Measles-mumps-rubella (MMR) vaccines

Abstract

The application generally relates to recombinant genetic constructs comprising a recombinant measles virus expressing at least one rubella virus polypeptide, protein, antigen, or antigenic fragment thereof, and one mumps virus polypeptide, protein, antigen, or antigenic fragment. The application also relates to the uses of genetic constructs or viruses, and more particularly their applications for inducing immune responses and protection against rubella infection, mumps infection, and measles infection.

Description

[0001] TITLE

[0002] Measles-Mumps-Rubella (MMR) vaccines

[0003] FIELD OF THE INVENTION

[0004] The application generally relates to recombinant genetic constructs comprising a recombinant measles virus expressing at least one Rubella virus polypeptide, protein, antigen, or antigenic fragment thereof, and one Mumps virus polypeptide, protein, antigen, or antigenic fragment. The application also relates to the uses of genetic constructs or viruses, and more particularly their applications for inducing immune responses and protection against rubella infection, mumps infection, and measles infection.

[0005] BACKGROUND OF THE INVENTION

[0006] Measles virus (MeV or MV) has been isolated in 1954 (Enders, J. F., and T. C. Peebles. 1954. Propagation in tissue cultures of cytopathogenic agents from patients with measles. Proc. Soc. Exp. Biol. Med.86:277-286). Measles virus is a member of the order mononegavirales, i.e. viruses with a non-segmented negative-strand RNA genome. The non-segmented genome of MeV has an antisense polarity which results in a genomic RNA which is neither translated in vivo or in vitro nor infectious when purified. Transcription and replication of nonsegmented (-) strand RNA viruses and their assembly into virus particles have been studied and reported especially in Fields virology (3rdedition, vol. 1 , 1996, Lippincott - Raven publishers - Fields BN et al.). Transcription and replication of the measles virus do not involve the nucleus of the infected cells but rather take place in the cytoplasm of host cell, just like the Lassa virus. The genome of the MeV comprises genes encoding six major structural proteins designated N, P, M, F, H and L, and an additional two non-structural proteins from the P gene, C and V. The gene order is the following: from the 3’ end of the genomic RNA; N, P (including C and V), M, F, H and L large polymerase at the 5’ end. The genome furthermore comprises non-coding regions in the intergenic region M / F. This non- coding region contains approximatively 100 nucleotides of untranslated RNA. The cited genes respectively encode the proteins of the nucleocapsid of the virus or nucleoprotein (N), the phosphoprotein (P), the large protein (L) which together assemble around the genome RNA to provide the nucleocapsid, the hemagglutinin (H), the fusion protein (F) and the matrix protein (M).

[0007] Attenuated viruses have been derived from MeV virus to provide vaccine strains, such as in particular the Schwarz strain. The Schwarz measles vaccine is a safe and efficient vaccine currently available for preventing measles. Besides providing vaccine, attenuated measles virus strains such as the Schwarz strain have shown to be stable and suitable for the design of efficient delivery vector for immunization against other viruses, like Zika virus or Chikungunya virus. Measles vaccines have been administered to hundreds of millions of children over the last 40 years and have proved its efficiency and safety. It is produced on a large scale in many countries and is distributed at low cost.

[0008] The rubella virus, also known as German measles, belongs to the family Matonaviridae and the genus Rubivirus. It is an enveloped, single-stranded RNA virus with a positive-sense RNA genome of approximately 9,762 nucleotides. The rubella virus genome encodes genes for its replication and pathogenesis. These include the Capsid protein (C), the Envelope glycoproteins (E1 and E2), nonstructural proteins (NSP1 and NSP2) involved in RNA replication, the large protein (L) that encodes the RNA-dependent RNA polymerase necessary for viral genome replication and mRNA transcription. The virus measures approximately 60-70 nanometers in diameter and has an icosahedral capsid. Within the envelope, the rubella virus contains glycoprotein spikes crucial for the virus's ability to infect host cells. The primary glycoproteins are E1 and E2. The E1 glycoprotein serves as the major attachment protein, facilitating the virus's binding to host cell receptors and mediating membrane fusion during entry. The E2 glycoprotein works in conjunction with E1 , also playing a role in receptor binding and membrane fusion. Together, these glycoproteins are vital for the virus's infectivity and its ability to induce an immune response in the host.

[0009] Beneath the envelope lies the capsid, which has icosahedral symmetry. The capsid encases the virus's single-stranded positive-sense RNA genome. The capsid protein (C protein) of the rubella virus encapsulates the RNA genome, facilitating the formation of the nucleocapsid, and plays a critical role in virion assembly and immune evasion. By interacting with host cell components and influencing immune responses, the C protein contributes to the virus's pathogenesis and ability to cause disease. The C protein is not only a structural protein but also has regulatory functions that can affect the virus's ability to replicate and spread within the host. Furthermore, the C protein has specific RNA-binding domains that facilitate the encapsulation of the viral RNA genome. Rubella virus causes rubella. It is characterized by a mild rash and fever, often lasting about three days. Rubella is highly contagious and primarily spreads through respiratory droplets when an infected person coughs or sneezes. The main challenge in rubella treatment lies in the absence of specific antiviral drugs, emphasizing the importance of prevention through vaccination.

[0010] The mumps virus is an enveloped virus that belongs to the family Paramyxoviridae and the genus Orthorubulavirus. It exhibits a pleomorphic shape, meaning it can appear either spherical or filamentous, with a typical diameter ranging from 100 to 600 nanometers. The envelope of the mumps virus is derived from the host cell membrane and contains embedded two main types of viral glycoproteins: Hemagglutinin-Neuraminidase (HN) Glycoprotein (or HN protein) and Fusion (F) Glycoprotein. HN protein facilitates attachment of the virus to sialic acid-containing receptors on the surface of host cells. It also possesses neuraminidase activity, which helps in the release of new virions from the host cell surface. NH protein extends outward from the viral envelope, forming spike-like projections. F protein mediates fusion of the viral envelope with the host cell membrane, allowing entry of the viral genome into the host cell. F protein exists as a trimer and undergoes conformational changes to facilitate membrane fusion.

[0011] Beneath the envelope lies the matrix (M) protein that links the envelope to the nucleocapsid, organizing the assembly and budding of new virions from the host cell. The core of the mumps virus contains the nucleocapsid, which is composed of the viral RNA genome encapsulated by nucleoprotein (N protein). The nucleocapsid has a helical symmetry and serves to protect the RNA genome. The genomic RNA of mumps if a single-stranded negative-sense RNA with a length of approximately 15,384 nucleotides. The genes encode for six main structural and non-structural proteins: Nucleoprotein (N), Phosphoprotein (P), Matrix protein (M), Fusion protein (F), Hemagglutinin-neuraminidase (HN), and Large polymerase protein (L).

[0012] Mumps virus causes mumps. It primarily affects the salivary glands, leading to painful swelling of the parotid glands. It is highly contagious and spreads through respiratory droplets from coughing, sneezing, or direct contact with infected saliva. There is no specific antiviral treatment for mumps. Management focuses on alleviating symptoms to reduce fever and pain, highlighting the importance of prevention through vaccination.

[0013] The Measles, Mumps, and Rubella (MMR) vaccine represents a cornerstone in global public health efforts, designed to protect against the three contagious viral diseases. The significance of the MMR vaccine cannot be overstated, given its role in preventing outbreaks, severe health complications, and fatalities associated with these diseases. The World Health Organization (WHO) has placed these vaccines in its high priority list for pre-qualification and set a goal to eradicate measles and rubella by 2030 (https: / / www.who. int / publications / i / item / measles-and-rubella-elimination-by- 2023), highlighting the important need for widespread vaccine coverage.

[0014] However, vaccination rates for measles and rubella have stagnated over the past several years, and despite enormous progress, the regional measles and rubella elimination targets for 2020 were not met, causing growing concern. Measles remains a major cause of morbidity and mortality worldwide, with an estimated 9.7 million annual cases and more than 140,000 measles-related deaths (https: / / www.who. int / publications / i / item / measles-and-rubella-strategic- framework-2021 -2030). The COVID-19 pandemic led to an interruption of routine vaccination services in many countries and cancellation or postponement of planned immunization programs. Measles is one of the most contagious diseases and requires maintaining high and equitable population immunity. As well, rubella has long-term health consequences for the estimated 103,000 infants bom with congenital rubella syndrome (CRS) (Vynnycky et al. 2016).

[0015] In this context, achieving WHO target faces numerous challenges, including disparities in vaccine coverage across different continents, temporary shortages in vaccine supply, and the complex and costly production processes involved. Globally, MMR vaccine coverage varies significantly, with developed countries achieving higher rates of immunization compared to developing countries. Factors contributing to this discrepancy include logistical challenges, resource limitations, and varying degrees of public health infrastructure. Additionally, the production of the MMR vaccine, primarily concentrated in a few regions, struggles to meet the global demand, exacerbating temporary shortages, particularly in African and Asian countries.

[0016] The current vaccines used are either monovalent measles vaccine or bi- and trivalent vaccines containing a combination of live attenuated measles, mumps, and rubella viruses (MR or MMR). The production complexities, requiring separate bioreactors and cell lines for each of the three viruses, coupled with the verification process for each virus and the final mixed formulation, contribute to the high costs and logistical hurdles associated with MMR manufacture.

[0017] Addressing these challenges requires innovative approaches to vaccine production that can reduce costs and enhance accessibility. Our laboratory has been deploying the measles virus Schwarz strain, renowned for its efficacy and safety profile, as a replicating vector able to express additional antigens (Combredet et al. 2003). The Schwarz vector platform was further used to create several recombinant vaccine candidates against several infectious diseases (Frantz, Teeravechyan, and Tangy 2018). Preclinical immunogenicity and protection from lethal challenges have been demonstrated in mice and / or NHPs for WNV (Brandler et al. 2012; Despres et al. 2005), CHIKV (Brandler et al. 2013), DENV (Brandler et al. 2007), HIV-1 (Lorin et al. 2004; Guerbois et al. 2009; Stebbings et al. 2013; Nzounza et al. 2021 ), SARS-CoV (Escriou et al. 2014), SARS-CoV2 (Frantz et al. 2021 ), and LASV (Mateo et al. 2019; Mateo et al. 2021 ; Mateo et al. 2023). The clinical safety and immunogenicity of several of these recombinant vaccines have been demonstrated in adults in the presence of preexisting measles immunity in phase I and II trials (Ramsauer et al. 2015; Reisinger et al. 2019; Tschismarov et al. 2023).

[0018] In the present disclosure, it is described the successful rescue of a single measles vector expressing antigens from both rubella and mumps viruses. This new vaccine virus candidate elicited immune responses comparable to the commercial MMR vaccine, Priorix®. This recombinant virus showcases the potential for a simplified, cost-effective single-virus MMR production process that could enable broader vaccine coverage, particularly in regions currently facing supply shortages. Furthermore, the promising results from our study lay the groundwork for advancing this recombinant virus into phase l / ll clinical trials, potentially transforming the landscape of MMR production and distribution on a global scale.

[0019] The MMR vaccine, comprising three live-attenuated viruses, has been proven safe and effective, protecting billions of children worldwide against measles, mumps, and rubella. Despite high vaccine coverage in developed countries, inadequate coverage in Africa and intermittent vaccine shortages in Asia hinder the WHO's goal of eradicating these diseases by 2030. A significant production challenge is that each vaccine strain is proprietary to major pharmaceutical companies. Moreover, the Jeryl Lynn mumps vaccine strain has become less effective against current field strains, leading to outbreaks among vaccinated populations (Barskey, Glasser, and LeBaron 2009; Westphal et al. 2019). The rubella strain Wistar 27 / 3 and measles Schwarz strain also present proprietary limitations.

[0020] The production of the MMR vaccine requires three separate bioreactors and qualified cell lines for each virus, significantly increasing GMP manufacturing costs and complicating batch control and cell line management. Therefore, developing a single-virus vaccine capable of protecting against at least two viruses selected from measles, mumps, and rubella, preferably the three viruses measles, mumps, and rubella, would offer a promising solution to reduce costs, facilitate vaccine production in low-income countries, and help improving global vaccine accessibility.

[0021] SUMMARY OF THE INVENTION

[0022] To address, at least partially, the drawbacks of the state of the art, the inventors achieved the production of active components (or ingredients) for vaccines based on recombinant genetic constructs, and especially based on recombinant nucleic acid constructs comprising, within an infectious replicative measles virus, cloned polynucleotide(s) encoding rubella or mumps or rubella and mumps virus polypeptides, proteins or antigens, or antigenic fragments thereof. Vaccines may be recovered when the recombinant measles virus replicates in the host after administration. The invention thus relates to a MV-Rubella vaccine, MV-Mumps vaccine and MV-Rubella-Mumps vaccine, especially a pediatric vaccine, and relates to active ingredient based on an attenuated measles virus strain such as a known vaccine strain commercially available, especially the widely used Schwarz measles vaccine. For all these reasons, the inventors used attenuated measles viruses to generate recombinant measles virus particles stably expressing structural antigens of rubella and / or mumps, in particular immunogenic particles thereof and / or virus-like particles (VLPs). The measles approach of the invention meets all the relevant criteria of a future rubella vaccine. One aim of the invention is to provide a genetic construct, in particular recombinant genetic constructs, in particular nucleic acid constructs, for recovering infectious virus from the nucleic acid construct, and in particular a measles virus allowing the expression of rubella particles or mumps or rubella and mumps particles, in particular Virus-like particles, and preferably Measles, Rubella and Mumps particles, in particular Virus-like particles.

[0023] The present invention provides a recombinant measles virus-based vaccine platform for delivering antigens of rubella and mumps, offering significant advantages over existing approaches, including both the commercial Priorix® vaccine and measles-based constructs disclosed in the prior art. In particular, the vaccine candidate of the invention elicits a strong immune response at markedly reduced doses. While measles-based constructs described in the prior art require higher doses to generate sufficient immunogenicity, the present construct achieves comparable or improved antibody responses at a ten- to thousand-fold lower dose (103— 104TCID50), demonstrating unexpectedly low dose efficiency.

[0024] Moreover, the prior art employs a measles-based vector encoding only rubella antigens and requires a dose of 106TCID50 to achieve a measurable antibody response at a serum dilution of 104. In contrast, the vaccine of the invention produces a comparable or stronger response at a higher serum dilution (105) and with a significantly lower dose (103TCID50). This superior performance is unexpected, particularly since high immunogenicity is generally correlated with higher doses in the field.

[0025] Sequence analysis further revealed that the antigen sequences used in the prior art contain A / T-rich stretches resembling known RNA editing motifs within the measles virus genome. These motifs are absent in the antigen sequences employed in the present invention. Without being limited to any particular theory, it is believed that the avoidance of such motifs contributes to improved antigen expression and genetic stability which in turn enhances immunogenicity. Western blot data confirm that the prior art construct leads to weak or undetectable antigen expression, whereas the invention enables robust expression levels.

[0026] Taken together, these results indicate that the present vaccine platform not only avoids the limitations associated with reduced expression in the prior art, but also achieves improved immunogenicity at unexpectedly low doses.

[0027] Further, while Priorix® and the vaccine of the invention may be administered at comparable doses in preclinical settings, the underlying vaccine technologies are fundamentally different. Priorix® is a trivalent live attenuated virus vaccine, comprising attenuated strains of measles, mumps, and rubella viruses. In contrast, the present invention utilizes a recombinant measles virus platform engineered to express selected antigens from rubella and / or mumps via inserted polynucleotides. This structural difference provides important functional and technical distinctions:

[0028] - The recombinant platform enables selective insertion and expression of antigens of interest. This modularity facilitates streamlined production, reduced reliance on strain attenuation processes, and potentially lower risk of reversion to pathogenic forms, which remains a concern with live attenuated vaccines like Priorix®;

[0029] - Antigen expression is tied to replication and behavior of the full viral genome of each attenuated strain. In contrast, the present invention allows antigen expression to be regulated and optimized independently by modifying the inserted polynucleotide sequences. This can lead to improved consistency in antigen production and tailored immune responses, - Live attenuated vaccines such as Priorix® are known to carry certain safety risks, particularly in immunocompromised individuals. Reported adverse events include mild viral shedding, febrile seizures, and very rare cases of vaccine-induced complications (e.g., mumps orchitis or rubellalike rash). In contrast, the recombinant measles platform reduces or eliminates replication of full rubella and mumps viral genomes, potentially improving the safety profile,

[0030] - Co-administration of three live attenuated viruses in Priorix® can result in viral interference, where one component affects the replication or immune response to the others. The recombinant measles-based approach allows co-expression of antigens in a single vector, potentially minimizing such interference and improving immunogenic balance across the targeted pathogens,

[0031] - The invention also benefits from deliberate antigen design, avoiding A / T- rich sequences that are known to trigger RNA editing in the measles genome. This contributes to more consistent expression and antigen integrity — parameters that cannot be as easily controlled in traditional attenuated virus vaccines.

[0032] Accordingly, the present invention provides a nucleic acid construct which comprises:

[0033] (1 ) a cDNA molecule encoding a full length antigenomic (+) RNA strand of a measles virus (MeV);

[0034] (2) a first heterologous polynucleotide or a plurality of first heterologous polynucleotides encoding at least the following rubella virus structural proteins: Capsid (C), Envelope 2 (E2) and Envelope (E1 ), or antigenic fragments thereof; and wherein the first heterologous polynucleotide or the plurality of first heterologous polynucleotides is (are) operatively cloned within an additional transcription unit (ATU) inserted within the cDNA of the antigenomic (+) RNA, in particular an ATU localized between the P gene and the M gene of the MeV,

[0035] (3) a second heterologous polynucleotide encoding at least the mumps virus hemagglutinin-neuraminidase protein (HN), or antigenic fragments thereof, the second heterologous polynucleotide being operatively cloned within an ATU at a location distinct from the location of the first heterologous polynucleotide or plurality of first heterologous polynucleotides, in particular an ATU localized between the H gene and the L gene of the MeV, in particular the ATLI3 inserted between the H gene and the L gene of the MeV.

[0036] The selected rubella antigens (C-E2-E1 proteins) are known to form virus-like particles (VLPs) (Hobman et al. 1994; Qiu et al. 1994; Garbutt, Chan, and Hobman 1999; Garbutt et al. 1999), similarly to the highly immunogenic rMV- CHIK, which was shown to be unaffected by pre-existing measles immunity. Verification of these VLPs' secretion was achieved by ultracentrifugation of the media from infected cultures, though electron microscopy is needed for definitive confirmation.

[0037] The immunogenicity of rMV-Rub1 E (recombinant Measle Virus - Rubella virus strain 1 E) or Rub2B (Rubella virus strain 2B) given at one log higher dose demonstrated a significant enhancement, with IgG titers more than one log higher than those elicited by the commercial MMR vaccine, Priorix®. Similarly, rMV- mumps-HN (recombinant Measle Virus - Mumps virus HN protein) induced significantly higher levels of mumps neutralizing antibodies than Priorix®. Notably, the mumps virus HN antigen was more effectively expressed in ATLI3, likely due to genomic similarities between mumps and measles viruses, providing an optimal expression site for the HN gene in the measles vector.

[0038] The nucleic acid constructs of the invention, and the products issued from the expression of the antigens coded herein contribute significantly to the successful development of a rMV-Rubella virus, a rMV-Mumps virus and a rMV-Rubella- Mumps virus as a single-virus vaccine candidate against multiple infections.

[0039] In comparative immunization studies with IFNAR - / - mice with given at the similar dose, the single-virus MMR vaccine elicited higher specific IgG to Rubella and comparable levels of Mumps neutralizing antibodies to those induced by Priorix®. These preliminary results, mirroring the immunogenicity profile of the commercial MMR vaccine, underscore the potential of our single-virus MMR vaccine for further clinical trials. Given the importance of neutralizing antibodies as a correlate of protection, our vaccine shows promising potential for advancing toward clinical evaluation.

[0040] The development of a single-virus MMR vaccine not only promises to reduce production costs significantly but also aims to enhance vaccine coverage in regions currently facing insufficient vaccine distribution, such as countries in Africa. Additionally, with the majority of MMR vaccines in Asia derived from the less efficacious Zagreb measles strain, the vaccine, based on the Schwarz strain, stands to improve efficacy and safety profiles. This initiative aligns with global health objectives, potentially accelerating the achievement of measles and rubella eradication by 2030, as targeted by the WHO.

[0041] DETAILED DESCRIPTION OF THE INVENTION

[0042] In a first aspect of the invention, it is provided a nucleic acid construct which comprises:

[0043] (1 ) a cDNA molecule encoding a full length antigenomic (+) RNA strand of a measles virus (MeV);

[0044] (1 ) a first heterologous polynucleotide or a plurality of first heterologous polynucleotides encoding at least the following rubella virus structural proteins: Capsid (C), Envelope 2 (E2) and Envelope 1 (E1 ), or antigenic fragments thereof; and wherein the first heterologous polynucleotide or the plurality of first heterologous polynucleotides is (are) operatively cloned within an additional transcription unit (ATU) inserted within the cDNA of the antigenomic (+) RNA, in particular an ATU localized between the P gene and the M gene of the MeV; and

[0045] (2) a second heterologous polynucleotide encoding at least the mumps virus hemagglutinin-neuraminidase protein (HN), or antigenic fragment thereof, the second heterologous polynucleotide being operatively cloned within an ATU at a location distinct from the location of the first heterologous polynucleotide or plurality of first heterologous polynucleotides, in particular an ATU localized between the H gene and the L gene of the MeV.

[0046] According to another aspect of the invention, it is provided a nucleic acid construct which comprises:

[0047] (1 ) a cDNA molecule encoding a full length antigenomic (+) RNA strand of a measles virus (MeV); (2) a first heterologous polynucleotide or a plurality of first heterologous polynucleotides encoding at least the following rubella virus structural proteins: Capsid (C), Envelope 2 (E2) and Envelope (E1 ), or antigenic fragments thereof; and wherein the first heterologous polynucleotide or the plurality of first heterologous polynucleotides is (are) operatively cloned within an additional transcription unit (ATU) inserted within the cDNA of the antigenomic (+) RNA, in particular an ATU localized between the P gene and the M gene of the MeV;

[0048] (3) at least one second heterologous polynucleotide encoding at least the mumps virus hemagglutinin-neuraminidase protein (HN), or antigenic fragments thereof, the at least one second heterologous polynucleotide being operatively cloned within an ATU at a location distinct from the location of the first heterologous polynucleotide or plurality of first heterologous polynucleotides, in particular an ATU localized between the H gene and the L gene of the MeV, in particular the ATU3 inserted between the H gene and the L gene of the MeV.

[0049] Particular nucleic acid constructs according to this aspect are illustrated in Fig.1, Fig.9a, Fig.11-12 and in Fig.15-16 and in the examples of the present invention.

[0050] In a particular aspect of the invention, it is provided a nucleic acid construct which comprises:

[0051] (1 ) a cDNA molecule encoding a full length antigenomic (+) RNA strand of a measles virus (MeV);

[0052] (2) a first heterologous polynucleotide or a plurality of first heterologous polynucleotides encoding at least the following rubella virus structural proteins: Capsid (C), Envelope 2 (E2) and Envelope (E1 ), or antigenic fragments thereof; and wherein the first heterologous polynucleotide or the plurality of first heterologous polynucleotides is (are) operatively cloned within an additional transcription unit (ATU) inserted within the cDNA of the antigenomic (+) RNA, in particular an ATU localized between the P gene and the M gene of the MeV.

[0053] In another aspect of the invention, it is provided a nucleic acid construct which comprises:

[0054] (1 ) a cDNA molecule encoding a full length antigenomic (+) RNA strand of a measles virus (MeV); (2’) a (second) heterologous polynucleotide encoding at least the mumps virus hemagglutinin-neuraminidase protein (HN), or antigenic fragment(s) thereof, the (second) heterologous polynucleotide being operatively cloned within an additional transcription unit (ATU) inserted within the cDNA of the antigenomic (+) RNA, in particular an ATU localized between the P gene and the M gene of the MeV, and / or in particular an ATU localized between the H gene and the L gene of the MeV, in particular the ATU3 inserted between the H gene and the L gene of the MeV.

[0055] More preferably, the nucleic acid construct comprises a plurality of (second) heterologous polynucleotides encoding at least the mumps virus hemagglutininneuraminidase protein (HN), or antigenic fragment(s) thereof, at least one (second) heterologous polynucleotides being operatively cloned within an additional transcription unit (ATU) inserted within the cDNA of the antigenomic (+) RNA, in particular an ATU localized between the P gene and the M gene of the MeV, and another (second) heterologous polynucleotides being operatively cloned within an ATU localized between the H gene and the L gene of the MeV, in particular the ATU3 inserted between the H gene and the L gene of the MeV. Particular nucleic acid constructs according to these aspects are illustrated in Fig.6, Fig.9, Fig.13-16 and in the examples of the present invention.

[0056] The expression “encodes” in the above definition encompasses the ability of the cDNA to allow transcription of a full length antigenomic (+) RNA, said cDNA serving especially as a template for transcription and where appropriate translation for product expression into cells or cell lines. Hence, when the cDNA is a double stranded molecule, one of the strands has the same nucleotide sequence as the antigenomic (+) RNA of the measles virus with the first heterologous polynucleotide cloned within, except “U” nucleotides that are substituted by “T” nucleotides in the cDNA. The nucleic acid construct of the invention may comprise regulatory elements controlling the transcription of the coding sequences, in particular promoters and termination sequences for the transcription, and possibly enhancer and other cis-acting elements. These regulatory elements may be heterologous with respect to the first heterologous polynucleotide(s) encoding the rubella proteins and / or mumps proteins, in particular may be the regulatory elements of the measles virus strain.

[0057] The expression “operatively cloned”, which can be substituted by the expression “operatively linked”, refers to the functional cloning, or insertion, of a heterologous polynucleotide within the nucleic acid construct of the invention such that said polynucleotide and nucleic acid construct are effectively, or efficiently, transcribed and if appropriate translated, in particular in cells, cell line, host cell used as a part of a rescue system for the production of recombinant infectious MeV particles or MeV expressing at least one polypeptide, or at least one protein, or at least one antigen, or at least an antigenic fragment thereof, of the rubella virus and / or mumps that is encoded by the first heterologous polynucleotide or the plurality of first heterologous polynucleotides. In other words, the nucleic acid construct of the invention allows the production, when placed in appropriate conditions, of an infectious antigenomic (+) RNA capable of producing at least one polypeptide, or at least one protein, or at least one antigen, or at least an antigenic fragment thereof, of rubella virus and / or mumps virus.

[0058] In a particular embodiment of the invention, the nucleic acid construct comprising the cDNA encoding the nucleotides of the full-length infectious antigenomic (+) RNA strand of MeV but without the operatively cloned heterologous polynucleotide complies with the rule of six (6) of the measles virus genome. In other words, the cDNA encoding the nucleotides of the full-length, infectious antigenomic (+) RNA strand of MeV is a polyhexameric cDNA.

[0059] The organization of the genome of measles viruses and their replication and transcription process have been fully identified in the prior art and are especially disclosed in Horikami S.M. and Moyer S. A. (Curr. Top. Microbiol. Immunol. (1995) 191 , 35-50) or in Combredet C. et al (Journal of Virology, Nov 2003, p11546- 11554) for the Schwarz vaccination strain of the virus or for broadly considered negative-sense RNA viruses, in Neumann G. et al (Journal of General Virology (2002) 83, 2635-2662).

[0060] The “rule of six” is expressed in the fact that the total number of nucleotides present in a nucleic acid representing the MeV (+) strand RNA genome or in the nucleic acid constructs comprising the same is a multiple of six. The “rule of six” has been acknowledged in the state of the art as a requirement regarding the total number of nucleotides in the genome of the measles virus, which enables efficient or optimized replication of the MeV genomic RNA. In the embodiments of the present invention defining a nucleic acid construct that meets the rule of six, said rule applies to the nucleic acid construct specifying the cDNA encoding the full-length MeV (+) strand RNA genome. In this regard, the rule of six applies individually to the cDNA encoding the nucleotide sequence of the full-length infectious antigenomic (+) RNA strand of the measles virus, possibly but not necessarily to the polynucleotide(s) cloned into said cDNA and encoding at least one polypeptide of rubella or one polypeptide of mumps.

[0061] The nucleic acid construct of the invention is in particular a purified DNA molecule, obtained or obtainable by recombination of at least one polynucleotide of MeV and at least one, or a plurality, polynucleotide(s) of the rubella virus, operably cloned or linked together, and / or at least one, or a plurality, polynucleotide(s) of the mumps virus.

[0062] - The first polynucleotide(s) encoding the rubella protein(s)

[0063] According to the invention, the nucleic acid construct is prepared by cloning a polynucleotide, or a plurality of polynucleotides, encoding at least one polypeptide, or a protein, or an antigen, or an antigenic fragment thereof, selected from the group consisting of the Capsid (C), Envelope 2 (E2) and Envelope 1 (E1 ) of rubella virus in the cDNA encoding the full-length antigenomic (+) RNA of the measles virus. Such a construct is illustrated on Fig.1 , wherein a first polynucleotide encoding the Capsid (C), Envelope 2 (E2) and Envelope 1 (E1 ) protein of rubella virus is inserted within the intergenic region between the P gene and the M gene of the MV. A nucleic acid construct of the invention may be prepared using steps of synthesis of nucleic acid fragments or polymerization from a template, including by PCR. The polynucleotide(s) and nucleic acid construct of the invention may rather be prepared in accordance with any known method in the art and in particular may be cloned, obtained by polymerization especially using PCR methods or may be synthesized.

[0064] The first heterologous polynucleotide may be issued from the fusion of several other polynucleotides, each encoding a particular polypeptide, or a particular protein, antigen or an antigenic fragment thereof, of rubella virus. For example, the first heterologous polynucleotide may be issued from the fusion of polynucleotides each encoding a single protein: the Capsid (C), Envelope 2 (E2) and Envelope 1 (E1 ), these three polynucleotides being linked within the nucleic acid construct by a linker sequence. The linker sequence may comprise a peptide signal. Linker sequences can be a short nucleotide sequence comprising or consisting in a regulatory sequence of the measles virus. Accordingly in a particular embodiment of the invention, the first heterologous polynucleotide or the plurality of first heterologous polynucleotides comprise(s) the E2 peptide signal, in particular upstream of a sequence encoding the E2 protein or the antigenic fragment thereof, and / or the E1 peptide signal, in particular upstream of a sequence encoding the E1 protein or the antigenic fragment thereof. The E2 peptide signal and the E1 peptide signal may be localized within linker sequences. In particular, the E2 peptide signal may be localized in the linker sequence localized between the nucleotide sequences encoding the Capsid protein and the Envelope 2 protein. In particular, the E1 peptide signal may be localized in the linker sequence localized between the nucleotide sequences encoding the Envelope 2 protein and the Envelope 1 protein.

[0065] The first heterologous polynucleotide or the plurality of first polynucleotides may encode three different polypeptides (i.e. the Capsid (C), Envelope 2 (E2) and Envelope 1 (E1 ), or three different polypeptides (i.e. the Capsid (C), Envelope 2 (E2) and Envelope 1 (E1 ) and may encode at least another different polypeptide. In an embodiment of the invention, the first heterologous polynucleotide or the plurality of first polynucleotides may encode three different polypeptides (i.e. the Capsid (C), Envelope 2 (E2) and Envelope 1 (E1 ), the rubella structural proteins C, E2 and E1 being encoded in this order from the 5’ end towards the 3’ end of the first heterologous polynucleotide or from the 5’ end towards the 3’ end of the plurality of first heterologous polynucleotides put end to end (e.g., 5’ C - E2 - E1 3’). Alternatively, the rubella structural proteins C, E2 and E1 being encoded in a different order from the 5’ end towards the 3’ end of the first heterologous polynucleotide or from the 5’ end towards the 3’ end of the plurality of first heterologous polynucleotides put end to end (e.g., 5’ E2 - E1 - C 3’ ; 5’ E1 - E2 - C 3’ , 5’ E2 - C - E1 3’ ; 5’ E1 - C - E2 3’ ; 5’ C - E1 - E2 3’). The first heterologous polynucleotide or the plurality of first heterologous polynucleotides may correspond to the genes of rubella virus encoding the Capsid (C), Envelope 2 (E2) and Envelope (E1 ) proteins.

[0066] In a particular embodiment of the invention, the first heterologous polynucleotide or the plurality of first heterologous polynucleotides is(are) originate or are derived from the rubella strain 1 E or 2B or Wistar RA 27 / 3. Information about rubella strains can be found in Brooke A. et al. (Phylogeography of rubella virus in Asia: Vaccination and demography shape synchronous outbreaks, Epidemics, Volume 28, 2019, 100346, ISSN 1755-4365, https: / / doi.Org / 10.1016 / j. epidem.2019.100346). The first heterologous polynucleotide or the plurality of first heterologous polynucleotides may correspond to the genes encoding the Capsid (C), Envelope 2 (E2) and Envelope (E1 ) proteins found in rubella strain 1 E or 2B or Wistar RA 27 / 3.

[0067] - The second polynucleotide(s) encoding the mumps protein(s)

[0068] When a mumps protein is encoded within the nucleic acid construct of the invention, the nucleic acid construct is prepared by cloning a polynucleotide, or a plurality of polynucleotides, encoding at least the hemagglutinin-neuraminidase protein (HN) of mumps virus in the cDNA encoding the full-length antigenomic (+) RNA of the measles virus. Such a construct is illustrated on Fig.6 and Fig.13-14, wherein one second polynucleotide encoding the HN protein of mumps virus is inserted either within the intergenic region between the P gene and the M gene of the MeV or within the intergenic region between the H gene and the L gene of the MV. In an embodiment, two second polynucleotides encoding the HN protein of mumps virus are inserted: one within the intergenic region between the P gene and the M gene of the MeV and one within the intergenic region between the H gene and the L gene of the MV.

[0069] In another embodiment illustrated on Fig.9 and Fig.15-16, a first polynucleotide encoding the Capsid (C), Envelope 2 (E2) and Envelope 1 (E1 ) protein of rubella virus is inserted within the intergenic region between the P gene and the M gene of the MeV and a second polynucleotide encoding the HN protein of mumps virus is inserted within the intergenic region between the H gene and the L gene of the MV. A nucleic acid construct comprising at least one second heterologous polynucleotide may be prepared according to the details given for the preparation of a nucleic acid construct comprising at least a first heterologous polynucleotide. The at least one second heterologous polynucleotide may encode one or several HN protein(s) of mumps virus and may encode at least another different polypeptide.

[0070] The second heterologous polynucleotide may correspond to the gene of mumps virus encoding the hemagglutinin-neuraminidase protein (HN).

[0071] - The ATUs

[0072] The first heterologous polynucleotide or the plurality of first heterologous polynucleotides gene(s) and / or the at least one second heterologous polynucleotide is / are cloned within an additional transcription unit (ATU) inserted in the cDNA of the MV. It has been observed that the transcription of the viral RNA of MeV follows a gradient from the 5’ to the 3’ end. This explains that, depending on where the first heterologous polynucleotide(s) is / are inserted, its / their level of expression will vary and be more or less efficient if inserted within ATU1 (i.e. the ATU localized upstream the P gene of the measles), ATU2 (i.e. the ATU localized between the P gene and the M gene of the measles) or ATU3 (i.e. the ATU localized between the H gene and the L gene of the measles). ATU sequences comprise, for use in steps of cloning into cDNA of MV, cis-acting sequences necessary for MV-dependent expression of a transgene, such as a promoter of the gene preceding, in MeV cDNA, the insert represented by the first polynucleotide or the plurality of first polynucleotide encoding the rubella polypeptides inserted into a multiple cloning sites cassette. The ATU may be further defined as disclosed by Billeter et al. in WO 97 / 06270 or by Tangy et al. in WO 2022 / 008687. One ATU is represented on Fig.1. An ATU may also be defined as multiple cloning cassette inserted within the cDNA of the MV, in particular between the N-P intergenic region of the MeV genome, and / or between the intergenic P-M region of the MV, and / or between the intergenic H-L region of the MeV genome. An ATU may contain cis-acting sequences necessary for the transcription of the P gene of MeV. The different ATUs may be identical regarding their nucleic acid sequence. ATUs are generally localized between two CTT codons corresponding respectively to the start and stop codons of the polymerase. ATUs may further comprise a ATG and a TAG codon corresponding respectively to the start and stop codons for translation of the heterologous polynucleotide cloned within the ATU. Alternatively, ATUs are localized between a ATG and a TAG codon corresponding respectively to the start and stop codons for translation of the heterologous polynucleotide cloned within the ATU. ATUs may further comprise a ATG and a TAG codon corresponding respectively to the start and stop codons for translation of the heterologous polynucleotide cloned within the ATU.

[0073] In an embodiment of the invention, an ATU, in particular ATU1 , ATU2 and ATU3 as defined herein, has the nucleotide sequence set forth in SEQ ID No. 21 and SEQ ID No. 22. SEQ ID No. 21 corresponds to the nucleotides localized upstream the 5’ end of the inserted heterologous polynucleotide, while SEQ ID No. 22 corresponds to the nucleotides localized downstream the 3‘ end of the inserted heterologous polynucleotide (i.e. 5’ SEQ ID No. 21 - inserted heterologous polynucleotide encoding the protein(s) from rubella or mumps - SEQ ID No. 22 3’).

[0074] In an embodiment of the invention, an ATU comprises from its 5’ end to its 3’ end: a promoter sequence originating from intergenic regions of measles virus genome, a heterologous polynucleotide encoding at least one heterologous polypeptide as defined herein, wherein the heterologous polynucleotide is in particular inserted in a multiple cloning site, especially a multiple cloning site containing restriction sites not found in the MeV cDNA; a terminator sequence, wherein the promoter and terminator sequences are inserted respectively upstream and downstream of additional heterologous sequences in a way to allow their expression by the measles RNA-dependent RNA polymerase and wherein the promoter and terminator sequences can be selected from the functional sequences in any intergenic sequence of measles virus genome: N-P, P-M, M-F, F-H, H-L.

[0075] In an embodiment, the first heterologous polynucleotide or the plurality of first heterologous polynucleotides is (are) operatively cloned within the ATU localized between the P gene and the M gene of the MeV. In an embodiment, the first heterologous polynucleotide or the plurality of first heterologous polynucleotides is (are) operatively cloned within the ATLI2 inserted between the P gene and the M gene of the MeV.

[0076] In an embodiment, the at least one second heterologous polynucleotide is (are) operatively cloned within the ATU localized between the P gene and the M gene of the MeV.

[0077] In an embodiment, the at least one second heterologous polynucleotide is (are) operatively cloned within the ATU localized between the H gene and the L gene of the MeV.

[0078] In an embodiment, at least two second heterologous polynucleotides are present within the nucleic acid construct, one being operatively cloned within the ATU localized between the H gene and the L gene of the MeV, and another being operatively cloned within the ATU localized between the P gene and the M gene of the MeV.

[0079] In an embodiment, the first heterologous polynucleotide or the plurality of first heterologous polynucleotides is (are) operatively cloned within the ATU localized between the P gene and the M gene of the MeV and the at least one second heterologous polynucleotide is (are) operatively cloned within the ATU localized between the H gene and the L gene of the MeV.

[0080] In an embodiment, the first heterologous polynucleotide or the plurality of first heterologous polynucleotides is (are) operatively cloned within the ATU2 localized between the P gene and the M gene of the MeV and the at least one second heterologous polynucleotide is (are) operatively cloned within the ATU3 localized between the H gene and the L gene of the MeV.

[0081] When considering the embodiments wherein a second polynucleotide encoding a protein from mumps virus is present within the nucleic construct of the invention, the second polynucleotide is present within another ATU. In particular, another ATU known under reference ATU3 is advantageously located in the intergenic region between the H and L genes of MeV, while the other ATU (ATU2) is preferentially located between the P and M genes of the virus. Since the transcription of the viral RNA of MeV follows a gradient from the 5’ to the 3’ end, the inventors found that cloning two polynucleotides at different locations within the cDNA encoding the full-length antigenomic (+) RNA of the measles virus may lead to the production of higher yield of antigenic particles of rubella virus like particles (VLPs), while this production may be less important when the polynucleotides are all cloned within a single and same location. Furthermore, cloning the heterologous polynucleotides at different locations may reduce the attenuation of the expression of the encoded polypeptides. Indeed, when several genes are cloned within a single ATU, it may lead to reduction of the expression of the encoded polypeptides.

[0082] - The antigenic proteins and their coding sequences

[0083] The term “proteins” is used interchangeably with the terms “antigen” or “polypeptide” or “antigenic fragment” and defines a molecule resulting from a concatenation of amino acid residues. In particular, the polypeptides disclosed in the application originate from the rubella virus and / or mumps virus and are antigens, proteins, structural proteins, or antigenic fragments thereof, that may be identical to native proteins or alternatively that may be derived thereof by mutation, including by substitution (in particular by conservative amino acid residues) or by addition of amino acid residues or by secondary modification after translation or by deletion of portions of the native proteins(s) resulting in fragments having a shortened size with respect to the native protein of reference. Fragments are encompassed within the present invention to the extent that they bear epitopes of the native protein suitable for the elicitation of an immune response in a host, in particular in a human host, including a child host, preferably a response that enables the protection against a rubella virus infection and / or mumps virus infection or against a rubella virus and / or mumps virus associated disease. Epitopes are in particular of the type of T epitopes involved in elicitation of Cell Mediated Immune response (CMI response). T epitopes are involved in the stimulation of T cells through presentation of some parts of the T-cell epitope which can bind on MHC class II molecules, leading to the activation of T cells. Epitopes may alternatively be of type B, involved in the activation of the production of antibodies in a host to whom the protein has been administered or in whom it is expressed following administration of the infectious replicative particles of the invention. Fragments may have a size representing more than 50% of the amino-acid sequence size of the native proteins C, E2 and E1 , in particular the native proteins that originate or that are derived from rubella virus strain 1 E or 2B or Wistar RA 27 / 3, preferably at least 90%, more preferably at least 95%. Polypeptide may have at least 50% identity with the native protein of rubella virus strain 1 E or 2B or Wistar RA 27 / 3, preferably at least 60%, preferably at least 70%, preferably at least 85%, preferably at least 90%, and more preferably at least 95%. Fragments may have a size representing more than 50% of the amino-acid sequence size of the native protein HN, in particular the native protein HN that originates or that is derived from mumps virus strain of genotype G or C, or from a mosaic protein that originates or that is derived from mumps virus strains of genotype G and C preferably at least 90%, more preferably at least 95%. Polypeptide may have at least 50% identity with the native protein of mump strain of genotype G or C, preferably at least 60%, preferably at least 70%, preferably at least 85%, preferably at least 90%, and more preferably at least 95%.

[0084] In a particular embodiment of the invention, each polynucleotide operatively cloned within the cDNA of the antigenomic (+) RNA encodes polypeptides comprising epitopes localized within any one of the rubella protein(s) and / or mumps protein. According to this embodiment, the epitope sequence(s) share(s) 100% identity with the epitope sequence(s) of the native rubella proteins and / or mumps protein. Such epitopes are listed in the Immune Epitope database and analysis resource (www.iedb.org). Within the polypeptide(s) of the rubella encoded by the polynucleotide and having an epitope sequence(s) as defined herein, amino acid residue that does not belong to any epitope may be different from the sequence of the native rubella protein(s). Within the HN polypeptide of the mumps virus encoded by the polynucleotide and having an epitope sequence(s) as defined herein, amino acid residue that does not belong to any epitope may be different from the sequence of the native mumps HN protein.

[0085] By “protein of rubella” or “protein of mumps” is meant a “polypeptide” as defined herein (either a polypeptide, an antigen, a protein, or an antigenic fragment thereof), the amino acid sequence of which is identical to a counterpart in a strain of rubella, especially rubella strain 1 E or 2B or Wistar RA 27 / 3, or a strain of mumps, including a polypeptide which is a native mature or precursor of protein of rubella or mumps or is an antigenic fragment thereof or a mutant thereof as defined herein in particular an antigenic fragment or a mutant having at least 50%, at least 80%, in particular advantageously at least 90% or preferably at least 95% amino acid sequence identity to a naturally occurring rubella C protein, E2 protein or E1 protein or mumps HN protein. Amino acid sequence identity can be determined by alignment by one of skill in the art using manual alignments or using the numerous alignment programs available (for example, BLASTP - http: / / blast.ncbi.nlm.nih.gov / ). Fragments or mutants of rubella polypeptides of the invention may be defined with respect to the particular amino acid sequences illustrated herein, especially the amino acid sequences from the group consisting of SEQ ID No. 3, SEQ ID No. 23 (which correspond to Capsid C protein of rubella strain 1 E and 2B, respectively), SEQ ID No. 5, SEQ ID No. 25 (which correspond to Envelope E2 protein of rubella strain 1 E and 2B, respectively), SEQ ID No. 7, SEQ ID No. 27 (which correspond to Envelope E1 protein of rubella strain 1 E and 2B, respectively), SEQ ID No. 33 and SEQ ID No. 35 (which correspond to polyprotein encompassing Capsid C protein, Envelope E2 protein and Envelope E1 protein of rubella strain 1 E and 2B, respectively). In a particular embodiment of the invention, the polypeptides share at least 50%, at least 80%, in particular advantageously at least 90% or preferably at least 95% amino acid sequence identity with their native proteins of the rubella strain 1 E or 2B, or with the polypeptides of SEQ ID No. 3, SEQ ID No. 23, SEQ ID No. 5, SEQ ID No. 25, SEQ ID No. 7 or SEQ ID No. 27. Fragments or mutants of mumps polypeptides of the invention may be defined with respect to the particular amino acid sequences illustrated herein, especially the amino acid sequences from the group consisting of SEQ ID No. 13 (which corresponds to HN protein).

[0086] In an embodiment of the invention, the nucleic acid construct of the invention comprises a first heterologous polynucleotide or a plurality of first heterologous polynucleotides that encode(s) the rubella protein C comprising or consisting of the amino acid sequence set forth in SEQ ID No. 3 or SEQ ID No. 23 or an antigenic fragment thereof, and / or the rubella protein E2 comprising or consisting of the amino acid sequence set forth in SEQ ID No. 5 or SEQ ID No 25 or an antigenic fragment thereof, and / or the rubella protein E1 comprising or consisting of the amino acid sequence set forth in SEQ ID No. 7 or SEQ ID No 27 or an antigenic fragment thereof, in particular which further encode(s) the E2 peptide signal comprising or consisting of the amino acid sequence set forth in SEQ ID No. 9 or SEQ ID No.29 (which corresponds to the E2 peptide signal of E2 protein of rubella strain 1 E and 2B, respectively), and / or the E1 peptide signal comprising or consisting of the amino acid sequence set forth in SEQ ID No. 1 1 or SEQ ID No. 31 (which corresponds to the E1 peptide signal of E1 protein of rubella strain 1 E and 2B, respectively).

[0087] In an embodiment of the invention, the nucleic acid construct of the invention comprises a first heterologous polynucleotide or a plurality of first heterologous polynucleotides that encode(s) the rubella protein C consisting of the amino acid sequence set forth in SEQ ID No. 3 or SEQ ID No. 23 or an antigenic fragment thereof, and the rubella protein E2 consisting of the amino acid sequence set forth in SEQ ID No. 5 or SEQ ID No. 25 or an antigenic fragment thereof, and the rubella protein E1 consisting of the amino acid sequence set forth in SEQ ID No. 7 and SEQ ID No. 27 or an antigenic fragment thereof. The first heterologous polynucleotide or a plurality of first heterologous polynucleotides may further comprise the linker sequences comprising the E2 peptide signal consisting of the amino acid sequence set forth in SEQ ID No. 9 or SEQ ID No. 29, and the E1 peptide signal consisting of the amino acid sequence set forth in SEQ ID No. 11 or SEQ ID No. 31.

[0088] According to one aspect of the invention, a polynucleotide encoding at least one protein of rubella originates or is derived from the genome of isolated and purified wild strain(s) of rubella, including any Rubella strain whose genome has been fully or partially sequenced. At least some of these sequences may be found in the NCBI nucleotide database. In particular, the polynucleotide encoding at least one rubella protein may originate of be derived from any Rubella strain sequenced and referenced, for example in GenBank, in particular the Rubella virus strain Wistar RA 27 / 3, 1 E or 2B.

[0089] Preferentially, the polynucleotide originates or is derived from the rubella strain 1 E or 2B or RA 27 / 3 whose genomic sequences may be found under GenBank accession No. KT962864 for strain 1 E or GenBank accession No. MN786647 for strain 2B or GenBank accession number No. FJ211588.1 for strain Wistar RA 27 / 3. According to one aspect of the invention, a polynucleotide encoding at least one protein of mumps originates or is derived from the genome of isolated and purified wild strain(s) of mumps, including any mumps strain whose genome has been fully or partially sequenced. At least some of these sequences may be found in the NCBI nucleotide database. In particular, the polynucleotide encoding at least one mumps protein may originate or be derived from any mumps genotype G sequenced and referenced in GenBank accession No. JX287385 or C strain sequenced and referenced in GenBank accession No. EU370206.

[0090] The first heterologous polynucleotide or the plurality of first heterologous polynucleotides may comprise any one of the following sequences, or a plurality of the following sequences, or at least two of the following sequences, or at least three of the following sequences:

[0091] - SEQ ID No. 4 and / or SEQ ID No. 24 which encodes the rubella C protein, and / or

[0092] - SEQ ID No. 6 and / or SEQ ID No. 26 which encodes the rubella protein E2, and / or

[0093] - SEQ ID No. 8 and / or SEQ ID No. 28 which encodes the rubella protein E1 , or

[0094] - SEQ ID No.34 and / or SEQ ID No.35 which encodes the polyprotein comprising rubella proteins Capsid, E2 and E1.

[0095] Within the first heterologous polynucleotide or the plurality of first heterologous polynucleotides, each sequence defined here above may be present a single time, or a plurality of times. In a preferred embodiment of the invention, each sequence is present a single time within a single heterologous polynucleotide, or is present a single time within the heterologous polynucleotides taken together.

[0096] Alternatively, or complementarily, the first heterologous polynucleotide(s) may encode any one of the following polypeptides, or an antigenic fragment thereof, or at least two of the following polypeptides, or at least three of the following polypeptides:

[0097] - the Capsid C protein of SEQ ID No. 3 and / or SEQ ID No. 23 or an antigenic fragment thereof; and,

[0098] - the Envelope E2 of SEQ ID No. 5 and / or SEQ ID No. 25 or an antigenic fragment thereof; and - the Envelope E1 protein of SEQ ID No. 7 and / or SEQ ID No. 27 or an antigenic fragment thereof, or

[0099] - the Rubella structural polyprotein precursor protein of SEQ ID No. 33 and / or SEQ ID No. 35 or an antigenic fragment thereof.

[0100] The polynucleotide(s) may encode a polypeptide as defined here above a single time or a several times. In a preferred embodiment, each polypeptide is encoded a single time within a single heterologous polynucleotide, and more preferentially, each polypeptide is encoded a single time within the plurality of polypeptides. According to a particular embodiment of the invention, several polynucleotides wherein each polynucleotide encodes at least one rubella protein are combined or fused to form a polynucleotide encoding several proteins of the rubella. These polynucleotides may distinguish from each other by the fact that they code for proteins of various strains of the rubella, or for different proteins of a rubella strain. In an embodiment, the first heterologous polynucleotide(s) may encode at least the following polypeptides:

[0101] - the Capsid C protein of SEQ ID No. 3 or an antigenic fragment thereof; and,

[0102] - the Envelope E2 of SEQ ID No. 5 or an antigenic fragment thereof; and

[0103] - the Envelope E1 protein of SEQ ID No. 7 or an antigenic fragment thereof; or

[0104] - the Rubella structural polyprotein precursor protein of SEQ ID No. 33.

[0105] In an embodiment, the first heterologous polynucleotide(s) may encode at least the following polypeptides:

[0106] - the Capsid C protein of SEQ ID No. 23 or an antigenic fragment thereof; and,

[0107] - the Envelope E2 of SEQ ID No. 25 or an antigenic fragment thereof; and,

[0108] - the Envelope E1 protein of SEQ ID No. 27 or an antigenic fragment thereof; or,

[0109] - the Rubella structural polyprotein precursor protein of SEQ ID No. 35. In a particular aspect of the invention, the first heterologous polynucleotide or the plurality of first heterologous polynucleotides comprise(s) at least the following sequences:

[0110] SEQ ID No. 4 and / or SEQ ID No. 24 which encodes the rubella C protein; and,

[0111] SEQ ID No. 6 and / or SEQ ID No. 26 which encodes the rubella protein E2; and,

[0112] SEQ ID No. 8 and / or SEQ ID No. 28 which encodes the rubella protein E1 . More particularly, the first heterologous polynucleotide or the plurality of first heterologous polynucleotides may further comprise:

[0113] SEQ ID No. 10 and / or SEQ ID No. 30 which encodes the E2 peptide signal; and / or,

[0114] SEQ ID No. 12 and / or SEQ ID No. 32 which encodes the E1 peptide signal.

[0115] In a particular aspect of the invention, the first heterologous polynucleotide or the plurality of first heterologous polynucleotides comprise(s) at least the following sequences:

[0116] SEQ ID No. 4 which encodes the rubella C protein; and,

[0117] SEQ ID No. 6 which encodes the rubella protein E2; and,

[0118] SEQ ID No. 8 which encodes the rubella protein E1 ; or,

[0119] SEQ ID No. 34 which encodes the polyprotein comprising the C protein, the E2 protein, and the E1 protein.

[0120] More particularly, the first heterologous polynucleotide or the plurality of first heterologous polynucleotides may further comprise:

[0121] SEQ ID No. 10 which encodes the E2 peptide signal; and / or,

[0122] SEQ ID No. 12 which encodes the E1 peptide signal.

[0123] In a particular aspect of the invention, the first heterologous polynucleotide or the plurality of first heterologous polynucleotides comprise(s) at least the following sequences:

[0124] SEQ ID No. 24 which encodes the rubella C protein; and,

[0125] SEQ ID No. 26 which encodes the rubella protein E2; and,

[0126] SEQ ID No. 28 which encodes the rubella protein E1 ; or, SEQ ID No. 36 which encodes the polyprotein comprising the C protein, the E2 protein, and the E1 protein.

[0127] More particularly, the first heterologous polynucleotide or the plurality of first heterologous polynucleotides may further comprise:

[0128] SEQ ID No. 30 which encodes the E2 peptide signal; and / or,

[0129] SEQ ID No. 32 which encodes the E1 peptide signal.

[0130] More particularly, the first heterologous polynucleotide or the plurality of first heterologous polynucleotides has(have) codon-optimized open reading frame(s) (ORF).

[0131] The first polynucleotide that comprises a sequence encoding the C protein of rubella may have the sequence set forth in SEQ ID No. 4 and / or SEQ ID No. 24 or may be derived from such a sequence.

[0132] The first polynucleotide that comprises a sequence encoding the E2 protein of rubella may have the sequence set forth in SEQ ID No. 6 and / or SEQ ID No. 26 or may be derived from such a sequence.

[0133] The first polynucleotide that comprises a sequence encoding the E1 protein of rubella may have the sequence set forth in SEQ ID No. 8 and / or SEQ ID No. 28 or may be derived from such a sequence.

[0134] In an embodiment, the nucleic acid construct according to the invention comprises a first heterologous polynucleotide or a plurality of first heterologous polynucleotides comprising at least SEQ ID No. 4 or SEQ ID No. 24, and SEQ ID No. 6 or SEQ ID No. 26, and SEQ ID No. 8 or SEQ ID No. 28.

[0135] In an embodiment, the nucleic acid construct according to the invention comprises a first heterologous polynucleotide or a plurality of first heterologous polynucleotides comprising at least SEQ ID No. 4 or SEQ ID No. 24, and SEQ ID No. 6 or SEQ ID No. 26, and SEQ ID No. 8 or SEQ ID No. 28, and SEQ ID No. 10 or SEQ ID No. 30, and SEQ ID No.12 or SEQ ID No. 32.

[0136] In a particular embodiment of the invention, the nucleic acid construct comprises within the first heterologous polynucleotide a nucleic acid encoding the C protein of rubella virus, preferentially the C protein of SEQ ID No. 3 and / or SEQ ID No. 23, or an antigenic fragment thereof; and a nucleic acid encoding the E2 protein of rubella virus, preferentially the E2 protein of SEQ ID No. 5 and / or SEQ ID No. 25, and a nucleic acid encoding the E1 protein of rubella virus, preferentially the E1 protein of SEQ ID No. 7 and / or SEQ ID No. 27, this first heterologous polynucleotide being cloned between the P and M genes of the MeV, preferentially within ATLI2 as defined here above.

[0137] In a particular embodiment of the invention, the nucleic acid construct comprises: - within the first heterologous polynucleotide a nucleic acid encoding the C protein of rubella virus, preferentially the C protein of SEQ ID No. 3 and / or SEQ ID No. 23, or an antigenic fragment thereof; and a nucleic acid encoding the E2 protein of rubella virus, preferentially the E2 protein of SEQ ID No. 5 and / or SEQ ID No. 25, and a nucleic acid encoding the E1 protein of rubella virus, preferentially the E1 protein of SEQ ID No. 7 and / or SEQ ID No. 27, this first heterologous polynucleotide being cloned between the P and M genes of the MeV, preferentially within ATLI2 as defined here above, and within the at least on second heterologous polynucleotide a nucleic acid encoding the HN protein of mumps virus, preferentially the HN protein of SEQ ID No. 13, this at least one second heterologous polynucleotide being cloned between the H gene and the L gene of the MeV, preferentially within ATLI3 as defined here above.

[0138] In a particular embodiment of the invention, the nucleic acid construct comprises within the first heterologous polynucleotide, and in particular in the following order, a nucleic acid encoding the C protein of rubella virus, preferentially the C protein of SEQ ID No. 3 and / or SEQ ID No. 23, or an antigenic fragment thereof; and a nucleic acid encoding the E2 peptide signal of the rubella virus, in particular the E2 peptide signal of SEQ ID No. 9 and / or SEQ ID No. 29, and a nucleic acid encoding the E2 protein of rubella virus, preferentially the E2 protein of SEQ ID No. 5 and / or SEQ ID No. 25, and a nucleic acid encoding the E1 peptide signal of the rubella virus, in particular the E1 peptide signal of SEQ ID No. 11 and / or SEQ ID No. 31 , and a nucleic acid encoding the E1 protein of rubella virus, preferentially the E1 protein of SEQ ID No. 7 and / or SEQ ID No. 27, this first heterologous polynucleotide being cloned between the P and M genes of the MeV, preferentially within ATLI2 as defined here above.

[0139] The term “derived” appearing in relation to the polynucleotides merely specifies that the sequence of said polynucleotide may be identical to the corresponding sequence in a rubella strain (or mumps virus) or may vary to the extent that it encodes polypeptides, antigens, proteins, or fragments thereof, of rubella or mumps that meet(s) the definition of the “polypeptide” according to the present invention. In particular, a polynucleotide derives from the nucleic acid of a rubella strain or mumps strain when it is codon-optimized with respect to such sequence. Accordingly, the term does not restrict the production mode of the polynucleotide. In a particular embodiment, the nucleic acid construct of the invention comprises from the 5’ to 3’ end the following polynucleotides:

[0140] (a) a polynucleotide encoding the N protein of the MeV;

[0141] (b) a polynucleotide encoding the P protein of the MeV;

[0142] (c) the first heterologous polynucleotide or a plurality of first heterologous polynucleotides encoding at least the following rubella virus structural proteins: Capsid (C), Envelope 2 (E2) and Envelope 1 (E1 ), or antigenic fragments thereof; and optionally comprise(s) the E2 peptide signal, in particular upstream of a sequence encoding the E2 protein or the antigenic fragment thereof, and / or the E1 peptide signal, in particular upstream of a sequence encoding the E1 protein or the antigenic fragment thereof;

[0143] (d) a polynucleotide encoding the M protein of the MeV;

[0144] (e) a polynucleotide encoding the F protein of the MeV;

[0145] (f) a polynucleotide encoding the H protein of the MeV;

[0146] (g) a polynucleotide encoding the L protein of the MeV; and wherein said polynucleotides are operatively linked within the nucleic acid construct and are in particular under the control of the viral replication and transcriptional regulatory elements of the MeV, more particularly the leader and trailer sequence(s).

[0147] Such a construct is illustrated in Fig.1, Fig.11-12, and Fig.15-16.

[0148] The expressions "N protein", "P protein", "M protein", "F protein", "H protein" and "L protein" refer respectively to the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the fusion protein (F), the hemagglutinin protein (H) and the RNA polymerase large protein (L) of a measles virus and encompass reference to the respective polypeptides or antigenic fragments thereof. These components have been identified in the prior art and are especially disclosed in Fields, Virology (Knipe & Howley, 2001 ). The at least one second heterologous polynucleotide may comprise any number of the sequence set forth in SEQ ID No. 14 that encodes the HN protein of mumps virus. In particular, the at least one second polynucleotide that comprises a sequence coding the HN protein of mumps virus has the sequence set forth in SEQ ID No. 14. In particular embodiment, the second heterologous polynucleotide has codon-optimized open reading frame (ORF).

[0149] Alternatively, or complementarily, a second heterologous polynucleotide(s) may encode the HN protein of mumps virus of SEQ ID No. 13.

[0150] In a particular embodiment, when the nucleic acid construct of the invention comprises polynucleotide sequences encoding rubella proteins and mumps protein, the nucleic acid construct comprises from 5’ to 3’ end the following polynucleotides:

[0151] (a) a polynucleotide encoding the N protein of the MeV;

[0152] (b) a polynucleotide encoding the P protein of the MeV;

[0153] (c) the first heterologous polynucleotide or a plurality of first heterologous polynucleotides encoding at least the following rubella virus structural proteins: Capsid (C), Envelope 2 (E2) and Envelope 1 (E1 ), or antigenic fragments thereof; and optionally comprise(s) the E2 peptide signal, in particular upstream of a sequence encoding the E2 protein or the antigenic fragment thereof, and / or the E1 peptide signal, in particular upstream of a sequence encoding the E1 protein or the antigenic fragment thereof;

[0154] (d) a polynucleotide encoding the M protein of the MeV;

[0155] (e) a polynucleotide encoding the F protein of the MeV;

[0156] (f) a polynucleotide encoding the H protein of the MeV;

[0157] (g) the second heterologous polynucleotide encoding at least the mumps virus HN protein, or antigenic fragment thereof;

[0158] (h) a polynucleotide encoding the L protein of the MeV; and wherein said polynucleotides are operatively linked within the nucleic acid construct and are in particular under the control of the viral replication and transcriptional regulatory elements of the MeV, more particularly the leader and trailer sequence(s).

[0159] Such a construct is illustrated on Fig.6, Fig.9, Fig 13-14 and Fig.15-16. The MeV genome

[0160] The cDNA molecule encoding the full-length antigenomic (+) RNA strand of the MeV may be characteristic of or may be obtained from an attenuated strain of MeV. An “attenuated strain” of MeV is defined as a strain that is avirulent or less virulent than the parent strain in the same host, while maintaining immunogenicity and possibly adjuvanticity when administered in a host for preserving immunodominant T and B cell epitopes and possibly the adjuvanticity such as the induction of T cell costimulatory proteins or cytokine IL-12.

[0161] An attenuated strain of a measles virus accordingly refers to a strain which has been serially passaged on selected cells and, possibly, adapted to other cells to produce seed strains suitable for the preparation of human vaccine strains, harboring a stable genome which would not allow reversion to pathogenicity nor integration in host chromosomes. As a particular “attenuated strain”, an approved strain for a vaccine is an attenuated strain suitable for the invention when it meets the criteria defined by the FDA (US Food and Drug Administration); i.e. it meets safety, efficacy, quality and reproducibility criteria, after rigorous reviews of laboratory and clinical data (www.fda.gov / cber / vaccine / vacappr.htm).

[0162] In particular, the cDNA molecule encoding the full-length antigenomic (+) RNA strand of the MeV is obtained from an attenuated virus strain selected from the group comprising of consisting of the Schwarz strain, the Zagreb strain, the AIK- C strain, the Moraten strain, the Philips strain, the Beckenham 4A strain, the Beckenham 16 strain, the Edmonston seed A strain, the Edmonston seed B strain, the CAM-70 strain, the TD 97 strain, the Leningrad-16 strain, the Shanghai 191 strain and the Belgrade strain. The invention uses in particular strains that have been allowed for use as commercial vaccines. In particular, the cDNA molecule encoding the full length antigenomic (+) RNA strand of the MeV is obtained from the Schwarz strain.

[0163] In a particular embodiment, the cDNA molecule encoding the full-length antigenomic (+) RNA strand of the MeV has the sequence set forth in SEQ ID No. 19 (pTM-Sch) or SEQ ID No. 37 (pTM-MVSchw) or SEQ ID No. 20 (pSMAC). According to a particular embodiment of the invention, the cDNA molecule is placed under the control of heterologous expression control sequences. The insertion of such a control for the expression of the cDNA, is favorable when the expression of this cDNA is sought in cell types which do not enable full transcription of the cDNA with its native control sequences.

[0164] According to a particular embodiment of the invention, the heterologous expression control sequence comprises the T7 promoter and T7 terminator sequences. These sequences are respectively located 5’ and 3’ of the coding sequence for the full length antigenomic (+) RNA strand of MeV and from the adjacent sequences around this coding sequence.

[0165] In a particular embodiment of the invention, the cDNA molecule, which is defined here above is modified, i.e. comprises additional nucleotide sequences or motifs. In a preferred embodiment, the cDNA molecule used according to the invention further comprises, at its 5’-end, adjacent to the first nucleotide of the nucleotide sequence encoding the full-length antigenomic (+) RNA strand of the MeV approved vaccine strain, a GGG motif followed by a hammerhead ribozyme sequence and comprises, at its 3’-end, adjacent to the last nucleotide of said nucleotide sequence encoding the full-length anti-genomic (+) RNA strand, the sequence of a ribozyme. The Hepatitis delta virus ribozyme (5) is appropriate to carry out this preferred embodiment.

[0166] The GGG motif placed at the 5’ end, adjacent to the first nucleotide of the above coding sequence improves the efficiency of the transcription of said cDNA coding sequence. As a requirement for the proper assembly of measles virus particles is the fact that the cDNA encoding the antigenomic (+) RNA complies with the rule of six, when the GGG motif is added, a ribozyme is also added at the 5’ end of the coding sequence of the cDNA, 3’ from the GGG motif, in order to enable cleavage of the transcript at the first coding nucleotide of the full-length antigenomic (+) RNA strand of MeV.

[0167] In order to prepare the nucleic acid construct of the invention, the preparation of a cDNA molecule encoding the full-length antigenomic (+) RNA of a measles virus disclosed in the prior art is achieved by known methods. The obtained cDNA provides especially the basis for the genome vector involved in the rescue of recombinant measles virus particles when it is inserted in a vector such as a plasmid. A particular cDNA molecule suitable for the preparation of the nucleic acid construct of the invention is the one obtained using the Schwarz strain of measles virus. Plasmid pTM-MVSchw, which contains an infectious MeV cDNA corresponding to the anti-genome of the Schwarz MeV vaccine strain and is used for preparation of recombinant vectors encompassing the heterologous polynucleotides of the invention, has been described elsewhere (Combredet C., et al., A molecularly cloned Schwarz strain of measles virus vaccine induces strong immune responses in macaques and transgenic mice. J Virol, 2003. 77(21 ): p. 11546-54). Accordingly, the cDNA used within the present invention may be obtained as disclosed in WO 2004 / 000876 or may be obtained from plasmid pTM-MVSchw deposited by Institut Pasteur at the CNCM under No I- 2889 on June 12, 2002, the sequence of which is disclosed in WO 2004 / 000876 incorporated herein by reference. The plasmid pTM-MVSchw has been obtained from a Bluescript plasmid and comprises the polynucleotide coding for the full- length measles virus (+) RNA strand of the Schwarz strain placed under the control of the promoter of the T7 RNA polymerase. It has 18967 nucleotides and a sequence represented as SEQ ID No. 37. cDNA molecules (also designated cDNA of the measles virus or MeV cDNA for convenience) from other MeV strains may be similarly obtained starting from the nucleic acid purified from viral particles of attenuated MeV such as those described herein. An additional transcription unit may be a multiple-cloning site cassette previously inserted in the vector, as explained in Combredet et al., 2003. An ATU may comprise c / s-acting sequences necessary for the transcription of the inserted rubella genes. The heterologous polynucleotide(s) are cloned or inserted within additional transcription units (ATU) as defined here above.

[0168] According to a particular embodiment of the invention, it is provided a nucleic acid construct that does not comprise A / T-rich RNA editing motifs. A / T-rich RNA editing motifs refer to nucleotide sequences within viral RNA genomes that are characterized by a high proportion of adenine (A) and thymine (T) (or uracil (U) in RNA). In the context of the invention, these motifs are particularly relevant because they are associated with site-specific RNA editing events that occur during viral transcription. In the measles virus, the RNA editing sites are usually located in A / U-rich regions of the viral genome. A A / T-rich RNA editing motif can correspond to a string of consecutive As or Us (e.g., 5 -AAAAA-3' or 5 -UUUUU- 3' or 5’ AAATTT 3’ or 5’ AAAAT 3’). A conserved sequence surrounding the editing site, often including a 3' G immediately downstream can also be part of a A / T-rich RNA editing motif.

[0169] According to a preferred embodiment, the invention also concerns modification and in particular optimization of the polynucleotides to allow an efficient expression of the rubella proteins and / or mumps protein, polypeptides, antigens, or fragments thereof, in a host cell.

[0170] Accordingly, optimization of the polynucleotide sequence can be operated avoiding cis-active domains of nucleic acid molecules: internal TATA-boxes, chisites and ribosomal entry sites; AT-rich or GC-rich sequence stretches; ARE, INS, CRS sequence elements; repeat sequences and RNA secondary structures; cryptic splice donor and acceptor sites, branch points.

[0171] The optimized polynucleotides may also be codon optimized for expression in a specific cell type, in particular may be modified for the Macaque (?) codon usage or for the human codon usage. This optimization allows increasing the efficiency of chimeric infectious particles production in cells without impacting the amino acid composition of the expressed protein(s).

[0172] In particular, the optimization of the polynucleotide encoding the rubella proteins or mumps protein may be performed by modification of the wobble position in codons without impacting the identity of the amino acid residue translated from said codon with respect to the original one.

[0173] Optimization is also performed to avoid editing-like sequences from Measles virus. The editing of transcript of measles virus is a process which occurs in particular in the transcript encoded by the P gene of measles virus. This editing, by the insertion of extra G residues at a specific site within the P transcript, gives rise to a new protein truncated compared to the P protein. Addition of only a single G residue results in the expression of the V protein, which contains a unique carboxyl terminus (Cattaneo R et al., Cell. 1989 Mar 10;56(5):759-64).

[0174] In the polynucleotides according to this particular embodiment of the invention, the following editing-like sequences from measles virus can be mutated: AAAGGG, AAAAGG, GGGAAA, GGGGAA, as well as their complementary sequence: TTTCCC, TTTTCC, CCCTTT, CCCCTT. For example, AAAGGG can be mutated in AAAGGC, AAAAGG can be mutated in AGAAGG or in TAAAGG or in GAAAGG, and GGGAAA in GCGAAA.

[0175] According to any one of the particular embodiments of the invention, it is provided nucleic acid constructs comprising polynucleotide(s) which increase the efficiency of chimeric recombinant MeV-rubella infectious particles production.

[0176] In a particular embodiment of the invention, the nucleic acid construct comprises within a first heterologous polynucleotide a nucleic acid of SEQ ID No. 4 or SEQ ID No. 24 encoding the C protein of rubella, a nucleic acid of SEQ ID No: 6 or SEQ ID No. 26 encoding the E2 protein of rubella virus, and a nucleic acid of SEQ ID No. 8 or SEQ ID No. 28 encoding the E1 protein of rubella virus, preferentially these three nucleic acids are separated by two linker sequences, in particular of SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 30 and SEQ ID No. 32, as illustrated on Fig.1. In a preferred embodiment, the nucleic acid of SEQ ID No. 4 or SEQ ID No. 24 is localized upstream the nucleic acid of SEQ ID No. 6 or SEQ ID No. 26, which is localized upstream of the nucleic acid of SEQ ID No 8 or SEQ ID No. 28. This is for example illustrated on Fig.11 and Fig.12 with the construct named pTM2-Rub-1 E and pTM2-Rub-2B.

[0177] In a particular embodiment of the invention, the nucleic acid construct comprises within a first heterologous polynucleotide a nucleic acid of SEQ ID No. 4 encoding the C protein of rubella, a nucleic acid of SEQ ID No: 6 encoding the E2 protein of rubella virus, and a nucleic acid of SEQ ID No. 8 encoding the E1 protein of rubella virus, preferentially these three nucleic acids are separated by two linker sequences, in particular of SEQ ID No. 10 and SEQ ID No. 12, as illustrated on Fig.1. In a preferred embodiment, the nucleic acid of SEQ ID No. 4 is localized upstream the nucleic acid of SEQ ID No. 6, which is localized upstream of the nucleic acid of SEQ ID No 8.

[0178] In a particular embodiment of the invention, the nucleic acid construct comprises within a first heterologous polynucleotide a nucleic acid of SEQ ID No. 24 encoding the C protein of rubella, a nucleic acid of SEQ ID No: 26 encoding the E2 protein of rubella virus, and a nucleic acid of SEQ ID No. 28 encoding the E1 protein of rubella virus, preferentially these three nucleic acids are separated by two linker sequences, in particular of SEQ ID No. 30 and SEQ ID No. 32, as illustrated on Fig.1 In a preferred embodiment, the nucleic acid of SEQ ID No. 24 is localized upstream the nucleic acid of SEQ No. 26, which is localized upstream of the nucleic acid of SEQ ID No 28.

[0179] In a particular embodiment of the invention, the nucleic acid construct comprises the first heterologous polynucleotide and the second heterologous polynucleotide, and:

[0180] - the first heterologous polynucleotide comprises a nucleic acid of SEQ ID No. 4 or SEQ ID No. 24 encoding the C protein of rubella, a nucleic acid of SEQ ID No. 6 or SEQ ID No. 26 encoding the E2 protein of rubella virus, and a nucleic acid of SEQ ID No. 8 or SEQ ID No. 28 encoding the E1 protein of rubella virus, preferentially these three nucleic acids are separated by two linker sequences, in particular of SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 30 and SEQ ID No. 32, as illustrated on Fig.1 ; the first heterologous polynucleotide being preferentially cloned within ATLI2 as defined here above; and,

[0181] - the second heterologous polynucleotide comprises a nucleic acid, in particular of sequence SEQ ID No. 14, encoding the HN protein of mumps virus, or an antigenic fragment thereof, preferentially the HN protein of SEQ ID No.13, the second heterologous polynucleotide being preferentially cloned within ATLI3 as defined here above.

[0182] In a particular embodiment of the invention, the nucleic acid construct comprises two second heterologous polynucleotides, each comprising a nucleic acid of SEQ ID No. 13 encoding the HN protein of mumps virus, the second heterologous polynucleotides being cloned within ATLI2 and ATLI3 as defined here above.

[0183] In a particular embodiment of the invention, the nucleic acid construct comprises a recombinant cDNA whose sequence is selected from the group consisting of:

[0184] SEQ ID No. 1 (pTM2-Rub1 E);

[0185] SEQ ID No. 2 (pTM2-Rub2B);

[0186] SEQ ID No. 15 (pSMAC-Rub1 E-mumps-HN);

[0187] SED ID No. 16 (pSMAC-Rub2B-mumps-HN);

[0188] SEQ ID No. 17 (pTM2-mumps-HN); and,

[0189] SEQ ID No. 18 (pTM3-mumps-HN), wherein said sequences are described as follows:

[0190] SEQ ID NO. 1 SEQ ID No. 1 is the sequence of a nucleic acid construct according to a particular embodiment of the invention wherein said construct contains the pTM2- MVSchwarz vector wherein the sequences encoding the C, the E2 and the E1 proteins of rubella strain 1 E have been cloned within the Additional Transcription Unit 2.

[0191] SEQ ID NO. 2

[0192] SEQ ID No. 2 is the sequence of a nucleic acid construct according to a particular embodiment of the invention wherein said construct contains the pTM2- MVSchwarz vector wherein the sequences encoding the C, the E2 and the E1 proteins of rubella strain 2B have been cloned within the Additional Transcription Unit 2.

[0193] SEQ ID NO. 17

[0194] SEQ ID No. 17 is the sequence of a nucleic acid construct according to a particular embodiment of the invention wherein said construct contains the pTM2- MVSchwarz vector wherein the sequence encoding the HN protein of rubella has been cloned within the Additional Transcription Unit 2.

[0195] SEQ ID NO. 18

[0196] SEQ ID No. 18 is the sequence of a nucleic acid construct according to a particular embodiment of the invention wherein said construct contains the pTM3- MVSchwarz vector wherein the sequence encoding the HN protein of rubella has been cloned within the Additional Transcription Unit 3.

[0197] SEQ ID NO. 15 and SEQ ID NO. 16

[0198] SEQ ID No. 15 and SEQ ID No. 16 are two sequences of a nucleic acid construct according to a particular embodiment of the invention wherein said construct contains the pSMAC-MVSchwarz vector wherein the sequences encoding the C, the E2 and the E1 proteins of rubella strain 1 E have been cloned within the Additional Transcription Unit 2 and wherein the sequence encoding the HN protein of rubella has been cloned within the Additional Transcription Unit 3.

[0199] The invention also relates to a transfer vector, which may be used for the preparation of recombinant MeV-rubella particles or MeV-rubella-mumps particles or MeV-mumps particles when rescued from helper cells or production cells. Several transfer vectors are illustrated on Fig.11 to 16. In a preferred embodiment of the invention, the transfer vector is a transfer vector plasmid suitable for the transfection of helper cells or of production cells, and comprising the nucleic acid construct according to the invention. The transfer vector plasmid may be obtained from a Bluescript plasmid and may be obtained by cloning the heterologous polynucleotide(s) of the invention in the pTM-MVSchw or pSMAC plasmid described here above. In particular embodiments of the invention, the transfer plasmid vector has the sequence of SEQ ID No. 1 , SEQ ID No. 2, SEQ ID No. 15 or SEQ ID No. 16. Alternatively, the transfer plasmid vector has the sequence or SEQ ID No. 17 or SEQ ID No. 18.

[0200] According to an embodiment, it is provided a nucleic acid construct according to any embodiment disclosed herein, wherein the construct is capable of inducing an immune response comparable to that of Priorix® at a dose of 103to 104TCID50.

[0201] The invention also concerns the use of a transfer plasmid vector or the use of the nucleic acid construct according to the invention to transform cells suitable for the rescue of recombinant viral MeV-rubella particles or MeV-rubella-mumps particles or MeV-mumps particles, in particular to transfect or to transduce such cells respectively with plasmids or with viral vectors harboring the nucleic acid construct of the invention, said cells being selected for their capacity to express required measles virus proteins for appropriate replication, transcription and encapsidation of the recombinant genome of the virus corresponding to the nucleic acid construct of the invention in recombinant, infectious, replicative recombinant MeV-rubella particles or MeV-rubella-mumps particles or MeV- mumps particles.

[0202] The nucleic acid construct of the invention and the transfer plasmid vector are suitable and intended for the preparation of recombinant infectious replicative recombinant measles - Rubella virus (MeV-rubella) or MeV-rubella-mumps virus or MeV-mumps virus and accordingly said nucleic acid construct and transfer plasmid vector are intended for insertion in a transfer genome vector that as a result comprises the cDNA molecule of the measles virus, especially of the Schwarz strain, for the production of said recombinant MeV-rubella virus ) or MeV-rubella-mumps virus or MeV-mumps virus and expression of rubella polypeptide(s), possibly as rubella VLPs when the C, the E2 and the E1 proteins are encoded by at least one first heterologous polynucleotide, and / or by expression of mumps particles, possibly as mumps VLPS when the HN protein is encoded by at least one second heterologous polynucleotide. The pTM- MVSchw plasmid or the pSMAC plasmid are suitable to prepare the transfer vector, by insertion of the heterologous polynucleotide(s) as described herein necessary for the expression of rubella proteins, antigen(s), or antigenic fragment(s) thereof and / or the mumps proteins. As used herein, the term "viruslike particle" (VLP) refers to a structure that in at least one attribute resembles a virus but which has not been demonstrated to be infectious as such. Virus Like Particles in accordance with the invention do not carry genetic information encoding the proteins of the Virus Like Particles, in general, virus-like particles lack a viral genome and, therefore, are noninfectious and non- replicative. In accordance with the present invention, Virus Like Particles can be produced in large quantities and are expressed together with MeV-rubella recombinant particles or MeV-rubel la-mumps particles or MeV-mumps particles.

[0203] The invention also relates to the cells or cell lines thus transformed by the transfer vector of the invention and by further polynucleotides providing helper functions and proteins. Polynucleotides are thus present in said cells, which encode proteins that include in particular the N, P and L proteins of a measles virus ( / .e., native MeV proteins or functional variants thereof capable of forming ribonucleoprotein (RNP) complexes), preferably as stably expressed proteins at least for the N and P proteins functional in the transcription and replication of the recombinant viral MeV-rubella particles. The N and P proteins may be expressed in the cells from a plasmid comprising their coding sequences or may be expressed from a DNA molecule inserted in the genome of the cell. The L protein may be expressed from a different plasmid. It may be expressed transitory. The helper cell is also capable of expressing a RNA polymerase suitable to enable the synthesis of the recombinant RNA derived from the nucleic acid construct of the invention, possibly as a stably expressed RNA polymerase. The RNA polymerase may be the T7 phage polymerase or its nuclear form (nlsT7).

[0204] In an embodiment, the cDNA clone of a measles virus is from the same measles virus strain as the N protein and / or the P protein and / or the L protein. In another embodiment, the cDNA clone of a measles virus is from a different strain of virus than the N protein and / or the P protein and / or the L protein. The cells transformed or transfected with a nucleic acid construct according to the invention are able to produce recombinant measles viruses and and / or rubella VLPs and / or rubella-mumps VLPS and / or mumps VLPs, depending on whether the C, E2 and E1 proteins of rubella are expressed and / or the HN protein of mumps is expressed. Accordingly, the recombinant measles virus comprises in its genome a nucleic acid construct of the invention and is able to express at least one polypeptide, protein or antigenic fragment thereof, of the rubella or mumps. Preferably, the measles virus of the invention is able to express the C protein, or an antigenic fragment thereof; the E2 protein, or an antigenic fragment thereof; and the E2 protein, or an antigenic fragment thereof. In a preferred embodiment, the HN protein is also expressed. The rubella VLPs may comprise the C protein, or an antigenic fragment thereof, the E2 protein, or an antigenic fragment thereof, the E1 protein, or an antigenic fragment thereof, and the HN protein, of an antigenic fragment thereof.

[0205] Furthermore, according to some embodiments of the invention, the recombinant measles virus also expresses at least one polypeptide or protein, or an antigenic fragment thereof, of the measles virus. In other words, the recombinant measles virus expresses at least one of the following polypeptides: the N protein, the P protein, the M protein, the F protein, the H protein and the L protein of the MeV. According to this embodiment, the recombinant virus expresses recombinant antigenic particles of the measles virus and the rubella virus, and possibly the mumps virus, allowing the elicitation of cellular response, or a humoral response, or a cellular and humoral response against:

[0206] - polypeptides of the rubella; or,

[0207] - polypeptides of the measles; or,

[0208] - polypeptides of the mumps; or,

[0209] - polypeptides of the measles and the rubella; or,

[0210] - polypeptides of the measles and the mumps; or,

[0211] - polypeptides of the mumps and the rubella; or,

[0212] - polypeptides of the measles, the rubella and the mumps.

[0213] In particular embodiments of the invention, the elicitation of the cellular response comprises elicitation of a T cell response, in particular CD4+ and / or CD8+ T cells response. The invention thus relates to a process for the preparation of recombinant infectious measles virus particles comprising:

[0214] (a) transfecting cells, in particular helper cells, in particular HEK293 helper cells, stably expressing T7 RNA polymerase and measles N and P proteins with the nucleic acid construct according to the invention or with the transfer plasmid vector according to the invention;

[0215] (b) maintaining the transfected cells in conditions suitable for the production of recombinant measles-rubella VLPs or recombinant rubella-mumps VLPs or recombinant measles-rubella-mumps VLPs;

[0216] (c) infecting cells enabling propagation of the measles-rubella VLPs or recombinant rubella-mumps VLPs or recombinant measles-rubella- mumps VLPs by co-cultivating them with the transfected cells of step (b);

[0217] (d) harvesting the recombinant measles virus expressing at least one rubella protein, or at least one mumps protein, or at least one mumps protein and one rubella protein, preferentially the C protein, the E2 protein, the E1 protein of rubella and optionally the HN protein of mumps.

[0218] According to a particular embodiment, the invention relates to a process for the preparation of recombinant infectious measles virus particles comprising: a) transferring, in particular transfecting, the nucleic acid construct of the invention or the transfer vector containing such nucleic acid construct in a helper cell line which also expresses proteins necessary for transcription, replication and encapsidation of the antigenomic (+) RNA sequence of MeV from its cDNA and under conditions enabling viral particles assembly; and, b) recovering the recombinant infectious MeV-rubella virus or MeV- rubella-mumps virus or MeV-mumps virus expressing at least one polypeptide or protein of rubella, or an antigenic fragment thereof, and optionally mumps, or expressing at least one polypeptide or protein of mumps.

[0219] According to a particular embodiment of the invention, the process comprises: a) transfecting helper cells with a nucleic acid construct according to the invention with a transfer plasmid vector, wherein said helper cells are capable of expressing helper functions to express an RNA polymerase, and to express the N, P and L proteins of a MeV virus; b) co-cultivating said transfected helper cells of step 1 ) with passaged cells suitable for the passage of the MeV attenuated strain from which the cDNA originates; c) recovering the recombinant infectious MeV-rubella virus or MeV-rubella- mumps virus or MeV-mumps virus expressing at least one polypeptide of the rubella and / or one polypeptide of mumps.

[0220] According to another particular embodiment of the invention, the method for the production of recombinant infectious MeV-rubella or MeV-rubella-mumps or MeV-mumps comprises: a) recombining a cell or a culture of cells stably producing a RNA polymerase, the nucleoprotein (N) of a measles virus and the polymerase cofactor phosphoprotein (P) of a measles virus, with a nucleic acid construct of the invention and with a vector comprising a nucleic acid encoding a RNA polymerase large protein (L) of a measles virus; and, b) recovering the infectious, MeV-rubella virus or MeV-rubella-mumps virus or MeV-mumps virus from said recombinant cell or culture of recombinant cells.

[0221] According to a particular embodiment of the process, recombinant MeV are produced, which express rubella protein(s) and / or mumps protein(s) comprising at least the C protein, the E2 protein and the E1 protein of rubella virus, or antigenic fragments thereof. As an illustration, a process to rescue recombinant MeV expressing rubella proteins, in particular rubella VLPs comprises the steps of:

[0222] 1 ) co-transfecting helper cells, in particular HEK293 helper cells, that stably express T7 RNA polymerase, and measles N and P proteins with (i) a transfer vector, in particular a plasmid, comprising cDNA encoding the full-length antigenomic (+) RNA of a measles virus recombined with at least one polynucleotide encoding the C protein, the E2 protein and the E1 protein of rubella protein, and with (ii) a vector, especially a plasmid, encoding the MeV L polymerase cDNA;

[0223] 2) cultivating said co-transfected helper cells in conditions enabling the production of MV-rubella recombinant virus;

[0224] 3) propagating the thus produced recombinant virus by co-cultivating said helper cells of step 2) with cells enabling said propagation such as Vero cells;

[0225] 4) recovering replicating MeV-rubella recombinant virus and rubella protein(s), in particular rubella Virus Like Particles.

[0226] As used herein, “recombining” means introducing at least one polynucleotide into a cell, for example under the form of a vector, said polynucleotide integrating (entirely or partially) or not integrating into the cell. According to a particular embodiment, recombination can be obtained with a first polynucleotide, which is the nucleic acid construct of the invention. Recombination can, also or alternatively, encompasses introducing a polynucleotide, which is a vector encoding a RNA polymerase large protein (L) of a measles virus, whose definition, nature and stability of expression has been described herein.

[0227] In accordance with the invention, the cell or cell lines or a culture of cells stably producing a RNA polymerase, a nucleoprotein (N) of a measles virus and a polymerase cofactor phosphoprotein (P) of a measles virus is a cell or cell line as defined in the present specification or a culture of cells as defined in the present specification, i.e., are also recombinant cells to the extent that they have been transformed by the introduction of one or more polynucleotides as defined above. In a particular embodiment of the invention, the cell or cell line or culture of cells, stably producing the RNA polymerase, the N and P proteins, does not produce the L protein of a measles virus or does not stably produce the L protein of a measles virus, e.g., enabling its transitory expression or production. The production of recombinant MeV-rubella virus or MeV-rubella-mumps virus or MeV-mumps virus of the invention may involve a transfer of cells transformed as described herein. “Transfer” as used herein refers to the plating of the recombinant cells onto a different type of cells, and particularly onto monolayers of a different type of cells. These latter cells are competent to sustain both the replication and the production of infectious recombinant MeV-rubella virus or MeV-rubella-mumps virus or MeV-mumps virus i.e., respectively the formation of infectious viruses inside the cell and possibly the release of these infectious viruses outside of the cells possibly with release of rubella immunogenic particles and / or rubella VLPs and / or mumps VLPs and / or mumps immunogenic particles. This transfer results in the co-culture of the recombinant cells of the invention with competent cells as defined in the previous sentence. The above transfer may be an additional, i.e., optional, step when the recombinant cells are not efficient virus-producing culture i.e., when infectious recombinant MeV-rubella virus or MeV-rubella-mumps virus or MeV-mumps virus cannot be efficiently recovered from these recombinant cells. This step is introduced after further recombination of the recombinant cells of the invention with any nucleic acid construct of the invention, and optionally a vector comprising a nucleic acid encoding a RNA polymerase large protein (L) of a measles virus.

[0228] In a particular embodiment of the invention, a transfer step is required since the recombinant cells, usually chosen for their capacity to be easily recombined are not efficient enough in the sustaining and production of recombinant infectious MeV-rubella virus. In said embodiment, the cell or cell line or culture of cells of step 1 ) of the above-defined methods is a recombinant cell or cell line or culture of recombinant cells according to the invention.

[0229] Cells suitable for the preparation of the recombinant cells of the invention are prokaryotic or eukaryotic cells, particularly animal or plant cells, and more particularly mammalian cells such as human cells or non-human mammalian cells or avian cells or yeast cells. In a particular embodiment, cells, before recombination of its genome, are isolated from either a primary culture or a cell line. Cells of the invention may be dividing or non-dividing cells.

[0230] According to a preferred embodiment, helper cells are derived from human embryonic kidney cell line 293, which cell line 293 is deposited with the ATCC under No. CRL-1573. Particular cell line 293 is the cell line disclosed in W02008 / 078198 and referred to in the following examples as 293T7 / N / P. Thus, the invention also relates to a host cell, in particular an avian cell or a mammalian cell, transfected or transformed with the nucleic acid construct according to any embodiment of the invention, or transfected with a transfer plasmid vector. Suitable cells are the VERO NK cells (African green monkey kidney cells), and MRC5 cells (Medical Research Council cell strain 5). According to another aspect of this process, the cells suitable for passage are CEF cells (chick embryo fibroblasts). CEF cells can be prepared from fertilized chicken eggs as obtained from EARL Morizeau (8 rue Moulin, 28190 Dangers, France) or from any other producer of fertilized chicken eggs.

[0231] The process which is disclosed according to the present invention is used advantageously for the production of infectious replicative recombinant MeV- rubella virus or MeV-rubella-mumps virus or MeV-mumps virus appropriate for use as immunization compositions. The invention thus relates to a composition, in particular an antigenic composition, whose active principle comprises infection replicative recombinant MeV-rubella virus or MeV-rubella-mumps virus or MeV- mumps virus rescued from the nucleic acid construct of the invention and in particular obtained by the process disclosed. The composition may be a vaccine composition for administration to a human in need thereof, especially children. Said composition may be used for the treatment against rubella infection. Said composition may be used for the protection against rubella. Said composition may be used for the treatment against mumps infection. Said composition may be used for the protection mumps rubella. Said composition may be used for the treatment against rubella infection and mumps infection. Said composition may be used for the protection against rubella and mumps. Said composition may be used for the treatment against measles infection and rubella infection. Said composition may be used for the protection against measles and rubella. Said composition may be used for the treatment against measles infection, mumps infection and rubella infection. Said composition may be used for the protection against measles, mumps and rubella. Said composition may be used for the treatment against measles infection and mumps infection. Said composition may be used for the protection against measles and mumps rubella.

[0232] Thus, the composition may be an immunogenic or antigenic composition for the protective or prophylactic treatment against a rubella infection and / or a measles infection and / or a mumps infection. In particular, the active ingredients or active principles within the composition comprise recombinant MeV-rubella particles and / or MeV-mumps particles and / or MeV-rubella-mumps particles, said recombinant MeV-rubella particles being rescued from a transfer plasmid vector according to the invention and being optionally associated with VLPs of the invention. In the context of the invention, the terms “associated” or “in association” refer to the presence, in a single composition, of both MeV-rubella recombinant viral particles and rubella polypeptides or proteins, in particular as VLPs, usually as physically separate entities. In a particular embodiment of the invention, the composition is a vaccine.

[0233] The invention also concerns the recombinant MeV-rubella or MeV-rubella- mumps infectious replicating virus particles in association with rubella protein(s), or antigenic fragment(s) thereof, possibly associated rubella VLPs, or any composition according to the invention, for the use in the treatment or the prevention of an infection by rubella virus in a subject, in particular a human subject, in particular a child.

[0234] The invention also concerns the recombinant MeV-rubella or MeV-rubella- mumps infectious replicating virus particles in association with rubella protein(s), or antigenic fragment(s) thereof, possibly associated rubella VLPs, or any composition according to the invention, for the use in the treatment or the prevention of an infection by measles virus in a subject, in particular a human subject, in particular a child.

[0235] The invention also concerns the recombinant MeV-rubella or MeV-rubella- mumps infectious replicating virus particles in association with rubella protein(s), or antigenic fragment(s) thereof, possibly associated rubella VLPs, or any composition according to the invention, for the use in the treatment or the prevention of an infection by rubella and measles virus in a subject, in particular a human subject, in particular a child.

[0236] The invention also concerns the recombinant MeV-rubella-mumps infectious replicating virus particles in association with rubella protein(s), or antigenic fragment(s) thereof, possibly associated rubella VLPs, or any composition according to the invention, for the use in the treatment or the prevention of an infection by rubella, measles and mumps virus in a subject, in particular a human subject, in particular a child.

[0237] In a particular embodiment of the invention, the composition or the use of the composition is able to elicit immunization of a subject, in particular a human subject, in particular a child, after a single injection. In other words, the composition or the use of the composition may require a single administration of a selected dose of the recombinant MeV-rubella or MeV-rubella-mumps or Mev- mumps infectious replicative virus. Alternatively, it may require multiple doses administration in a prime-boost regimen. Priming and boosting may be achieved with identical active ingredients consisting of recombinant MeV-rubella or MeV- rubella-mumps or Mev-mumps infectious, replicative virus and associated rubella protein(s) and / or mumps protein(s), or antigenic fragment(s) thereof, and / or rubella VLPs and / or mumps VLPs.

[0238] The invention also concerns an assembly of different active ingredients including as one of these ingredients recombinant MeV-rubella or MeV-rubella-mumps or Mev-mumps infectious, replicative virus and associated rubella protein(s) and / or mumps protein(s), and / or rubella VLPs and / or mumps VLPs. The assembly of active ingredients is advantageously for use in immunization of a host, in particular a human host.

[0239] It has been shown that administration of the active ingredients according to the invention elicits immunization of the host. The composition according to the invention is able to elicit production of rubella-specific immunoglobulins and mumps-specific immunoglobulins, especially IgG. The composition according to the invention is a safe vaccine, immunogenic and efficacious in a host. The compositions and their use confer at least T cell response and confer immunity against a rubella virus infection in a vaccinated host and / or against a measles virus infection in a vaccinated host and / or against a mumps virus infection in a vaccinated host.

[0240] The composition according to the invention may also be able to elicit production of MeV-specific immunoglobulins, especially IgM and IgG, and neutralizing antibodies. The composition according to the invention is a safe vaccine, immunogenic and efficacious in a host. The compositions and their use may confer at least T cell response and may confer immunity against a measles virus infection in a vaccinated host.

[0241] The invention also relates to a method for preventing a rubella virus infection, said method comprising the immunization of a mammalian, especially a human, in particular a child, by the injection, in particular by subcutaneous injection, of recombinant rubella virus VLPs as disclosed herein, and / or a recombinant measles virus as disclosed herein. The invention also relates to a method for treating a rubella virus infection, said method comprising the immunization of a mammalian, especially a human, in particular a child, by the injection, in particular subcutaneous injection, of recombinant rubella virus VLPs as disclosed herein, and / or a recombinant measles as disclosed herein.

[0242] DESCRIPTION OF THE FIGURES

[0243] Some of the figures, to which the present application refers, are in color. The application as filed contains the color print-out of the figures, which can therefore be accessed by inspection of the file of the application at the patent office.

[0244] Figure 1. Schematic diagram of recombinant measles virus (MV) with an insertion of rubella virus structural genes. MeV non-segmented negative strand RNA genome is shown (N, nucleoprotein; P, phosphoprotein; M, matrix protein; F, fusion protein; H, hemagglutinin; L, Large polymerase, accessory proteins C and V). T7, T7RNA polymerase promoter; hh, hammerhead ribozyme; 5, hepatitis delta virus (HDV) genome ribozyme; T7t, T7RNA polymerase terminator. The pTM-MVSchw vector contains the structural genes of rubella virus (RubV), Capsid, Envelope E1 and E2 inserted into the additional transcription unit 2 (ATU2). The additional gene encodes a total of 1063 amino acids in which the position 277-301 and 562-583 are signal peptide of E2 and E1 , respectively. The structural genes of RubV were either from RubV strain 1 E or 2B.

[0245] Figure 2. Growth comparison and expression of rubella structural proteins in rMV. Growth kinetics of rMV expressing the structural proteins of rubella virus strain 1 E (a), and 2B (b) comparing to that of MV-Schwarz. c) Western blot analysis of rubella virus proteins expression in Vero cells infected with rMV expressing the structural proteins of rubella virus strain 1 E or 2B. The capsid protein, E1 and E2 were detected at 36 kDa, 30 kDa and 50 kDa.

[0246] Figure 3. Immunofluorescence visualization of rubella virus capsid protein expression in Vero cells infected with rMV. Vero cells were infected with MV- Rub1 E or MV-Rub2B or empty MeV Schwarz. Rubella virus capsid protein was detected 24-hours after infection in saponin-permeabilized cells using mouse anti-Capsid antibody followed by Cy3-conjugated goat anti-mouse IgG (red). Measles virus nucleoprotein N was detected using rabbit anti-N antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Nuclei were stained with DAPI (blue). Images were acquired using fluorescence microscope; 50 x magnification (scale bar, 50 pm). The experiments were conducted using two or three biologically independent Vero cell batches.

[0247] Figure 4. Western blot detection of rubella virus capsid protein in supernatant of Vero cells infected with rMV after ultracentrifugation onto 20% sucrose cushion.

[0248] Figure 5. ELISA detection of antibody response specific to rubella virus in sera from mice prime-boost immunized with 105TCID50 of rMV-Rub1E or rMV-Rub2B. a) Antibodies specific to rubella virus antigen, b) Normalized value with MeV antibody level.

[0249] Figure 6. Schematic diagram of recombinant measles virus vector with additional mumps virus Hemagglutinin (HN) gene inserted in either ATU2 or ATU3. MeV non-segmented negative strand RNA genome is shown with the structural proteins (N, nucleoprotein; P, phosphoprotein; M, matrix protein; F, fusion protein; H, hemagglutinin; L, Large polymerase, and accessory proteins C and V. T7, T7RNA polymerase promoter; hh, hammerhead ribozyme; 5, hepatitis delta virus (HDV) ribozyme; T7t, T7RNA polymerase terminator. The pTM- MVSchw vector contains mumps virus HN gene inserted at either ATLI2 or ATLI3. Figure 7. Growth kinetics of rMV-mumps-HN and expression of HN protein, a) Growth kinetics of rMV expressing mumps virus HN compared to that of MV- Schwarz. b) Hemagglutination assay to detect hemagglutinin activity of the rMV- Mump-HN both in ATLI2 and ATLI3 compared to MeV Schwarz empty virus.

[0250] Figure 8. Humoral response in hCD46-IFNAR- / - mice after immunization of rMV-mumps-HN. a) Immunization schedule of recombinant MeV (rMV) expressing HN. b) Specific IgG to whole mumps virus antigen and c) to MeV antigen. Plaque Reduction Neutralizing Test titer of sera from the immunized mice to the mumps virus; d) PRNT50 and e) PRNT90.

[0251] Figure 9. Schematic diagram of recombinant measles virus (MV) with an insertion of rubella virus antigens (C-E2-E1) and mumps virus Hemagglutinin (HN) genes. MeV genome and its non-segment negative RNA; a) The vector (pTM-MVSchw) contains Rubella antigens (C-E2-E1 ) at the ATLI2 and HN gene of Mumps virus at the ATLI3. b) Growth kinetics of the rMV- expressing RuV and MeV antigen either with Ruv 1 E or 2B strain, c) Hemagglutinin activity of the rMV expressing either HN or both C-E2-E1 at ATLI2 and HN at ATLI3. d) Western blot analysis of the Vero cell lysates infected with rMVs probing with either anti-mumps viral antigen, anti-rubella capsid or anti-N measles.

[0252] Figure 10. Humoral response in IFNAR- / - mice after immunization with rMV- Rub-Mumps. a) Immunization schedule of recombinant MeV (rMV) expressing RuV (C-E2-E1 ) antigens and Mumps HN antigen, b) Specific IgG to MeV antigen and c) to RuV antigen, d) Focus Reduction Neutralizing Test titer to MuV of sera from the immunized mice.

[0253] Figure 11. Schematic representation of transfer vector plasmid according to a first embodiment of the invention. The transfer vector has the sequence of SEQ ID No. 1 . It corresponds to the vector referenced pTM2-Rub1 E. Antigens from rubella virus are encoded within the MeV genome.

[0254] Figure 12. Schematic representation of transfer vector plasmid according to a first embodiment of the invention. The transfer vector has the sequence of SEQ ID No. 2. It corresponds to the vector referenced pTM2-Rub2B. Antigens from rubella virus are encoded within the MeV genome.

[0255] Figure 13. Schematic representation of transfer vector plasmid according to a first embodiment of the invention. The transfer vector has the sequence of SEQ ID No. 17. It corresponds to the vector referenced pTM2-mumps-HN. Antigens from mumps virus are encoded within the MeV genome.

[0256] Figure 14. Schematic representation of transfer vector plasmid according to a first embodiment of the invention. The transfer vector has the sequence of SEQ ID No. 18. It corresponds to the vector referenced pTM3-mumps-HN. Antigens from mumps virus are encoded within the MeV genome.

[0257] Figure 15. Schematic representation of transfer vector plasmid according to a first embodiment of the invention. The transfer vector has the sequence of SEQ ID No. 15. It corresponds to the vector referenced pSMAC-Rub1 E- mumps-HN. Antigens from rubella virus and mumps virus are encoded within the MeV genome. Figure 16. Schematic representation of transfer vector plasmid according to a first embodiment of the invention. The transfer vector has the sequence of SEQ ID No. 16. It corresponds to the vector referenced pSMAC-Rub1 B- mumps-HN. Antigens from rubella virus and mumps virus are encoded within the MeV genome.

[0258] Figure 17. Immune response in mice after immunization with rMV-Rub- Mumps. a) Immunization schedule of recombinant MeV (rMV) expressing RuV (C-E2-E1 ) antigens and Mumps HN antigen, b) ELISA of RubV after administration of 103particles of rMV-Rub-Mumps. c) ELISA of RubV after administration of 104particles of rMV-Rub-Mumps. d) ELISA of MV after administration of 103particles of rMV-Rub-Mumps. e) ELISA of MV after administration of 104particles of rMV-Rub-Mumps. Grey round: MV-Schwarz (control that does not express any Rubella or mumps antigens). Blue squares: MV-RubV that express both rubella and mumps antigens. Purple triangles: Priorix® vaccine (live attenuated viruses). ELISA are performed at day 0, and 28 and 42 days after prime immunization.

[0259] EXAMPLES

[0260] Materials and methods

[0261] Construction of vector plasmids

[0262] The sequences corresponding to the structural proteins of rubella virus, including the capsid and envelope genes (C-E2-E1 ) (positions 6512-9700 in the gene), were codon-optimized for expression in mammalian cells and synthesized to respect the rule of six for measles virus (Calain and Roux 1993). The rubella virus genotypes I (strain 1 E, accession number: KT962864), and II (strain 2B, accession number: MN786647), were chosen as these are they are currently the most prevalent in the world distribution of rubella virus. The synthetic sequences were cloned into additional transcription unit 2 (ATU2) of pTM-MVSchwarz vector (Combredet et al. 2003) between BsiWI and BssHII restriction sites.

[0263] Similarly, the Hemagglutinin gene (HN) of mumps virus was synthesized as a codon-optimized mosaic sequence mixing genotypes G and C, the most prevalent circulating strains. While genotype G is found in North America and Europe, genotype C is predominantly found in China. The synthetic sequence was cloned into either ATLI2 or ATLI3 of pTM-MVSchwarz vector between BsiWI and BssHII restriction sites.

[0264] To construct the vector plasmid expressing both rubella and mumps viruses’ antigens, the rubella virus C-E2-E1 gene was inserted into ATLI2, and the mumps virus HN gene into ATLI3 of pSMAC-MVSchwarz vector plasmid (PCT / EP2021 / 069070).

[0265] Virus Rescue, Propagation, and Titration

[0266] Rescue of the recombinant measles (rMV) was performed using a helper-cell- based system. Briefly, Helper HEK293-T7-NP cells were transfected with 3 pg of the plasmid either pTM-Rub1 E, pTM-Rub2B, pTM2-HN, pTM3-HN, or with 4 pg of pSMAC-Rub1 E-HN, pSMAC-Rub2B-HN and 0.02 pg of pEMC-La expressing the MeV polymerase L gene. After overnight incubation at 37°C, the transfection medium was replaced by fresh medium, and a heat shock was applied for 3 h at 42°C, then returned to 37°C. After two days of incubation at 37°C, transfected cells were transferred to 100-mm dishes with monolayers of Vero-NK cells (ATCC, CCL-81 ). Syncytia that appeared after 2-3 days of co-culture were singly picked and transferred onto Vero cells seeded in 6-well plates. Infected cells were trypsinized and expanded in 75-cm2and then 150-cm2flasks, in DMEM with 5% FBS. To collect viruses, cells were scraped into a small volume of OptiMEM (Thermo Fisher), lysed by a single freeze-thaw cycle, and cell lysates clarified by low-speed centrifugation. The infectious supernatant was then collected and stored at -80°C. Titers of rMVs were determined on Vero cells seeded in 96-well plates infected with serial ten-fold dilutions of virus in DMEM with 5% FBS. After incubation for 7 days, cells were stained with crystal violet, and TCID50 values were calculated using the Karber method.

[0267] Detection of additional antigens expression by rMVs

[0268] To assess the expression of additional antigens by the rMV vectors, Vero cells were infected by each rMV and total RNA was extracted from the infected cells using the RNeasy Mini Kit (Qiagen). The cDNA synthesis and PCR steps were performed using the RNA LA PCR kit (Takara Bio) with primers targeting ATLI2 and ATLI3, according to the manufacturer’s instructions. RT-PCR products were analyzed by Sanger sequencing (Eurofins Genomics).

[0269] To detect antigen expression at the protein level, Vero cells in 6-well plates were infected with various rMVs at an MOI of 0.1 . At 36-48 h post-infection, infected cells were lysed in RIPA lysis buffer (Thermo Fisher). Samples were briefly centrifuged and subjected to 4-12% gradient SDS-PAGE gel (Invitrogen). After transfer to a nitrocellulose membrane (GE Healthcare), the membranes were probed with mouse monoclonal anti-RubV capsid protein (9B11 Santa Cruz sc- 65935) at a 1 : 10000 dilution, anti-gpE1 RubV (1 .B.3. Santa Cruz sc-58013) at a 1 :1000 dilution, and anti-gpE2 RubV (D92G Invitrogen MA5-18255) at a 1 :2000 dilution. For mumps antigen detection, a rabbit polyclonal anti-mumps virus (Enders Alpha Diagnostic International, MUMS11 -S) was used at a 1 :1000 dilution. The membranes were then washed and incubated with either horseradish peroxidase (HRP)-conjugated anti-mouse IgG (NA931V, GE Healthcare), at a 1 :10000 dilution or HRP-conjugated swine anti-rabbit IgG antibody (P0399, Dako) at a 1 :3000 dilution. Bands were visualized using the SuperSignal West Pico Plus chemiluminescent HRP substrate (Thermo Fisher). For loading controls, membranes were stripped with 5% NaOH for 5 mins and reprobed with a mouse monoclonal anti-MV-N antibody (ab9397, Abeam) at a 1 :20000 dilution followed by an HRP-conjugated anti-mouse IgG (NA931V, GE Healthcare) at a 1 :10000 dilution.

[0270] To assess the mumps HN protein expression, a Sheep Hemagglutination kit (Rockland KPA-3913) was used. Briefly, 5 mL (normalized to 106TCIDso / mL) of rMV-HN were layered onto a 20% sucrose cushion in PBS and ultracentrifuged at 160,000 x g for 3h in a SW41 rotor. Pellets were resuspended in PBS with protease inhibitors (Roche) and mixed with 50 pL of serial dilutions with 50 pL of 0.5% sheep red blood cell (RBC) stock solution (prepared according to the manufacturer's instruction). The plates were incubated at room temperature for 90 min, and the hemagglutination reaction was observed comparing to the positive control of serial dilution of rabbit anti-sheep RBC in the range of 800 pg / mL to 25 pg / mL.

[0271] Immunofluorescence Assay This assay was performed with rMV-RubV only. Vero cells were infected with various rMVs at an MOI of 0.1. At 24-36 h post-infection, cells were fixed with 4% paraformaldehyde, blocked with 2% goat serum overnight, and then treated with or without 0.1 % saponin A (Sigma). Fixed cells were probed with a mouse monoclonal anti-gpE1 RubV (1.B.3. from Santa Cruz, sc-58013) at a 1 :1000 dilution, followed by Cy3-conjugated goat anti-rabbit (A10520, Jackson ImmunoResearch) at a 1 :1000 dilution. Staining for measles virus nucleoprotein was performed using a rabbit polyclonal anti-MV-N (Covalab, pab0035.1 ) at a 1 :1000 dilution followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (A- 11008, Thermo Fisher) to detect MeV antigen in infected cells. Nuclei were stained with DAPI. Images were collected using an inverted Leica DM IRB fluorescence microscope with a 20x objective.

[0272] Mice Immunizations

[0273] Transgenic mice expressing the human CD46 receptor and deficient for the type- I IFN receptor (IFNAR’ / _) are susceptible to MeV infection and were used for immunization studies. We used 129sv IFNAR / _mice that share the same MHC haplotype as the classical C57BL / 6 mice: MHC-I (H-2Kb / H-2Db) and MHC-II (I- Ab). To evaluate the immunogenicity of single antigen-added rMVs (rMV-Rub1 E, rMV-Rub2B, and rMV-HN-mumps either ATLI2 or ATLI3), groups of 6 to 8-week- old mice were intraperitoneally (IP) injected with 105TCID50 of rMV. Control animals received the empty MeV Schwarz or the commercial MMR vaccine, Priorix®. Two immunizations were performed at a four-week interval. Sera were collected before the first immunization (day -1 ), then before (day 28), and after (day 42) the second immunization. All serum samples were heat-inactivated for 30 min at 56°C.

[0274] ELISA

[0275] Edmonston strain-derived MeV antigens (Jena Bioscience) or Jeryl Lynn strain- derived mumps virus antigen (antibody.com ref A33335) were coated on NUNC MAXISORP 96-well immuno-plates (Thermo Fisher) at 1 pg / ml. For rubella virus, the anti-rubella ELISA IgG test kit (Euroimmun) was used. Coated or ELISA kit plates were incubated overnight at 4°C. After washing and blocking, sera from immunized mice were serially diluted in binding buffer and incubated on plates for 1 h at 37°C. After washing steps, an HRP-conjugated goat anti-mouse IgG (H+L) antibody (Jackson ImmunoResearch, 115-035-146 was added at a 1 :5000 dilution for 1 h at 37°C. Antibody binding was detected by the addition of the TMB substrate (Eurobio), and the reaction was stopped with 100 pL of 30% H2SO4. Optical densities were recorded at 450 and 620 nm wavelengths using the EnSpire 2300 Multilabel Plate Reader (Perkin Elmer). Endpoint titers for each individual serum sample were calculated as the reciprocal of the last dilution giving twice the absorbance of the negative control sera.

[0276] Plaque reduction neutralization test (PRNT) for mumps virus

[0277] Two-fold serial dilutions of heat-inactivated serum samples were incubated at 37°C for 1 h with 50 PFU of mumps virus (llrabe strain, reference code: 90 / 534, NIBSC) in DMEM medium without FBS and added to a monolayer of Vero cells seeded in 24-well plates. The virus was allowed to adsorb for 2 h at 37°C. The supernatant was removed, and the cells were overlaid with 500 pL of plaque assay overlay media (DMEM supplemented with 5% FBS and 1.5% carboxymethylcellulose). Plates were incubated at 37°C with 5% CO2 for 3 days. The virus was inactivated, cells fixed and stained with a 30% crystal violet solution containing 20% ethanol and 10% formaldehyde (Sigma). Serum neutralization titers were determined based on the dilution that reduced mumps virus plaques by 50% (PRNT50) or by 90% (PRNT90).

[0278] Focus reduction neutralization test (FRNT)

[0279] One hundred microliters of two-fold serial dilutions of heat-inactivated serum samples from immunized mice were added to an equal volume of 50 PFU of mumps virus in wells of a 96-well round bottom plate and incubated at 37°C with 5% CO2 for 1 h. The virus control was performed similarly but using PBS instead of serum. After incubation, 100 pL of each reaction mix was added in triplicate to a 96-well plate containing a Vero cell monolayer, and virus adsorption was performed at 37°C with 5% CO2 for 2-3 h. The virus inoculum was then gently removed by aspiration, and the wells were overlaid with 150 pL / well of DMEM supplemented with 5% FBS and 1.5% carboxymethylcellulose. The plates were incubated at 37°C with 5% CO2 for a further 2-3 days for mumps and measles viruses, while the plates for rubella virus were incubated for 10 days. The viral foci were detected using a modified immuno-colorimetric procedure previously described for measles and rubella plaque detection. Briefly, the media was removed by careful aspiration, then cells were fixed with 100 pL / well of 4% formaldehyde in PBS for 30 min. After washing with PBS, cells were permeabilized with 100 pL / well of 95% ethanol. Following two washes with PBS, cells were blocked with 200 pL / well blocking buffer (1 % BSA, 0.5% FBS, and 0.1 % Tween-20 in PBS) for 30 min at room temperature. The blocking buffer was then removed, and 100 pL / well of anti-measles nucleoprotein monoclonal antibody diluted 1 :2000 (Clone 3E1 , Abeam) in blocking buffer was added for 1 h at room temperature. After washing three times with PBST (PBS plus 0.1 % Tween-20) buffer, 100 pL / well of a 1 :2000 dilution of goat anti-mouse IgG horse radish peroxidase-labelled antibody (Molecular Probes, Eugene, OR, USA) was added and incubated for 1 h at room temperature. Plates were washed with PBST, and residual liquid was removed by tapping on a paper towel. Then, 100 pL / well of TMB stabilized substrate (Promega) was added for 30 min at room temperature. The substrate solution was then removed; plates were blotted and air-dried. The blue viral foci were counted based on the dilution that reduced virus plaques by 50% (PRNTso) or by 90% (PRNT90).

[0280] Results

[0281] Generation of recombinant measles vectors expressing rubella virus antigens (MR)

[0282] Recombinant measles viruses expressing rubella virus antigens (rMVs-Rub) were generated from the pTM2-Rub1 E or pTM2-Rub2B vector plasmids. These plasmids contain the capsid (C) gene and envelope genes, E2 and E1 , from either rubella virus strain 1 E or 2B, which were inserted into the measles genome at the additional transcription unit 2 (ATU2) (Fig.1 ). Following successful rescue, these rMVs demonstrated growth kinetics similar to the MV-Schwarz strain, though with a slight initial delay in the lag phase (Fig.2a, b). The maximum titer for both viruses reached over 5 x 106TCIDso / mL, indicating a titer level that is both relatively high and sufficient for production of a live-replicative vaccine normally administered at doses of 103-104TCID50. The expression of rubella virus antigens, specifically the capsid (C) and envelope proteins E2 and E1 , was detected in the lysates of infected Vero cells via Western blot analysis (Fig.2c). Additionally, immunofluorescence assays demonstrated the expression of the rubella virus C protein in Vero cells infected with either rMV- Rub1 E or Rub2B (Fig.3). These three structural proteins of rubella virus typically auto-assemble into virus-like particles (VLPs). To investigate the secretion of these proteins in the form of VLPs, the supernatant medium of infected Vero cells was subjected to ultracentrifugation through a 20% sucrose cushion. Western blot analysis of the resulting pellet demonstrated the presence of rubella virus capsid protein (Fig.4), indicating protein secretion in the form of high density VLPs. The assembly of these proteins into VLPs will be confirmed by electron microscopy.

[0283] Immunogenicity of rMV-Rub1 E and MV-Rub2B in mice

[0284] To evaluate the immunogenicity of these recombinant vectors, genetically modified mice expressing hCD46 (the human MeV receptor) and lacking the INF- I receptor (CD46-IFNAR’ / ’) were immunized with 105TCID50 of MV-Rub1 E or MV- Rub2B. Following a prime-boost regimen, the rMV-Rub viruses elicited high levels of specific anti-rubella virus IgG, with ELISA titers approximately reaching 106, a log higher titer than that elicited by the commercial vaccine, Priorix®, (Fig.5a and b). It is important to note that Priorix® contains a total live attenuated MV, RuV and MuV dose of approximately 104CCID50. This difference in dosing likely accounts for the observed one-log higher IgG titer. Regardless, these first results demonstrate that rMV-Rub vectors elicited a robust humoral response specific to the RuV.

[0285] Generation of recombinant measles vectors expressing mumps virus hemagglutinin (MM)

[0286] A mosaic mumps virus hemagglutinin (HN) gene composed of genotypes G and C prevalent in Europe and Asia, was designed, synthesized, and cloned into ATU2 or ATU3 of the measles plasmid vector pTM (Fig.6). Following the successful rescue of the corresponding rMV-HN virus, we observed the growth kinetics of both rMV-ATU2-HN and rMV-ATU3-HN, which reached titers of 106 TCIDso / mL. The rMV-ATU3-HN showed a slight delay in the lag phase compared to the control MeV (Fig.7a).

[0287] Due to closely related genomes of mumps and measles paramyxoviruses, the commercial antibodies for mumps virus detection cross-react with measles virus antigens (data not shown). Therefore, hemagglutination assay was used as alternative method to detect mumps virus HN expression in rMV-infected cells. Hemagglutination activity was measured on ultracentrifuged rMV-HN in comparison to empty MV. The results indicated that both rMV-ATU2 and ATLI3 exhibit 2-4 times higher hemagglutinin activity than that of the empty MV, particularly when HN was expressed from ATLI3 (Fig.7b).

[0288] Immunogenicity of rMV-HN in mice

[0289] CD46-IFNAR’ / ’ mice were immunized with 105TCID50 of rMV-HN in a prime-boost regimen and compared to the commercial Priorix® vaccine (Fig.8a). The antibody responses elicited against measles virus was similar (Fig.8c). To detect the specific HN antibody response in mice sera, we used a mumps virus ELISA in which the plates were coated with whole mumps virus antigens (Fig.8b). This assay detected a higher response after immunization with the commercial Priorix® vaccine as compared to rMV-HN. This observation highlights the difference between the rMV-HN that contains only the HN protein while the commercial vaccine contains the whole virus. Thus, to assess the efficacy of rMV- HN, we measured anti-mumps virus neutralizing antibody titers using a plaque reduction neutralization test (PRNT) on Vero cells (Fig.8d and e). Post primeboost immunization, both rMV-ATU2-HN and rMV-ATU3-HN induced significantly higher neutralization titers (3 to 10 times higher PRNT50 and PRNT90) compared to Priorix®. Control mice immunized with empty MeV vector produced some IgG non-specifically recognizing mumps virus (Fig.8b) but they were not neutralizing. Interestingly, the neutralizing antibodies elicited by rMVs persisted.

[0290] Generation of recombinant measles vector expressing both rubella and mumps antigens in a single virus (MMR)

[0291] To design a triple MMR vaccine candidate using the MeV Schwarz vector, we need to choose in which ATU rubella and mumps antigens should be cloned. Given our previous observation that rMV-ATU3-HN showed enhanced HN expression and elicited higher neutralizing antibodies against mumps virus compared to rMV-ATU2-HN, we cloned the rubella virus antigens C-E2-E1 gene into ATLI2 and the mumps virus HN gene into ATLI3 to achieve optimal expression of the antigens (Fig.9a). Following successful virus rescue, the recombinant viruses (rMV-Rub1 E-mumps-HN and rMV-Rub2B-mumps-HN) were tested for their growth kinetics compared to the control MeV Schwarz. These recombinant viruses, containing both rubella virus C-E2-E1 and mumps virus HN antigens, exhibited slower growth during the lag phase but achieved similar titers of 106-107TCIDso / mL (Fig.9b). The expression of the rubella virus capsid protein was detected by Western blot analysis of infected Vero cells lysates (Fig.9c) and the hemagglutinin activity test confirmed the expression of HN (Fig.9d)

[0292] Immunogenicity of rMV-Rub-Mumps in mice

[0293] CD46-IFNAR’ / ’ mice were immunized with the bivalent rMV-Rub-Mumps virus. To ensure comparability with the commercial MMR vaccine, Priorix®, mice were immunized with rMV-Rub-Mumps at a similar titer of 104TCID50. Priorix® contains 103CCID50 of Schwarz measles, at least 1 O3 7CCIDso of RIT4385 mumps, and no less than 103CCID50 of Wistar RA 27 / 3 rubella virus strains, totaling approximately 104CCID50, which aligns with the administered dose of our rMV- Rub-Mumps recombinant vaccine. The control rMV-ATU2 / 3-HN was used at a titer of 105TCIDso / mL for comparison with prior immunization data. After primeboost immunization, the rMV-mumps yielded results consistent with previous results (Fig.10b). The result shown on Fig. 10c was done with ELISA plates coated with 1 pg / mL of each of the three proteins (C, E2, and E1 ), which rendered lower overall IgG titer compared to commercial rubella ELISA kit. Our previous results indicated that the bivalent rMV elicited significantly higher specific rubella virus IgG than the commercial MMR vaccine, approximately one log higher at the similar immunization titer. Interestingly, immunization with the recombinant rMVs elicited higher neutralizing antibody titers against mumps virus (Fig.10c). Furthermore, both bivalent rMVs induced mumps neutralizing antibodies at 103titers, relatively at the same levels elicited by the commercial MMR, Priorix®, after prime and 3-fold higher after boost (Fig.lOd). The efficiency of the construct at low doses is illustrated on Fig. 17A-E. These figures demonstrate the effectiveness of the vaccine candidate. Although the standard immunization dose in mice is 105TCID50, the dose was reduced to 103and 104TCID50 in order to assess the immunogenic potential at lower levels. As shown in Fig. 17B-E, even at these reduced doses, the vaccine candidate elicited a measurable immune response specifically against measles and rubella. The antibody titers observed at a serum dilution of 105confirm that the immune response remains robust even at a dose as low as 103TCID50. These results are provided to illustrate the efficiency of the vaccine construct at low doses, particularly in comparison with prior art platforms that require higher doses to achieve similar immunogenicity.

[0294] In addition, Western blot analysis confirms that antigen expression is clearly detectable in cells infected with the vaccine candidate, supporting the conclusion that the construct enables efficient antigen production and a consistent immune response, even at reduced dosing levels.

[0295] BIBLIOGRAPHIC REFERENCES

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Claims

CLAIMS1. A nucleic acid construct which comprises:(1 ) a cDNA molecule encoding a full length antigenomic (+) RNA strand of a measles virus (MeV);(2) a first heterologous polynucleotide or a plurality of first heterologous polynucleotides encoding at least the following rubella virus structural proteins: Capsid (C), Envelope 2 (E2) and Envelope 1 (E1 ), or antigenic fragments thereof; and wherein the first heterologous polynucleotide or the plurality of first heterologous polynucleotides is (are) operatively cloned within an additional transcription unit (ATU) inserted within the cDNA of the antigenomic (+) RNA, in particular an ATU localized between the P gene and the M gene of the MeV; and,(3) a second heterologous polynucleotide encoding at least the mumps virus hemagglutinin-neuraminidase protein (HN), or antigenic fragment thereof, the second heterologous polynucleotide being operatively cloned within an ATU at a location distinct from the location of the first heterologous polynucleotide or plurality of first heterologous polynucleotides, in particular an ATU localized between the H gene and the L gene of the MeV.

2. The nucleic acid construct according to claim 1 , wherein the first heterologous polynucleotide or the plurality of first heterologous polynucleotides comprise(s) the E2 peptide signal, in particular upstream of a sequence encoding the E2 protein or the antigenic fragment thereof, and / or the E1 peptide signal, in particular upstream of a sequence encoding the E1 protein or the antigenic fragment thereof.

3. The nucleic acid construct according to claim 1 or 2, wherein the rubella structural proteins C, E2 and E1 are encoded in this order from the 5’ end towards the 3’ end of the first heterologous polynucleotide or from the 5’end towards the 3’ end of the plurality of first heterologous polynucleotides put end to end.

4. The nucleic acid construct according to any one of claims 1 to 3, wherein the first heterologous polynucleotide or the plurality of first heterologous polynucleotides is(are) originate or are derived from the rubella strain 1 E or 2B.

5. The nucleic acid construct according to any one of claims 1 to 4, wherein the first heterologous polynucleotide or the plurality of first heterologous polynucleotides encode(s) the rubella protein C comprising or consisting of the amino acid sequence set forth in SEQ ID No. 3 or SEQ ID No. 23, or an antigenic fragment thereof, and / or the rubella protein E2 comprising or consisting of the amino acid sequence set forth in SEQ ID No. 5 or SEQ ID No. 25, or an antigenic fragment thereof, and / or the rubella protein E1 comprising or consisting of the amino acid sequence set forth in SEQ ID No. 7 or SEQ ID No. 27, or an antigenic fragment thereof, and optionally further encode(s) the E2 peptide signal comprising or consisting of the amino acid sequence set forth in SEQ ID No. 9 or SEQ ID No. 29, and / or the E1 peptide signal comprising or consisting of the amino acid sequence set forth in SEQ ID No. 11 or SEQ ID No. 31 .

6. The nucleic acid construct according to any one of claims 1 to 5, wherein the first heterologous polynucleotide or the plurality of first heterologous polynucleotides has(have) codon-optimized open reading frame(s) (ORF) and comprise(s) at least the following sequences:- SEQ ID No. 4 or SEQ ID No. 24 which encodes the rubella C protein, and / or- SEQ ID No. 6 or SEQ ID No. 26 which encodes the rubella protein E2, and / or- SEQ ID No. 8 or SEQ ID No. 28 which encodes the rubella protein E1 , or- SEQ ID No. 34 or SEQ ID No. 36 which encodes the polyprotein comprising the rubella proteins C protein, E2 and E1. and optionally further comprise(s):- SEQ ID No. 10 or SEQ ID No. 30 which encodes the E2 peptide signal, and / or- SEQ ID No. 12 or SEQ ID No. 32 which encodes the E1 peptide signal.

7. The nucleic acid construct according to any one of claims 1 to 6 comprising from 5’ to 3’ end the following polynucleotides:(a) a polynucleotide encoding the N protein of the MeV;(b) a polynucleotide encoding the P protein of the MeV;(c) the first heterologous polynucleotide or a plurality of first heterologous polynucleotides encoding at least the following rubella virus structural proteins: Capsid (C), Envelope 2 (E2) and Envelope 1 (E1 ), or antigenic fragments thereof; and optionally comprise(s) the E2 peptide signal, in particular upstream of a sequence encoding the E2 protein or the antigenic fragment thereof, and / or the E1 peptide signal, in particular upstream of a sequence encoding the E1 protein or the antigenic fragment thereof;(d) a polynucleotide encoding the M protein of the MeV;(e) a polynucleotide encoding the F protein of the MeV;(f) a polynucleotide encoding the H protein of the MeV;(g) a polynucleotide encoding the L protein of the MeV; and wherein said polynucleotides are operatively linked within the nucleic acid construct and are in particular under the control of the viral replication and transcriptional regulatory elements of the MeV, more particularly the leader and trailer sequence(s).

8. The nucleic acid construct according to any one of claims 1 to 7, wherein the second heterologous polynucleotide encodes the mumps virus HN protein comprising or consisting of the amino acid sequence set forth in SEQ ID No. 13.

9. The nucleic acid construct according to any one of claims 1 to 8, wherein the second heterologous polynucleotide has codon-optimized open reading frame (ORF) and comprises at least the nucleotide sequence set forth in SEQ ID No. 14 which encodes the mumps virus HN protein.

10. The nucleic acid construct according to any one of claims 1 to 9 comprising from 5’ to 3’ end the following polynucleotides:(a) a polynucleotide encoding the N protein of the MeV;(b) a polynucleotide encoding the P protein of the MeV;(c) the first heterologous polynucleotide or a plurality of first heterologous polynucleotides encoding at least the following rubella virus structural proteins: Capsid (C), Envelope 2 (E2) and Envelope 1 (E1 ), or antigenic fragments thereof, and optionally comprise(s) the E2 peptide signal, in particular upstream of a sequence encoding the E2 protein or the antigenic fragment thereof, and / or the E1 peptide signal, in particular upstream of a sequence encoding the E1 protein or the antigenic fragment thereof;(d) a polynucleotide encoding the M protein of the MeV;(e) a polynucleotide encoding the F protein of the MeV;(f) a polynucleotide encoding the H protein of the MeV;(g) the second heterologous polynucleotide encoding at least the mumps virus HN protein, or antigenic fragment thereof;(h) a polynucleotide encoding the L protein of the MeV; and wherein said polynucleotides are operatively linked within the nucleic acid construct and are in particular under the control of the viral replication and transcriptional regulatory elements of the MeV, more particularly the leader and trailer sequence(s).

11. The nucleic acid construct according to any one of claims 1 to 10, wherein the sequence of the first heterologous polynucleotide or of the plurality of first heterologous polynucleotides comprise(s) at least SEQ ID No. 4 or No. 24, SEQ ID No. 6 or No. 26, SEQ ID No. 8 or No. 28, and optionally SEQ ID No. 10 or No. 30, and / or SEQ ID No. 12 or No. 32.

12. The nucleic acid construct according to any one of claims 1 to 11 , wherein the measles virus is an attenuated virus strain selected from the group consisting of the Schwarz strain, the Zagreb strain, the AIK-C strain, the Moraten strain, the Philips strain, the Beckenham 4A strain, the Beckenham 16 strain, the Edmonston seed A strain, the Edmonston seed B strain, the CAM-70 strain, the TD 97 strain, the Leningrad-16 strain, the Shanghai 191 strain and the Belgrade strain, in particular the Schwarz strain.

13. The nucleic acid construct according to any one of claims 1 to 12, whose recombinant cDNA sequence is SEQ ID No. 1 , SEQ ID No. 2, SEQ ID No. 15 or SEQ ID No. 16.

14. A recombinant measles virus, said virus comprising in its genome a nucleic acid construct according to any one of claims 1 to 12, or whose genome consists of nucleic acid construct of claim 13.

15. The recombinant measles virus according to claim 14 expressing at least the rubella proteins C, E1 and E2, or antigenic fragments thereof, and mumps virus HN, or antigenic fragment thereof.

16. A host cell transfected with the nucleic acid construct according to any one of claims 1 to 13 or infected with the recombinant measles virus according to claim 14 or 15, in particular a mammalian cell, a VERO NK cells, CEF cells, human embryonic kidney cell line 293 or MRC5 cells.

17. Recombinant virus like particles (VLPs) comprising the rubella virus proteins selected from the group consisting of C, E2 and / or E1 , or antigenic fragments thereof, and mumps virus HN, or antigenic fragment thereof, wherein the proteins or antigenic fragments thereof are encoded by the first and second heterologous polynucleotides of the nucleic acidconstruct according to claims 1 to 13, or the recombinant measles virus according to claim 14 or 15, or produced within the host cell of claim 16.18.An immunogenic composition, especially a virus vaccine composition, comprising the recombinant VLPs according to claim 17, or the recombinant measles virus according to claim 14 or 15, or the recombinant VLPs according to claim 17 and the recombinant measles virus according to claim 14 or 15, and a pharmaceutically acceptable vehicle.

19. The composition according to claim 18 for use in the elicitation of a protective, and preferentially prophylactic, immune response against the rubella virus by the elicitation of antibodies directed against rubella protein(s) C, E2 and / or E1 , or antigenic fragment(s) thereof, and / or a cellular and / or humoral and cellular response against the rubella virus, in a host in need thereof, in particular a human host, in particular a child.

20. The composition according to claim 18 for use in the elicitation of a protective, and preferentially prophylactic, immune response against the mumps virus by the elicitation of antibodies directed against mumps protein HC, or antigenic fragment(s) thereof, and / or a cellular and / or humoral and cellular response against the mumps virus, in a host in need thereof, in particular a human host, in particular a child.

21. The composition according to claim 18, for use in the elicitation of a protective, and preferentially prophylactic, immune response against:- the rubella virus by the elicitation of antibodies directed against rubella protein(s) C, E2 and / or E1 , or antigenic fragment(s) thereof, and / or a cellular and / or humoral and cellular response against the rubella virus- against mumps protein HC, or antigenic fragment(s) thereof, and / or a cellular and / or humoral and cellular response against the mumps virus, in a host in need thereof, in particular a human host, in particular a child.

22. The composition of any one of claims 18 to 21 for use in the elicitation of a protective, and preferentially prophylactic, immune response against measles virus by the elicitation of antibodies directed against measles virus protein(s), and / or a cellular and / or humoral and cellular response against the measles virus, in a host in need thereof, in particular a human host, in particular a child.

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