Rhinotracheitis Virus (IBR), strain CEDDEL; o 320 HAU (hemagglutination units) inactivated bovine Parainfluenza 3 (PI-3) strain SF4.

[0427] Group B: 10 calves were vaccinated intramuscularly in the neck muscle with two doses, separated three weeks apart, on Day 0 (DO) and Day 21 (D21 ) and then, revaccinated 6 months later, on Day 203 (D203) of the study with a 2 mL mockvaccine composition comprising sterile PBS (phosphate buffer saline). The vaccine composition of Group A was formulated 50% v / v with the commercial adjuvant Montanide™ IMS (SEPPIC).

[0428] The attenuated strain of BRSV and the inactivated strain of PI-3 were obtained from the commercial vaccine HIPRABOVIS® 4 (Laboratorios HIPRA, S.A., Avda. La Selva, 135, 17170 Amer, Girona, Spain). The attenuated strain of IBR was obtained from the commercial vaccine HIPRABOVIS® IBR Marker Live (Laboratorios HIPRA, S.A., Avda. La Selva, 135, 17170 Amer, Girona, Spain). Recombinant E2 proteins from BVDV-1 and BVDV- 2 were obtained by recombinant DNA technology and expressed in mammalian cells (CAP cells), as described in Example 1.

[0429] To assess the efficacy of the vaccine composition against BVDV-2 transplacental infection, all animals were synchronized and bred by artificial insemination after revaccination. Artificial insemination was conducted on day 262 of the study and pregnancy check on day 294 and at different time points after challenge. At the 85thday of gestation, corresponding to Day 347 (D347) of the study, pregnant animals in both Groups, A and B, were experimentally challenged with BVDV-2 by the intranasal route. Animals received a 10 mL suspension comprising 104CCID5o / ml of BVDV-2 non-cytopathic strain V- 1201 (half of the dose volume was administered into each nostril through a disposable nasal applicator aerosol generator coupled to a syringe).

[0430] All animals were monitored on daily basis for general clinical signs during the entire study (from day -1 (D-1 ) until the end of the study (D442). Blood samples were collected from heifers at different sampling points from DO to D442 to assess anti-BVDV-2 antibodies and seroneutralizing response against BVDV-2 as well as for BVDV-2 detection and titration by PCR and by cell culture from nasal swabs, buffy coats and fetal tissues. Heparinized blood samples were also collected to obtain peripheral blood mononuclear cells (PBMCs) to determine BVDV-specific IFN-y. Blood samples for white blood cell (WBC) counts were collected in EDTA tubes one day prior to challenge and different days post-challenge- Viremia was monitored from the day of challenge (D347) up to day 21 after challenge. Pregnancy monitoring and checks were performed at different time points before and after challenge. If abortions occurred during the study, any fetal tissue, placenta or liquid available from the aborted fetus was collected in order to check the presence of BVDV-2 in said samples. Heifers were humanely euthanized at the end of the study on Day 442 (D442) and each fetus was recovered to obtain fetal’s serum and fetal tissues (brain, Peyer’s patches, liver, spleen and thymus gland) for virus isolation. Overall, the following parameters were evaluated after BVDV-2 challenge:

[0431] - General clinical signs (monitored all over the study).

[0432] - Serology: Analysis of BVDV-2 antibodies (BVDV ELISA kit, IDEXX), BVDV-2 seroneutralization antibodies (SN test) and interferon gamma (IFNg) (Bovigam ELISA Kit).

[0433] - White blood cell (WBC) counts analyzed using a semi-automated electronic cell counting device (XN-100 Sysmex).

[0434] - BVDV-2 virus detection and titration from buffy coats on heifers (RT-qPCR).

[0435] - BVDV-2 virus detection on fetal tissue (virus isolation in cell culture and IHC).

[0436] Total anti-BVDV-2 antibodies and Seroneutralization results in heifers:

[0437] From D21 until challenge (D347) vaccinated animals (Group A) had a significantly higher average titre of ELISA anti-BVDV-2 antibodies (Figure 25). Similarly, from D43 until challenge (D347), vaccinated animals had significantly higher average titre of seroneutralizing anti-BVDV-2 antibodies compared to the control group (Group B) (Figure 26). On challenge day (D42) vaccinated group (Group A) had a mean antibody log? titer of 7.5 (GMT, 177.4), whereas control group (Group B) had no neutralizing antibodies prior to challenge. Regarding the percentage of animals with neutralizing antibodies, at least 75% of the vaccinated animals (Group A) were seropositive from D43 and reached 100% on D228. The percentage of seropositive animals, either total anti-BVDV-2 antibody titres (ELISA) and neutralizing antibodies (SN assay) was significantly higher in the vaccinated group (Group A) compared to the control group (Group B) from D43 to the day of challenge (D347) either by ELISA or SN assay. All vaccinated animals became seropositive after the second dose, and after the third dose, anti-BVDV-2 antibodies measured by ELISA or SN assay increased in the vaccinated group A, indicating a booster immune response on these animals.

[0438] After challenge, the total anti-BVDV antibody titers (ELISA) and seroneutralizing anti- BVDV-2 antibody titres (SN assay) of vaccinated group (Group A) remained significantly higher compared to control group (Group B) (Figure 25 and 26). All control animals (Group B) had anti-BVDV-2 antibodies 21 days after challenge, indicating that challenge was correctly conducted.

[0439] In terms of immune cellular response, the measured by interferon-gamma analysis, the study showed that vaccinated animals (Group A) had significantly higher values on the day of challenge (D347) compared to control group (Group B) (Figure 27). This difference was also observed 7 days later (D354), when vaccinated animals had a higher average on interferon-gamma compared to the values obtained in the challenge day, whereas control animal had no IFNg expression at all.

[0440] Viremia:

[0441] The detection of BVDV-2 on blood samples by PCR was performed in order to assess the viremia on the animals. The differences in viremia between control animals (Group B) and vaccinated animals (Group A) are depicted in Figure 28. The total BVDV-2 virus titer was significantly lower from day 7 to day 9 in vaccinated animals (Group A) compared to control group (Group B). The percentage of viremic animals was also significantly lower in the vaccinated group (Group A) in comparison with the control group (Group B) on days 7 and 8 after challenge. Regarding the number of days with viremia, the vaccinated group (Group A) had a significantly lower average of viremic days, which was practically zero days (0.18 days), in comparison with the control group (Group B), with an average of viremic days of 2 days.

[0442] White Blood Cell (WBC) count:

[0443] After the BVDV-2 challenge, control group (Group B) showed a significant decrease in white blood cells on Day 5 post-challenge. Vaccinated group (Group A) did not show a decrease. Furthermore, the average WBC count in vaccinated group was higher than in control group on Days 5 to 8 post-challenge. Thus, the vaccine composition of the invention prevents immunosuppression caused by BVDV-2 infection.

[0444] BVDV-2 virus detection on fetal tissues by virus isolation in cell culture and IHC:

[0445] All fetal samples from control animals (Group B) were positive for presence of BVDV- 2 in all tissues assessed (brain, spleen, thymus gland, Peyer’s patches and liver) (Figure 30). BVDV-2 was detected in 100% of the fetal samples from the control group (Group B); whereas it was only detected in 5.9% of the fetal samples from the vaccinated group (Group A). Hence, the control group (Group B) showed a significantly higher percentage of animals with presence of BVDV-2 virus in all the collected fetal samples compared to the vaccinated group (Group A). The average BVDV-2 virus titration was also significantly lower in the vaccinated group (Group A) compared to the control group (Group B) in all the collected samples (Figure 29).

[0446] Overall, the results demonstrate that the vaccine composition of the invention provides an immune response against BVDV-2 that is protective for reproductive parameters. The vaccine compositions clearly reduced the number of BVDV-2 viremic animals and the duration of BVDV-2 viremia caused by experimental infection with BVDV- 2. The reduction in the number of days of viremia and in the total viral titer observed in vaccinated animals prevented transplacental infection and birth of persistently infected offspring. Furthermore, the results also demonstrated that the vaccine compositions of the invention administered to heifers in a two-dose vaccination protocol followed by a revaccination with a third dose is effective to protect from births of persistently infected animals and from transplacental infection against BVDV-2.

[0447] When a two-dose vaccination regime is followed, the results obtained also demonstrated the efficacy of the vaccine compositions of the invention to protect from births of persistently infected animals and from transplacental infection against BVDV-2, although a superior response was obtained with the basic two-dose protocol plus a revaccination booster (third dose) at 6 months.

[0448] EXAMPLE 7: Passive immunity in calves by the colostrum of vaccinated dams

[0449] The aim of this study was to assess the effect of maternal immunization against BVD (Bovine Viral Diarrhoea) and IBR (Infectious Bovine Rhinotracheitis) of vaccine compositions of the invention comprising recombinant E2 proteins of BVDV-1 and BVDV-2 on the transfer of passive immunity to calves.

[0450] Cows and heifers at different pregnancy stages from two dairy cattle herds were included in the study. Animals were randomly distributed in two groups stratified according to pregnancy stage.

[0451] Group A was vaccinated intramuscularly) with 2 mL of a vaccine composition comprising: o 60 pg / dose of recombinant E2 protein from BVDV-1 and 60 pg / dose of recombinant E2 protein from BVDV-2; o 1060CCID5o / dose (cell culture infectious dose 50%) of live attenuated Bovine Respiratory Syncytial Virus (BRSV) strain Lym-56; o 107 3CCID5o / dose of live modified Bovine herpesvirus type 1 (BoHV-1 ), also called Infectious Bovine Rhinotracheitis Virus (IBR), strain CEDDEL; o 480 HAU (hemagglutination units) inactivated bovine Parainfluenza 3 (PI-3) strain SF4.

[0452] The vaccine composition of Group A was formulated 50% v / v with the commercial adjuvant Montanide™ IMS (SEPPIC). The attenuated strain of BRSV and the inactivated strain of PI-3 were obtained from the commercial vaccine HIPRABOVIS® 4 (Laboratorios HIPRA, S.A., Avda. La Selva, 135, 17170 Amer, Girona, Spain). The attenuated strain of IBR was obtained from the commercial vaccine HIPRABOVIS® IBR Marker Live (Laboratorios HIPRA, S.A., Avda. La Selva, 135, 17170 Amer, Girona, Spain). Recombinant E2 proteins from BVDV-1 and BVDV- 2 were obtained by recombinant DNA technology and expressed in mammalian cells (CAP cells), as described in Example 1.

[0453] The other group, Control group B, was administered intramuscularly a 2 mL mockvaccine composition comprising sterile PBS (phosphate buffer saline).

[0454] All animals received the basic vaccination schedule (two doses of the corresponding-study product 21 days apart) prior to calving. Animals at the inclusion day (day 0, first dose vaccination) found in the first trimester of pregnancy, received an additional dose 6 months after, making a total of three doses before calving.

[0455] The immunological response was assessed at calving. For that purpose, colostrum and serum samples from the dams were collected within 24 hours of calving. Serum samples from the newborns were collected at birth (between 2 and 7 days after birth; D2 / 7), after colostrum feeding of their own dam, and one month later (D30). Colostrum and serum samples were analyzed for BVDV and BoHV-1 (IBR) antibody detection by means of ELISA (IDEXX BVDV Total Ab and CIVTEST®BOVIS IBRgB for serum / CIVTEST®BOVIS IBR for colostrum). Newborn serum samples were also analyzed for neutralizing antibodies detection against BVDV-1 , BVDV-2 and BoHV-1 by virus neutralization test.

[0456] Overall, samples from 37 animals in the Control group B and 36 animals in Group A were collected.

[0457] Colostrum samples from the Group A presented a significantly higher mean antibody titers both for BVDV and BoHV-1 compared to the Control group B (Figure 31 ).

[0458] Serum BVDV ELISA antibody titers from Group A were significantly higher in comparison to the Control group B. Serum samples from the Control group B remained seronegative and below the cut-off value for all samplings. Newborns from Group A presented greater values than dam serum samples. Specifically, the highest titers were observed at birth timepoint (D2 / 7), while remaining significantly high one month after (D30) (Figure 32). In the same line, the antibody response against BoHV-1 (IBR) in serum was statistically significantly higher in Group A compared to the Control group B for all sampling timepoints in both dams and newborn animals (Figure 32).

[0459] Neutralizing antibodies observed in newborn animals of Group A against BVDV-1 , BVDV-2 and BoHV-1 (IBR) were statistically significantly higher than the Control group B, both at birth and at D30 timepoints (Figure 33).

[0460] The results demonstrate that the vaccine composition of the invention induces a significantly high antibody response against BVD (and IBR) in vaccinated dams at different pregnancy stages, which provides passive immunity to newborn calves fed with colostrum from vaccinated dams.

Claims

CLAIMS1. An immunogenic or vaccine composition for use in a method of treatment and / or prevention of Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or Bovine Viral Diarrhoea Virus type 2 (BVDV-2) infection in cattle, where the immunogenic or vaccine composition comprises a recombinant E2 protein of a BVDV-1 strain and a recombinant E2 protein of a BVDV-2 strain.

2. The immunogenic or vaccine composition for use according to claim 1 , wherein the treatment and / or prevention of BVDV-1 and / or BVDV-2 infection is the reduction of the clinical signs associated with BVDV-1 and / or BVDV-2 infection.

3. The immunogenic or vaccine composition for use according to claim 2, wherein the clinical signs associated with BVDV-1 and / or BVDV-2 infection are selected from a group consisting of: viremia, virus shedding, leukopenia, lung lesions, hyperthermia and any combination thereof.

4. The immunogenic or vaccine composition for use according to any one of claims 1 to 3, wherein the prevention of BVDV-1 and / or BVDV-2 infection is the prevention of transplacental BVDV-1 and / or BVDV-2 fetal infection.

5. The immunogenic or vaccine composition for use according to any one of claims 1 to 4, wherein the prevention of BVDV-1 and / or BVDV-2 infection is the prevention of the birth of persistently infected calves.

6. The immunogenic or vaccine composition for use according to any one of claims 1 to 5, wherein the prevention is the prevention of the progeny of a pregnant cow from a BVDV-1 and / or BVDV-2 infection by passive immunization by the colostrum.

7. The immunogenic or vaccine composition for use according to any one of claims 1 to 6, wherein the cattle has circulating maternally-derived antibodies (MDA) anti- Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or maternally-derived antibodies (MDA) anti-Bovine Viral Diarrhoea Virus type 2 (BVDV-2).

8. The immunogenic or vaccine composition for use according to any one of claims 1 to 7, wherein the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain comprises the sequence according to SEQ ID NO: 1 or a functionally equivalent variant thereof.

9. The immunogenic or vaccine composition for use according to claim 8, wherein the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain consists of the sequence according to SEQ ID NO: 1.

10. The immunogenic or vaccine composition for use according to any one of claims 1 to 7, wherein the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain comprises the sequence encoded by the sequence according to SEQ ID NO: 3 or a functionally equivalent variant of said sequence encoded by the sequence according to SEQ ID NO: 3.11 . The immunogenic or vaccine composition for use according to claim 10, wherein the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain consists of the sequence encoded by the sequence according to SEQ ID NO: 3.

12. The immunogenic or vaccine composition for use according to any one of claims 1 to 11 , wherein the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain comprises the sequence according to SEQ ID NO: 2 or a functionally equivalent variant thereof.

13. The immunogenic or vaccine composition for use according to claim 12, wherein the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain consists of the sequence according to SEQ ID NO: 2.

14. The immunogenic or vaccine composition for use according to any one of claims 1 to 11 , wherein the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain comprises the sequence encoded by the sequence according to SEQ ID NO: 4 or a functionally equivalent variant of said sequence encoded by the sequence according to SEQ ID NO: 4.

15. The immunogenic or vaccine composition for use according to claim 14, wherein the recombinant E2 protein of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain consists of the sequence encoded by the sequence according to SEQ ID NO: 4.

16. The immunogenic or vaccine composition for use according to any one of claims 1 to 15, wherein the immunogenic or vaccine composition further comprises an immunogenic active component derived from a virus selected from the group consisting of: Bovine Respiratory Syncytial Virus (BRSV), Parainfluenza-3 virus (Pl- 3), Bovine Herpesvirus type 1 (BoHV-1 ) and any combination thereof.

17. The immunogenic or vaccine composition for use according to claim 16, wherein the immunogenic or vaccine compositions treats and / or prevents the infection by a virus selected from the group consisting of: Bovine Respiratory Syncytial Virus (BRSV), Parainfluenza-3 virus (PI-3), Bovine Herpesvirus type 1 (BoHV-1 ) and any combination thereof.

18. The immunogenic or vaccine composition for use according to claims 16 or 17, wherein the immunogenic or vaccine composition comprises an immunogenic active component derived from a Bovine Respiratory Syncytial Virus (BRSV) strain and a Bovine Herpesvirus type 1 (BoHV-1 ) strain, or a Bovine Respiratory Syncytial Virus (BRSV) strain and a Parainfluenza-3 virus (PI-3) strain, or a Bovine Herpesvirus type 1 (BoHV-1 ) strain and a Parainfluenza-3 virus (PI-3) strain, or a Bovine Respiratory Syncytial Virus (BRSV) strain and a Bovine Herpesvirus type 1 (BoHV- 1 ) strain and Parainfluenza-3 virus (PI-3) strain.

19. The immunogenic or vaccine composition for use according to any one of claims 1 to 18, wherein the immunogenic or vaccine composition treats and / or prevents the clinical signs associated with Bovine Respiratory Disease (BRD) selected from a group consisting of: viremia, virus shedding, leukopenia, lung lesions, hyperthermia and any combination thereof.

20. The immunogenic or vaccine composition for use according to any one of claims 1 to 19, wherein the immunogenic or vaccine composition treats and / or prevents the clinical signs associated with Bovine Respiratory Disease (BRD), wherein the clinical signs are selected from a group consisting of: abortions, congenital defects, cerebellar hypoplasia, encephalitis, weak and abnormally small calves, unthrifty, persistently infected (PI) calves, mortality, failure to nurse, early embryonic death, fetal mummification, fetal malformations, fetal transfer, transplacental fetal infection, and transient infertility.21 . The immunogenic or vaccine composition for use according to any one of claims 16 to 20, wherein the immunogenic active component is selected from a group consisting of: attenuated viral strain, inactivated viral strain, and recombinant protein derived from a viral strain.

22. The immunogenic or vaccine composition for use according to claim 21 , wherein the attenuated viral strain is obtained by genetic modification of the viral genome, or by repeated virus passage in primary cell cultures.

23. The immunogenic or vaccine composition for use according to claim 21 or 22, wherein the inactivated viral strain is a whole inactive viral strain, a subunit derived of the viral strain, and / or an antigen derived of the viral strain.

24. The immunogenic or vaccine composition for use according to any one of claims 21 to 23, wherein the recombinant protein is a subunit of the viral strain, an antigen of a viral strain and / or an antigen fragment of a viral strain.

25. The immunogenic or vaccine composition for use according to claim 21 or 22, wherein the attenuated viral strain is an attenuated Bovine Respiratory Syncytial Virus (BRSV) strain, preferably is the attenuated Bovine Respiratory Syncytial Virus (BRSV) Lym-56 strain, and / or the attenuated viral strain is an attenuated Bovine Herpesvirus type 1 (BoHV-1 ) strain, preferably is the Bovine Herpesvirus type 1 (BoHV-1 ) CEDDEL strain.

26. The immunogenic or vaccine composition for use according to claim 21 or 23, wherein the inactivated viral strain is an inactivated Parainfluenza-3 virus (PI-3) strain.

27. The immunogenic or vaccine composition for use according to claim 26, wherein the inactivated Parainfluenza-3 virus (PI-3) strain is the SF4 strain.

28. The immunogenic or vaccine composition for use according to any one of claims 1 to 27, wherein the immunogenic or vaccine composition further comprises an acceptable pharmaceutical adjuvant.

29. The immunogenic or vaccine composition for use according to claim 28, wherein the acceptable pharmaceutical adjuvant is selected from a group consisting of: mineral oil, ginseng, chitosan, dimethylaminoethyl (DEAE), aluminum hydroxide, phosphate buffer, Montanide IMS and any combination thereof.

30. The immunogenic or vaccine composition for use according to claim 28 or 29, wherein the acceptable pharmaceutical adjuvant is an emulsion.31 . The immunogenic or vaccine composition for use according to any one of claims 28 to 30, wherein the acceptable pharmaceutical adjuvant is at a concentration of between about 25% v / v to about 75% v / v, preferably 50% v / v.

32. The immunogenic or vaccine composition for use according to any one of claims 1 to 31 , wherein the immunogenic or vaccine composition further comprises a freeze- drying excipient.

33. The immunogenic or vaccine composition for use according to any one of claims 1 to 32, wherein the cattle is a female, more preferably a pregnant or lactating female.

34. The immunogenic or vaccine composition for use according to any one of claims 1 to 33, wherein the immunogenic or vaccine composition is administered bysubcutaneous, intradermal, or intramuscular route, preferably by intramuscular route.

35. The immunogenic or vaccine composition for use according to any one of claims 1 to 34, wherein the immunogenic or vaccine composition is administered in at least two doses.

36. The immunogenic or vaccine composition for use according to claim 35, wherein the first dose is administered in a subject of at least 10 weeks old.

37. The immunogenic or vaccine composition for use according to claim 35 or 36, wherein the second dose is administered at least about three weeks after the first dose.

38. The immunogenic or vaccine composition for use according to any one of claims 35 to 37, wherein a third dose is administered no longer than about 6 months after the second dose as a booster.

39. The immunogenic or vaccine composition for use according to any one of claims 35 to 38, wherein a further dose is administered as an annual booster no longer than 12 months after the third dose.

40. The immunogenic or vaccine composition for use according to any one of claims 1 to 39, wherein the immunogenic or vaccine composition is administered at a dose of between about 15 pg per dose to about 75 pg per dose of each of the recombinant E2 protein of the BVDV-1 strain and of the recombinant E2 protein of the BVDV-2 strain.41 . The immunogenic or vaccine composition for use according to claim 40, wherein the immunogenic or vaccine composition is administered at a dose of about 30 pg, 40 pg, 50 pg or 60 pg per dose of each of the recombinant E2 protein of the BVDV-1 strain and of the recombinant E2 protein of the BVDV-2 strain.

42. The immunogenic or vaccine composition for use according to any one of claims 17 to 27 or any one of claims 28 to 39 whenever in the context of claims 17 to 27, wherein the immunogenic or vaccine composition is administered at a dose of between about 1046to about 1066cell culture infections dose 50% (CCID50) of attenuated Bovine Respiratory Syncytial Virus (BRSV), preferably about 1056CCID5o and / or a dose of between about 1057to about 107 7cell culture infections dose 50% (CCID50) of attenuated Bovine herpesvirus type 1 (BoHV-1 ), preferablyabout 1 O67CCID5O and / or a dose of between about 75 to 580 hemagglutination units (HAU) of inactivated bovine Parainfluenza 3 (PI-3), preferably 480 HAU PI-3.

43. The immunogenic or vaccine composition for use according to any one of claims 1 to 42, wherein the immunogenic or vaccine composition is administered in a volume of about 2 ml per dose.

44. The immunogenic or vaccine composition for use according to any one of claims 1 to 43, wherein the treatment and / or prevention of Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) and / or Bovine Viral Diarrhoea Virus type 2 (BVDV-2) infection in cattle is conferred for at least six months after administration of at least two doses.

45. The immunogenic or vaccine composition for use according to any one of claims 1 to 44, wherein the immunogenic or vaccine composition allows the differentiation of infected animals from vaccinated animals (DIVA).

46. The immunogenic or vaccine composition for use according to claim 45, wherein the DIVA test is performed by detecting the recombinant E2 protein of a Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain and / or the recombinant E2 protein of a Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain and the p80 protein of the Bovine Viral Diarrhoea Virus type 1 (BVDV-1 ) strain and / or of the Bovine Viral Diarrhoea Virus type 2 (BVDV-2) strain, wherein the detection of the E2 protein and the lack of detection of the p80 protein is indicative of the animal having been vaccinated by the immunogenic or vaccine composition.

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