Non-structural antigens expressing flaviviruses selected from group consisting of DENV, ZIKV and YFV, polynucleotides and lentiviral vectors for inducing immunization of protective CD8 + T cells in hosts

Abstract

The present invention relates to a recombinant polynucleotide encoding at least one recombinant polynucleotide expressing at least a first fusion polypeptide comprising an MHC class I T cell epitope adapted to elicit a T cell immune response in a host in need thereof wherein the MHC class I T cell epitope is derived from a plurality of antigens, wherein the antigen comprises at least a non-structural antigen and is from at least one flavivirus selected from the group consisting of dengue virus (DENV), Zika virus (ZIKV) and yellow fever virus (YFV). The invention also relates to a polypeptide comprising multiple epitopes of the antigen encoded by the recombinant polynucleotide.

Description

Technical Field

[0001] The present invention relates to recombinant polynucleotides encoding at least one recombinant polynucleotide expressing at least a first fusion polypeptide comprising an MHC class I T cell epitope adapted to elicit a T cell immune response in a host in need thereof, wherein the MHC class I T cell epitope is derived from a plurality of antigens, wherein the antigen comprises at least a non-structural antigen and is a flavivirus from at least one selected from dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV). The present invention also relates to polypeptides comprising multi-epitopes of the antigen encoded by the recombinant polynucleotide.

[0002] In another aspect, the present invention relates to a lentiviral vector designed to provide an immune response for resistance to infection or to the onset or development of a disorder or disease associated with infection by a flavivirus selected from dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV), particularly by inducing a CD8+ T cell response.

[0003] In particular, the present invention relates to such a lentiviral vector expressing one or more fusion polypeptides selected for their ability to elicit an immune response in a host in need thereof, particularly a mammalian host, particularly a human host, wherein the immune response encompasses a specific CD8+ T cell response. The one or more fusion polypeptides may be expressed in the lentiviral backbone of the vector as one or more neoantigens from one insert or multiple inserts, wherein the one or more inserts contain at least one polynucleotide encoding one fusion polypeptide or multiple fusion polypeptides or consist thereof, each fusion polypeptide comprising a selected MHC class I T cell epitope derived from a plurality of antigens of a defined virus selected from dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV). The neoantigen is produced by the fusion of the expression products of polynucleotide regions recombined or assembled from different genes of the virus.

[0004] Providing or expressing the recombinant polynucleotide, lentiviral vector, or fusion polypeptide of the present invention is for designing an immune composition suitable for a mammalian host, particularly a human host, preferably a vaccine candidate, particularly a vaccine, particularly a prophylactic vaccine. Background Art

[0005] Dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV) are common mosquito-borne viral pathogens. DENV causes 390 million infections annually, mainly in tropical and subtropical regions. Although most cases are asymptomatic, 96 million cases still present clinically each year, including 2 million severe cases: hemorrhagic dengue and dengue shock syndrome, which result in 12,500 deaths annually (1, 2).

[0006] Despite decades of research and vaccine development, the development of an effective vaccine against DENV has been an especially challenging task. The failure to develop such a vaccine can be explained by the specific epidemiology of DENV: the disease is caused by at least four different serotypes (DENV-1 to DENV-4) that co-circulate in endemic areas. Primary infection with one DENV serotype usually elicits durable immunity against that serotype, but immunity against other serotypes is shorter-lived, lasting only a few months. Because of the antigenic differences between DENV serotypes, reinfection with a different serotype after this period usually results in more severe disease. More particularly, antibodies produced in response to primary infection do not effectively neutralize DENV of different serotypes but instead bind to virus particles, increasing the uptake of the virus particles by antigen-presenting cells (the primary target cells of DENV in the human organism). This phenomenon, known as antibody-dependent enhancement (ADE), significantly increases the replication of DENV in the body, leading to severe disease. Because of the understanding of the role that antibody responses may play in dengue pathogenesis, DENV vaccine development has focused on trying to develop a vaccine that can simultaneously induce an immune response against the 4 DENV serotypes. To date, only one such vaccine (CYD-TDV, Sanofi Pasteur) has been developed, but its widespread use is hampered by several important drawbacks: the vaccine provides weak protection against some DENV serotypes and also causes an increased risk of infection in individuals who have not previously been infected with DENV (3). Other candidate vaccines that are currently in advanced clinical trials are all live attenuated vaccines (LAVs), and thus there are known problems associated with production and safety aspects of this approach. Another approach aimed at circumventing the problems presented by antibody responses in DENV pathogenesis is to focus on other elements of the immune response, namely, T cell responses. Although initially suspected of playing a harmful role in DENV disease via a mechanism known as "original antigenic sin," recent studies have shown that T cell responses are largely beneficial and can induce cross-serotype specific protection against DENV of different serotypes (2, 4, 5). Responses of CD8+ T cells appear to be particularly important for the control of DENV infection in mice and humans and have been shown to mainly target epitopes located in the conserved non-structural proteins NS3, NS5, and NS4B of DENV (5-9). T cell responses (particularly responses of cytotoxic CD8+ cells) have been shown to protect mice from ADE and reduce the severity of heterotypic DENV infection (10, 11). These results suggest that it may be possible to produce a pure "T cell" vaccine, i.e., a vaccine that avoids generating a humoral response against the structural proteins of DENV that could lead to ADE and instead relies on T cell responses to provide protection against different serotypes of DENV simultaneously (12).

[0007] Zika virus (ZIKV) has caused large-scale outbreaks of neonatal congenital syndrome and adult Guillain-Barre syndrome. Between 2007 and 2016, the virus spread throughout the Pacific Islands, reached South America and Southeast Asia, and finally had a large-scale outbreak in Brazil in 2016, involving at least 100,000 human cases. This situation prompted the WHO to declare the Zika virus epidemic a global health emergency in 2016. Although the number of Zika virus cases has decreased in subsequent years, the likelihood of its recurrence remains high, and to date, no licensed vaccine or effective treatment against this virus has been developed. The recent global spread of ZIKV has initiated intensive vaccine development efforts, and many vaccine candidates using various vaccine platforms have been tested in preclinical studies. These vaccines include nucleic acid-based vaccines (DNA and mRNA), virus-like particles (VLPs), inactivated virus vaccines, live attenuated vaccines, and viral vector vaccines (using platforms based on adenovirus, measles, and poxvirus vectors) (49, 50). Many of these vaccine candidates have shown protection against ZIKV in mouse and / or non-human primate (NHP) models, and some vaccines have entered clinical trials. Most of the vaccine candidates are currently in Phase I trials, and two are in Phase I / II trials (reviewed in (50)). The amino acid identity between ZIKV and DENV is approximately 55-56%, and ZIKV is more closely related to DENV compared to other flaviviruses (2). The degree of antigenic similarity between ZIKV and DENV raises concerns that antibody responses against ZIKV may render individuals susceptible to more severe DENV disease via the ADE mechanism. A large number of studies have confirmed this concern (51-56). Despite this concern, the vast majority of ZIKV vaccines currently in preclinical or clinical development are formulated to induce neutralizing antibody responses and are directed against surface proteins. Because DENV and ZIKV co-circulate in the same geographic regions (2), a protective vaccine against both viruses is needed that does not induce cross-specific antibody responses that may enhance the severity of DENV and ZIKV diseases.

[0008] Yellow fever virus (YFV) is endemic in the tropical and subtropical regions of South America and Africa. Although most human YFV infections are asymptomatic, approximately 12% of infected individuals develop severe YF, which can manifest as jaundice, hemorrhage, and multi-system organ failure (57). Since the mosquito control programs slackened in the 1970s, this disease, which had been controlled by vaccination and mosquito control measures, has re-emerged in South America. It is estimated that there are 200,000 infections and 30,000 deaths annually, and 400-500 million unvaccinated people live in endemic areas (58). Although an effective vaccine against YFV (YF-VAX, Sanofi Pasteur) has been in use since the 1930s and provides a high level of protection against the disease, the vaccine is based on a live attenuated virus strain of YFV (17D-204) and thus its safety profile is not optimal. Additionally, in rare cases, the vaccine can produce neurological complications. Several analyses of vaccinated travelers estimated the incidence of serious adverse events (i.e., death, life-threatening disease, hospitalization, or permanent disability) to be 1.1-4.7 per 100,000 doses (59). Additionally, the production of YF-VAX vaccine is limited by technical problems, and there are current vaccine shortages in some endemic areas (60). Therefore, there is a need for an easily producible vaccine with an improved safety profile to continuously protect people from YFV infection. A cell-culture inactivated virus vaccine candidate, XRX-001, developed as a potential alternative, was evaluated in a Phase I clinical trial. Although vaccination with this candidate vaccine induced 100% seroconversion in 24 human subjects and no serious adverse events occurred, the safety profile of this vaccine candidate could not be compared with that of the live attenuated vaccine due to the limited number of subjects participating in this study (59, 61).

[0009] Lentiviral vectors (LVs) are one of the most efficient vaccine platforms because of their excellent potential to transfer genes into the nuclei of host cells, particularly including antigen-presenting cells (APCs). These vectors can integrate into the genome of target cells, induce continuous antigen presentation by APCs (13), and strongly induce T cell immunity (13), and thus are widely used in gene therapy. To date, the application of these vectors in vaccine development has been limited by safety concerns because this technology implies inserting LV-derived genetic material into the genome of target cells. However, recent studies using integration-deficient LVs have shown that effective antigen presentation can be achieved without integrating LVs into the genome, thus greatly improving the safety of these vectors (14, 15). The nuclear transfer of genes by lentiviral vectors initiates the expression of antigens that easily enter the major histocompatibility complex class I (MHC-I) presentation machinery, namely the proteasome, further triggering CD8 +T cells. In sharp contrast to the great ability of viral vectors, including LVs, to direct endogenously produced antigens to the MHC-I pathway, they are not very efficient in triggering CD4 + T cell responses. The inventors have demonstrated that CD8 + T cells contribute significantly to the immune control of infectious diseases caused by flaviviruses selected from dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV). Thus, the potential of using LVs to induce CD8 + T cells against multiple epitopes derived from multiple non-structural antigens would provide new vaccine strategies, particularly prophylactic strategies, wherein the antigens are from at least one flavivirus selected from dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV). SUMMARY OF THE INVENTION

[0011] The inventors disclose the development of several candidate vaccines, particularly candidate LV vaccines, which are shown to simultaneously induce protection against infection with 4 serotypes of DENV in IFNAR-KO mice and / or induce protection against infection with Zika virus (ZIKV) and yellow fever virus (YFV) in said mice. The protection is mainly attributed to the induction of CD8+ T cell responses against conserved regions of the non-structural DENV proteins, or the induction of CD8+ T cell responses against conserved regions of ZIKV or YFV located in non-structural and structural proteins, respectively.

[0012] Although the tested vaccines only showed partial protection in the mouse model, it should be emphasized that this model does not fully recapitulate human disease and it only has some infection parameters (such as viremia). Although the mouse model does not fully recapitulate human flavivirus-mediated disease, it is the most commonly used small preclinical animal model in which flavivirus viremia can be measured. In addition, testing vaccines in mice that are designed to optimize human T cell responses may only induce limited protection in mice because the location of the epitopes targeted by the T cell responses is different between humans and mice. However, in this study, this preclinical model can be used to determine that three LV-based vaccine candidates have strong and statistically significant protective potential.

[0013] Thus, the present inventors have demonstrated for the first time that the recombinant polynucleotides of the present invention (especially when vectorized by LV to enable T cell responses to be elicited by the vector) can be used to develop multivalent vaccines that can simultaneously induce protection against different pathogens of the same class (such as different serotypes of DENV) or different pathogens of the same genus (i.e., flaviviruses). The proposed antigen design method initiated with DENV antigens allows for further modification of immunogenic fusion polypeptides, such as adding antigen modules designed to protect against other flaviviruses such as Zika virus (ZIKV) and yellow fever virus (YFV), thereby producing a multivalent vaccine that can simultaneously prevent several flaviviruses.

[0014] In one aspect, the present invention relates to a recombinant polynucleotide comprising at least one polynucleotide encoding a fusion polypeptide comprising an MHC class I T cell epitope suitable for eliciting a T cell response, wherein the MHC class I T cell epitope is derived from a plurality of conserved proteins, especially non-structural proteins, and wherein the antigen is from at least one flavivirus selected from dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV).

[0015] In a particular aspect of the present invention, a first recombinant polynucleotide encodes a first fusion polypeptide comprising an MHC class I T cell epitope derived from more than one non-structural DENV protein and forming a DENV-based assembled antigen exhibiting a common amino acid sequence of DENV-1, DENV-2, DENV-3, and DENV-4 virus strains.

[0016] In another aspect of the present invention, there is provided another second recombinant polynucleotide encoding a second fusion polypeptide comprising an MHC class I T cell epitope derived from more than one conserved ZIKV protein, especially from more than one non-structural ZIKV protein and correspondingly forming a ZIKV-based assembled antigen.

[0017] In another aspect of the present invention, there is provided another third polynucleotide encoding a third fusion polypeptide comprising an MHC class I T cell epitope derived from more than one conserved YFV protein, especially from more than one non-structural YFV protein and correspondingly forming a YFV-based assembled antigen.

[0018] The present invention also relates to a recombinant lentiviral vector genome comprising at least one of a plurality of recombinant polynucleotides disclosed herein encoding one or more fusion polypeptides, wherein each fusion polypeptide comprises an MHC class I T cell epitope derived from more than one conserved DENV, ZIKV, and / or YFV protein, especially from more than one non-structural DENV, ZIKV, and / or YFV protein.

[0019] The present invention further relates to a DNA plasmid comprising the recombinant lentiviral vector genome according to the present invention.

[0020] The present invention also relates to a recombinant lentiviral vector, namely, a recombinant lentiviral vector particle comprising the recombinant lentiviral vector genome according to the present invention.

[0021] The present invention also relates to a fusion polypeptide encoded by a recombinant polynucleotide and a fusion polypeptide expressed by a recombinant lentiviral vector.

[0022] The present invention further relates to a host cell transfected with the DNA plasmid according to the present invention, preferably a mammalian host cell, particularly a human host cell, particularly wherein the host cell is a HEK-293T cell line or a K562 cell line.

[0023] In another aspect, the present invention relates to a pharmaceutical composition, particularly a vaccine composition, suitable for administration to a mammalian host, particularly a human host, comprising the recombinant polynucleotide or recombinant lentiviral vector of the present invention and one or more pharmaceutically acceptable excipients suitable for administration to a host in need, particularly a mammalian host, particularly a human host.

[0024] In particular, the present invention relates to a pharmaceutical composition for eliciting a T cell response, particularly a CD8+ T cell response, in a host in need, particularly a mammalian host, particularly a human host, by an epitope contained in one or more antigen fusion polypeptides or immunogenic fragments thereof, thereby eliciting a protective, preferably prophylactic, immune response.

[0025] Another aspect of the present invention relates to a method for preparing recombinant lentiviral vector particles suitable for preparing a pharmaceutical composition, particularly a vaccine composition, the method comprising the following steps:

[0026] a) Transfecting a recombinant lentiviral transfer vector carrying the lentiviral vector genome according to the present invention or the DNA plasmid according to the present invention into a host cell, such as a HEK-293T cell line or a K562 cell line;

[0027] b) Co-transfecting the cells of step a) with (i) a plasmid vector encoding lentiviral GAG and POL or a mutant POL protein as a packaging construct; and (ii) a plasmid encoding an envelope protein of a virus (a non-HIV virus and advantageously a non-lentivirus), such as a VSV-G Indiana or New Jersey envelope;

[0028] c) Incubating the host cells under conditions suitable for producing recombinant lentiviral vector particles expressing the fusion polypeptide of the present invention;

[0029] d) Recovering the recombinant lentiviral particles expressing the fusion polypeptide of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0031] The inventors have designed and prepared a recombinant polynucleotide comprising at least one recombinant polynucleotide encoding a fusion polypeptide comprising an MHC class I T cell epitope suitable for eliciting a T cell response, wherein the MHC class I T cell epitope is derived from a plurality of antigens, particularly non-structural antigens, and wherein the antigen is from at least one flavivirus selected from dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV).

[0032] In one embodiment, the recombinant polynucleotide of the present invention is capable of expressing a fusion polypeptide comprising an epitope for eliciting a multivalent immune response against multiple serotypes of dengue virus (DENV), particularly against the 4 known serotypes of dengue virus. Accordingly, the present invention relates to a recombinant polynucleotide comprising a first polynucleotide encoding a first fusion polypeptide comprising an MHC class I T cell epitope, the MHC class I T cell epitope being derived from more than one non-structural DENV protein and forming a DENV-based assembled common antigen of DENV-1, DENV-2, DENV-3, and DENV-4 strains. The DENV-1, DENV-2, DENV-3, and DENV-4 strains are strains of DENV serotype 1 (DENV-1), DENV serotype 2 (DENV-2), DENV serotype 3 (DENV-3), and DENV serotype 4 (DENV-4), respectively.

[0033] In another embodiment, the recombinant polynucleotide of the present invention is capable of expressing a second fusion polypeptide comprising an epitope for eliciting an immune response against Zika virus. Accordingly, the present invention relates to a recombinant polynucleotide comprising a polynucleotide encoding a fusion polypeptide comprising an MHC class I T cell epitope, the MHC class I T cell epitope being derived from more than one ZIKV protein and forming a ZIKV-based assembled antigen, particularly a non-structural assembled antigen.

[0034] In another embodiment, the recombinant polynucleotide of the present invention is capable of expressing a third fusion polypeptide comprising an epitope for eliciting an immune response against yellow fever virus. Accordingly, the present invention relates to a recombinant polynucleotide comprising a polynucleotide encoding a fusion polypeptide comprising an MHC class I T cell epitope, the MHC class I T cell epitope being derived from more than one YFV protein and forming a YFV-based assembled antigen, particularly a non-structural assembled antigen.

[0035] In one embodiment, the first polynucleotide comprises or consists of a single open reading frame (ORF), particularly an ORF encoding a first fusion polypeptide, the first fusion polypeptide being a DENV-based assembly common antigen. In another embodiment, the recombinant polynucleotide comprises or consists of two or three ORFs, wherein each ORF encodes a fusion polypeptide, the fusion polypeptide being a DENV-based assembly common antigen, a ZIKV-based assembly antigen or a YFV-based assembly antigen as disclosed herein. In this case, the fusion polypeptides encompass a first fusion polypeptide, a second fusion polypeptide and a third fusion polypeptide.

[0036] The expression "T cell epitope" refers to an antigenic determinant involved in a T cell-driven adaptive immune response. In particular, the T cell epitope elicits T cells when delivered to a host under suitable conditions. According to the invention, the fusion polypeptide comprises one or more epitopes mediating a CD8+ T cell response. In particular, the T cell epitopes of the fusion polypeptides of the invention are MHC class I (MHC-I) epitopes, which are suitable for eliciting an immune response via the MHC class I presentation mechanism, i.e. the proteasome, to further trigger CD8+ T cells in the host, particularly CD8+ T cells specific for the virus targeted by the fusion polypeptide.

[0037] As used in this specification, the plural or singular form of the expression "originating / originates" when referring to MHC class I T cell epitopes means that the expressed epitope has viral antigenic characteristics, i.e., the epitope has immunogenic properties against which a targeted immune response can be determined for the identified viral antigen. For this reason, an MHC class I T cell epitope "originating" from a viral antigen may also be referred to herein as an MHC class I T cell epitope "derived from" such viral antigen, thereby indicating that the MHC class I T cell epitope is a region isolated from or contained within the entire viral antigen, or that this region in the viral antigen sequence is a model for designing the MHC class I T cell epitope. An epitope is also said to originate from a viral antigen when the fusion polypeptide according to the invention containing the epitope elicits a targeted immune response against the virus upon administration to a host, especially when the fusion polypeptide is expressed from an LV vector in the administered host. Known methods can be used to evaluate the immunogenic properties of T cell epitopes, and such methods include intracellular cytokine staining, ELISpot, in vitro stimulation, or proliferation of immune cells. In particular, epitopes derived from an identified viral antigen are selected starting from known available viral antigen sequences (amino acid sequences and / or nucleotide sequences). The epitopes used in the present invention are characterized by an amino acid sequence that reflects the native sequence of the identified antigen in the virus, or the amino acid sequence is derived by amino acid mutations from the native sequence containing a known or predicted viral T cell epitope. Thus, the T cell epitope may be identical to the sequence in the region containing the native epitope of the identified viral antigen, or may be designed relative to such native sequence as a mutant sequence or an optimized consensus sequence that defines, for example, a consensus sequence (such as SEQ ID NO:166 for DENV1-4 serotypes or SEQ ID NO:169 for ZIKV_all). Thus, tools available for determining epitopes can be used to design mutant sequences or consensus sequences, and HLA alleles, especially human MHC class I (MHC class I T cell epitopes) presentation, can be tested. Prediction tools for identifying epitopes are known to those skilled in the art and include T cell epitope prediction tools available at the IEDB database and analysis resources and at the website of the Technical University of Denmark (DTU) / Health Tech department, such as TepiTool, the Proteasome cleavage / TAP transport / MHC class I combination predictor, and netCTLpan (21, 25). In particular, the MHC class I T cell epitopes obtained from the expression of one or more polynucleotides of the present invention are octameric peptides, nonameric peptides, decameric peptides, and / or undecameric peptides. The examples disclosed herein provide a detailed description of identifying epitopes suitable for dengue virus and such description can be applied to other viruses of interest in the present invention, such as ZIKV or YFV, in order to be able to prepare fusion polypeptides covering such epitopes.Viral antigens selected for identification of T cell epitopes encompass non-structural proteins (from DENV, ZIKV and YFV), and optionally encompass structural proteins (especially from ZIKV and YFV).

[0038] As described above, in one embodiment, the present invention relates to a recombinant polynucleotide comprising a first polynucleotide encoding a first fusion polypeptide, the first fusion polypeptide comprising an MHC class I T cell epitope, the MHC class I T cell epitope being derived from more than one non-structural DENV protein and forming a DENV-based assembled common antigen of DENV-1, DENV-2, DENV-3 and DENV-4 strains, wherein the non-structural protein or antigen is selected from NS1, NS2A, NS2B, NS3, NS4A, NS4B or NS5. An epitope is said to be derived from a viral antigen when the fusion polypeptide containing the epitope elicits a targeted immune response against dengue virus, particularly against one or more strains selected from the DENV-1 serotype, DENV-2 serotype, DENV-3 serotype and DENV-4 serotype, preferably against all strains, and more preferably against the strains of the 4 DENV serotypes. Such an epitope is characterized by an amino acid sequence that reflects the native sequence of the antigen of a defined DENV strain, and / or the amino acid sequence is derived from the native sequence containing a known or predicted T cell epitope by mutation, particularly point mutations related to the native antigen sequences of one or more DENV serotypes. Thus, the T cell epitope may be identical to the sequence in the epitope-containing region of the DENV antigen of the non-structural protein selected from the DENV serotypes, or the T cell epitope may be a defined consensus sequence or an optimized consensus sequence, the sequence being designed using tools available for epitope determination and tested for presentation of HLA alleles, particularly human HLA class I alleles (MHC class I T cell epitopes). Examples herein provide a detailed description of the identification of DENV epitopes and the preparation of fusion polypeptides containing such epitopes. Thus, the design of the multi-epitope is based on the preparation of the consensus sequence (primary consensus sequence) of the target antigen of each of the 4 DENV serotypes and the alignment of these 4 consensus sequences, thereby preparing a higher-level consensus sequence (major consensus sequence) of these 4 consensus sequences. The resulting major consensus sequence is used to provide the epitope of the fusion polypeptide or is modified by point mutations or by adding other epitopes considered suitable to reflect the specific variations of the 4 serotypes. Point mutations at several positions may result in switching to the most representative amino acid residues in the dataset of one or more epitope-containing regions of the antigen for the identification of the 4 primary consensus sequences of the DENV serotypes. Optionally, particularly when the amino acid variations in the epitope-containing region of the original antigen are significant in the sequence dataset used for designing the primary consensus sequence, an additional short sequence of the antigen is advantageously added to the fusion polypeptide to represent the commonality of the remaining genotypes (relative to the genotypes reflected by the primary consensus sequence or the major consensus sequence) in the final multi-epitope.Prediction tools starting with the identification of epitopes are known to those skilled in the art and include T cell epitope prediction tools available at the IEDB database and analysis resources and at the website of the Technical University of Denmark (DTU) / Health Tech, such as TepiTool, the proteasome cleavage / TAP transport / MHC class I combination predictor, and netCTLpan (21, 25).

[0039] In a particular embodiment, the recombinant polynucleotide encodes a first fusion polypeptide comprising an MHC class I T cell epitope derived from at least 2 DENV antigens selected from NS3, NS4A, NS4B, and NS5 antigens and preferably from each of the NS3, NS4A, NS4B, and NS5 antigens. The wild-type antigens of various serotypes of DENV are well known in the art and can be obtained from public databases such as NCBI and IEDB.

[0040] In a particular embodiment, the recombinant polynucleotide encodes a second fusion polypeptide that comprises an MHC class I T cell epitope derived from at least 2 non-structural antigens of ZIKV, which are NS4B and NS5 antigens, and further comprises an MHC class I T cell epitope derived from the C and PrM antigens of ZIKV. The wild-type antigens of ZIKV are well known in the art and can be obtained from public databases such as NCBI and IEDB.

[0041] In a particular embodiment, the recombinant polynucleotide encodes a third fusion polypeptide comprising an MHC class I T cell epitope derived from at least 2 YFV antigens selected from NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 antigens and preferably from each of the NS2A, NS2B, NS3, NS4A, NS4B, and NS5 antigens. The wild-type antigens of YFV are well known in the art and can be obtained from public databases such as NCBI and IEDB.

[0042] The recombinant polynucleotides of the ZIKV and YFV constructs can be designed according to the description provided herein for the DENV serotypes, particularly with regard to the steps for preparing the common sequences of each of ZIKV and YFV.

[0043] In one embodiment, a recombinant polynucleotide encodes one or more fusion polypeptides as disclosed in other embodiments above and below, the one or more fusion polypeptides comprising an MHC class I T cell epitope, the MHC class I T cell epitope being selected based on its properties (e.g., using the NetCTLpan tool), the properties being the presence of a human epitope suitable for presentation by, or advantageously presented by, 12 human HLA supertype alleles (HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*26:01, HLA-B*07:02, HLA-B*08:01, HLA-B*27:05, HLA-B*39:01, HLA-B*40:01, HLA-B*58:01, HLA-B*15:01), particularly when the MHC class I T cell epitope is derived from a non-structural DENV antigen disclosed herein. The 12 HLA supertypes listed above were selected to best represent the global repertoire of HLA molecules (i.e., predictions based on this set should approximate representation of all possible HLA-A and HLA-B alleles) (36). The set of the 27 most prevalent human HLA-A and HLA-B alleles includes alleles that should be expressed by 97% of the population (37), i.e., includes the alleles most commonly found in the global population, but these alleles are not necessarily the most widely dispersed. Thus, these two sets do not completely overlap.

[0044] One or more recombinant polynucleotides of the invention are provided in the form of fusion polynucleotides, wherein fragments from different viral antigens or MHC class I T cell epitopes of such antigens are assembled or fused together via a linker region. In a preferred embodiment of the invention, the linker region formed does not contain non-specific epitopes or neo-epitopes that can trigger a non-specific immune response in the host.

[0045] The expression "linker region" refers to regions in an assembled recombinant polynucleotide, particularly a recombinant polynucleotide encoding a DENV-based common antigen, a ZIKV-based antigen, or a YFV-based antigen, that link contiguous protein domains when the contiguous protein domains derived from a virus, particularly from non-structural proteins of a virus, are not naturally contiguous in the native or common sequence of one or more viral proteins under consideration. In one embodiment, the linker region consists solely of a nucleic acid region of a recombinant polynucleotide encoding amino acid residues that belong to two different domains or protein fragments being fused and that are adjacent to the site at which the two domains or protein fragments are fused. In particular, the linker region encodes a region of 2 to 10 amino acid residues that are displayed around the fusion site of the two domains. In such an embodiment, the linker region does not add nucleotides or amino acid residues to the nucleotides or amino acid residues that constitute the antigen domain. In another embodiment, the linker region consists of a nucleic acid region encoding amino acid residues that belong to two different domains or protein fragments being fused and that are adjacent to the site at which the two domains or protein fragments are fused, wherein the fusion site further comprises nucleotides encoding a defined linker. According to this embodiment, the linker region may encode a region added to the antigen domain, such as 2 to 10 amino acid residues, particularly 2 to 9 amino acid residues, that function as a linker. The linker is determined by those skilled in the art based on the circumstances of the adjacent antigen domains and typically contains hydrophobic amino acid residues. Constructs disclosed herein provide examples of linkers, and such linkers are applied or suitable for avoiding the formation of new epitopes in the linker region formed by adjacent viral antigen domains.

[0046] In a preferred embodiment, the linker region comprises a hydrophobic amino acid linker and does not contain sequences encoding non-specific immunodominant epitopes. The present invention describes by its sequences specific linkers suitable for use according to the present invention. Such specific examples should not be considered limiting, as those skilled in the art should be able to design alternative linkers particularly in view of the conditions illustrated by the steps of assembling the complete antigens of DENV (DENV-Ag1) and YFV (YFV-Ag1 and YFV-Ag2):

[0047] 1. The selected antigenic regions are pairwise assembled and used to predict human MHC class I epitopes (using the NetCTLpan tool) that can be presented by 12 HLA class I supertypes (HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*26:01, HLA-B*07:02, HLA-B*08:01, HLA-B*27:05, HLA-B*39:01, HLA-B*40:01, HLA-B*58:01, HLA-B*15:01) to ensure that the predicted epitopes are correctly formed and no immunodominant neoepitopes are generated due to the regions being joined together. If the epitope prediction algorithm does not detect a neoepitope, the assembled regions are retained and the next region is joined to the N-terminus or C-terminus of the assembled regions, and then the prediction is repeated again. If this stepwise assembly method detects a predictable neoepitope after adding a specific region, an attempt is made to replace it with another region until all combinations of regions that can be assembled without generating neoepitopes are identified.

[0048] 2. In the second step (for regions that cannot be added to the antigen without generating neoepitopes), each adjacent antigenic region is extended by adding 3 - 4 amino acid residues (preferably a 3 - 4 amino acid long sequence that is adjacent (next to) each antigenic region in the "natural" (e.g., common) viral sequence). If this extension does not eliminate the nascent epitope, certain amino acids (mainly non-polar amino acids: M, L, F, V, I, A, T, Y) that are found (empirical observation) to often occur in MHC class I epitopes in a 6 - 8aa long "see-through" linker region are replaced one by one with less frequently detected amino acids (certain non-polar amino acids: G, C, N, P, W, and polar amino acids: D, E, K, R, Q, H, S).

[0049] 3. If the method outlined above results in the elimination of the predicted neoepitope, the ligation is further optimized by reducing the number of "extra" amino acids between the regions. Alternatively, if the outlined method is not successful, the linker region is extended (again by adding amino acids that are not frequently detected within MHC class I epitopes) until no neoepitope is detected at the ligation site.

[0050] The same strategy was also adopted in the design of ZIKV-Ag, except that in this case, the antigenic regions were not rearranged but assembled in the order in which they occur in the viral genome.

[0051] In one embodiment, the present invention relates to a recombinant polynucleotide, wherein the DENV-based assembled common antigen comprises a polynucleotide encoding MHC class I T cell epitopes: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 or a variant thereof that does not contain SEQ ID NO:2 and that contains SEQ ID NO:32, SEQ ID NO:34 and SEQ ID NO:36 starting at the 5' end, wherein optionally, the polynucleotides encoding the polypeptides listed above are provided 5' to 3' in the recombinant polynucleotide in the order of the amino acid sequences in the above list.

[0052] In a specific embodiment of this aspect of the invention, the recombinant polynucleotide is selected from:

[0053] a. A recombinant polynucleotide that comprises a polynucleotide encoding an MHC class I T cell epitope having the following sequences, the polynucleotides being arranged 5' to 3' in the recombinant polynucleotide in the following order: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:28, and wherein the linker region between the polynucleotides encoding the MHC class I T cell epitopes does not contain a sequence encoding a non-specific immunodominant epitope, particularly wherein a hydrophobic amino acid linker sequence is inserted as the linker region between all consecutive sequences above, except between SEQ ID NO:8 and SEQ ID NO:10, between SEQ ID NO:16 and SEQ ID NO:18 and between SEQ ID NO:18 and SEQ ID NO:20, or

[0054] b. A recombinant polynucleotide comprising a polynucleotide encoding an MHC class I T cell epitope having the following sequences, said polynucleotides being arranged 5' to 3' in the following order in the recombinant polynucleotide: SEQ ID NO:32, SEQ ID NO:34 and SEQ ID NO:36, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and wherein the linker region between said polynucleotides encoding the MHC class I T cell epitope does not contain a sequence encoding a non-specific immunodominant epitope, in particular a recombinant polynucleotide wherein a hydrophobic amino acid linker sequence is inserted as a linker region between all consecutive sequences listed above, except between SEQ ID NO:34 and SEQ ID NO:36, between SEQ ID NO:8 and SEQ ID NO:10, between SEQ ID NO:16 and SEQ ID NO:18, and between SEQ ID NO:18 and SEQ ID NO:20.

[0055] In one embodiment, the invention relates to a recombinant polynucleotide, wherein the ZIKV-based assembled antigen comprises a polynucleotide encoding an MHC class I T cell epitope: SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, wherein optionally, the polynucleotides encoding the polypeptides listed above are provided 5' to 3' in the recombinant polynucleotide according to the order of the amino acid sequences in the above list.

[0056] In a specific embodiment of this aspect of the invention, the recombinant polynucleotide comprises a polynucleotide encoding an MHC class I T cell epitope, which polynucleotides are arranged 5' to 3' in the recombinant polynucleotide in the following order: SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, and wherein the linker regions between said polynucleotides encoding MHC class I T cell epitopes do not contain sequences encoding non-specific immunodominant epitopes, in particular a recombinant polynucleotide wherein a hydrophobic amino acid linker sequence is inserted as a linker region between all consecutive sequences above, except between SEQ ID NO:44 and SEQ ID NO:46 and between SEQ ID NO:50 and SEQ ID NO:52.

[0057] In one embodiment, the invention relates to a recombinant polynucleotide, wherein an assembly antigen based on the non-structural proteins of YFV (YFV-Ag1) comprises a polynucleotide encoding MHC class I T cell epitopes: SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID No.78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, wherein optionally, the polynucleotides encoding the polypeptides listed above are provided 5' to 3' in the recombinant polynucleotide according to the order of the amino acid sequences in the above list.

[0058] In one embodiment, the present invention relates to a recombinant polynucleotide, wherein the YFV-based non-structural protein and the assembled antigen of non-structural protein NS1 (YFV-Ag2) comprise a polynucleotide encoding MHC class I T cell epitopes: SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, wherein optionally, the polynucleotides encoding the polypeptides listed above are provided 5' to 3' in the recombinant polynucleotide in the order of the amino acid sequences in the above list.

[0059] In a specific embodiment of this aspect of the present invention, the recombinant polynucleotide is selected from:

[0060] a. A polynucleotide comprising a polynucleotide encoding an MHC class I T cell epitope, said polynucleotides being arranged 5' to 3' in the following order in a recombinant polynucleotide: SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID No.78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID No 86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102 and SEQ ID NO:104, and wherein the linker region between said polynucleotides encoding the MHC class I T cell epitope does not contain a sequence encoding a non-specific immunodominant epitope, in particular a recombinant polynucleotide in which a hydrophobic amino acid linker sequence is inserted as a linker region between all consecutive said sequences, except between SEQ ID NO:60 and SEQ ID NO:62, between SEQ ID NO:66 and SEQ ID NO:68, between SEQ ID NO:74 and SEQ ID NO:76, between SEQ ID NO:78 and SEQ ID NO:80 and SEQ ID NO:82, and between SEQ ID NO:94 and SEQ ID NO:96, or

[0061] b. A polynucleotide comprising a polynucleotide encoding an MHC class I T cell epitope, wherein the polynucleotides are arranged 5' to 3' in the following order in the recombinant polynucleotide: SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, and SEQ ID NO:140, and wherein the linker region between the polynucleotides encoding the MHC class I T cell epitopes does not contain a sequence encoding a non-specific immunodominant epitope, particularly wherein a hydrophobic amino acid linker sequence is inserted as a linker region between all consecutive sequences as above, except between SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116 and SEQ ID NO:118 and between SEQ ID NO:124 and SEQ ID NO:126.

[0062] In another embodiment, the recombinant polynucleotide is a nucleic acid molecule whose sequence is modified relative to at least one of the SEQ ID No. sequences disclosed above with respect to the DENV fusion polynucleotide or relative to at least one of the SEQ ID No. sequences disclosed above with respect to the ZIKV polynucleotide or relative to at least one of the SEQ ID No. sequences disclosed above with respect to the YFV polynucleotide, wherein the modification consists of one or more point mutations of nucleotides, particularly substitutions or deletions of nucleotides, and the modified sequence encodes a fusion polypeptide having at least 90% sequence identity, at least 94% or at least 95% sequence identity or 94% to 99% sequence identity with the sequence of the original fusion polypeptide. The modified sequence as defined herein is considered a variant sequence relative to the reference sequence.

[0063] In one embodiment, the recombinant fusion polynucleotide (including the recombinant fusion polynucleotide having a variant sequence as defined herein) encodes an antigenic domain of a fusion polypeptide having the sequence disclosed above or a variant thereof, wherein the polynucleotide comprises the following operably linked nucleotide sequences:

[0064] (i) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or a variant thereof, wherein SEQ ID NO:1 is deleted and comprises SEQ ID NO:31 and / or SEQ ID NO:33 and / or SEQ ID NO:35 at the 5' end, and optionally, these polynucleotide fragments are provided 5' to 3' in a fusion polynucleotide in the order of the above list (from SEQ ID NO:1 to SEQ ID NO:27 or from SEQ ID NO:31 to SEQ ID NO:27 (excluding SEQ ID NO:1) according to the above disclosure); or

[0065] (ii) SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, and optionally, these polynucleotide fragments are provided 5' to 3' in a fusion polynucleotide in the order of the above list (from SEQ ID NO:39 to SEQ ID NO:51 according to the above disclosure); or

[0066] (iii) SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, and optionally, these polynucleotide fragments are provided 5' to 3' in a fusion polynucleotide in the order of the above list (from SEQ ID NO:57 to SEQ ID NO:103 according to the above disclosure); or

[0067] (iv) SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, where optionally, these polynucleotide fragments are provided 5' to 3' in the recombinant polynucleotide in the order according to the above list (from SEQ ID NO: 107 to SEQ ID NO: 139 according to the above disclosure).

[0068] In a particular embodiment, the recombinant polynucleotide further contains a sequence encoding a signal peptide at its 5' end.

[0069] In a particular embodiment, the recombinant polynucleotide is a nucleic acid molecule, the sequence of which consists of SEQ ID NO: 29 or SEQ ID NO: 37.

[0070] In a particular embodiment, the recombinant polynucleotide is a nucleic acid molecule, the sequence of which consists of SEQ ID NO: 53 or SEQ ID NO: 55.

[0071] In a particular embodiment, the recombinant polynucleotide is a nucleic acid molecule, the sequence of which consists of SEQ ID NO: 105 or SEQ ID NO: 141.

[0072] In a particular embodiment, the recombinant polynucleotide is a fusion of a polynucleotide selected from SEQ ID NO:29 and SEQ ID NO:37 with a polynucleotide selected from SEQ ID NO:53 and SEQ ID NO:55 and / or a polynucleotide selected from SEQ ID NO:105 and SEQ ID NO:141. In a particular embodiment, the recombinant polynucleotide is a fusion of a polynucleotide having SEQ ID NO:29 or SEQ ID NO:37 with a polynucleotide having SEQ ID NO:53 with a polynucleotide having SEQ ID NO:105 or SEQ ID NO:141. The order of occurrence of the polynucleotides defined by their SEQ ID No. in the recombinant polynucleotide sequence is chosen by the person skilled in the art. In a particular embodiment, in the 5' to 3' sense of the recombinant polynucleotide, the order is such that the polynucleotide encoding the MHC class I T cell epitope derived from DENV occurs first.

[0073] In a particular embodiment, the recombinant polynucleotides are selected from SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147 and SEQ ID NO:149.

[0074] In another embodiment, the recombinant polynucleotide is a nucleic acid molecule the sequence of which is modified relative to the sequence of SEQ ID NO:29 or SEQ ID NO:37 or relative to the sequence of SEQ ID NO:53 or SEQ ID NO:55 or relative to the sequence of SEQ ID NO:105 or SEQ ID NO:141 or relative to the sequence of SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147 and SEQ ID NO:149, wherein the modification consists of one or more point mutations of nucleotides, in particular substitutions or deletions of nucleotides, and the modified sequence encodes a fusion polypeptide having at least 90% sequence identity, at least 94% or at least 95% sequence identity or 94% to 99% sequence identity with the sequence of the original fusion polypeptide.

[0075] In one embodiment, the recombinant fusion polynucleotide (including the recombinant fusion polynucleotide having a variant sequence as defined herein) encodes a fusion polypeptide having SEQ ID NO:30 or SEQ ID NO:38 or SEQ ID NO:54 or SEQ ID NO:56 or SEQ ID NO:106 or SEQ ID NO:142 or a variant thereof as disclosed above.

[0076] In a specific embodiment, the sequence of the polynucleotide is modified relative to the sequence of SEQ ID NO:29 or SEQ ID NO:37, wherein the modification consists of replacing the antigen domain in the NH2-terminal sequence of the fusion polypeptide with a complementary antigen region representing the antigen domain of the selected DENV serotype subgroup.

[0077] In one embodiment, one or more point mutations include altering amino acid residues present in the DENV-1, DENV-2, DENV-3, and / or DENV-4 genotypes, particularly amino acid residues common to at least two of these genotypes.

[0078] In one embodiment, the recombinant fusion polynucleotide is a fusion ORF encoding an MHC class I T cell epitope disclosed herein, wherein the coding sequence is under the control of transcriptional and translational control elements, particularly within a single transcriptional regulatory unit under the control of a single promoter for at least the MHC class I T cell epitopes derived from the same viral group. The transcriptional and translational control elements may be the transcriptional and translational control elements disclosed below for the transfer vector of the vector genome.

[0079] In one embodiment, the recombinant polynucleotide can be a nucleic acid molecule that encodes an MHC class I T cell epitope derived from at least one of the DENV proteins, ZIKV proteins, and YFV proteins disclosed herein, wherein the nucleic acid sequences are operably linked. According to such an embodiment, the recombinant polynucleotide may comprise one or more expression cassettes for MHC class I T cell epitopes derived from different viruses (including at least one of DENV, ZIKV, and YFV). In a specific embodiment, the recombinant polynucleotide comprises an expression cassette for MHC class I T cell epitopes derived from dengue virus and Zika virus, wherein the nucleic acid sequence encoding the MHC class I T cell epitope derived from dengue virus and the nucleic acid sequence encoding the MHC class I T cell epitope derived from Zika virus are separated by a sequence encoding a self-cleaving peptide (such as the 2A self-cleaving peptide), and optionally combined with a spacer sequence (such as a spacer sequence encoding GSG located N-terminal to the 2A self-cleaving peptide). The 2A peptides are well known in the art and include peptides having 19 to 22 amino acid residues, such as a peptide having the sequence LLNFDLLKLAGDVESNPGP (SEQ ID NO:217) or 2A-like peptides, such as P2A (GSGATNFSLLKQAGDVEENPGPSEQ ID NO:218), T2A, E2A, F2A disclosed in the art. The 2A peptides are suitable for mediating the co-expression and cleavage of the fusion ORF and causing the secretion of the expressed polypeptides.

[0080] In a particular embodiment, a recombinant polynucleotide according to the invention comprises (i) a first polynucleotide encoding a first fusion polypeptide comprising an MHC class I T cell epitope derived from a non-structural DENV protein and forming a DENV-based assembled antigen; and further comprises (ii) a second polynucleotide encoding a second fusion polypeptide comprising an MHC class I T cell epitope derived from structural and non-structural ZIKV proteins and forming a ZIKV-based assembled antigen, or an ORF encoding the NS1 protein of ZIKV, preceded by a signal peptide derived from the E protein (SEQ ID No. 56); and further comprises (iii) a third polynucleotide encoding a third fusion polypeptide comprising an MHC class I T cell epitope derived from structural and / or non-structural YFV proteins and forming a YFV-based assembled antigen, wherein the first polynucleotide and the second polynucleotide and the third polynucleotide (when present) are operably linked in an expression cassette and separated by a sequence encoding a self-cleaving peptide such as a 2A self-cleaving peptide, and optionally associated with a spacer sequence such as a spacer sequence encoding GSG located N-terminal to the 2A self-cleaving peptide).

[0081] In a particular embodiment, the first polynucleotide and the second polynucleotide encoding an MHC class I T cell epitope derived from dengue virus and an MHC class I T cell epitope derived from Zika virus or an ORF encoding the NS1 protein of ZIKV (SEQ ID NO: 55), preceded by a signal peptide derived from the E protein, are assembled from the 5'-end to the 3'-end, i.e., the second polynucleotide follows the first polynucleotide (such as in the exemplary Flavi-2 or Flavi-4 constructs) or the first polynucleotide follows the second polynucleotide (such as in the exemplary Flavi-3 or Flavi-5 constructs).

[0082] In any of the disclosed embodiments, the recombinant polynucleotide construct may further comprise, at its 5'-end, a nucleic acid sequence encoding a signal peptide and / or additional nucleotides or codons necessary or advantageous for translating the polynucleotides disclosed herein into a fusion polypeptide fusion, such as nucleotides encoding DENV-Ag1 or MA amino acid residues or MA amino acid residues in YFV-Ag1 and YFV-Ag2 (as shown in the sequences of the transgenes disclosed herein). When the selected assembly region lacks an M (Met) codon, addition of such a codon enables translation. The additional codons are selected to have a stronger Kozak sequence (GCCACC at the 5'-end of the coding region ATGG - SEQ ID NO:172), thus enabling more efficient translation. The last 4 nucleotides of the stronger Kozak sequence are part of the coding region, and the last nucleotide (G) is at the first position of the codon for the second amino acid. Thus, the preferred amino acids at this position are A, V, D, E, or G. To select an amino acid from this list, the inventors sequentially performed MHC class I epitope prediction on sequences containing each amino acid to select the amino acid that least interferes with the correct processing of the first MHC class I epitope located at the N-terminal portion of the multi-epitope.

[0083] The nucleic acid of the recombinant polynucleotide disclosed herein can be DNA, particularly cDNA, or can be RNA, particularly stabilized RNA. The RNA sequence is deduced from the DNA sequence, wherein the thymine (T) nucleobase is replaced by the uracil (U) nucleobase. The RNA polynucleotide can be obtained by transcription of DNA or cDNA or can be synthesized.

[0084] The nucleic acid molecule can further comprise control nucleotide sequences for transcription or for expressing a fusion polypeptide. The nucleic acid molecule can also be modified so as to be operably linked to different polynucleotides, such as a plasmid or a vector genome, particularly a transfer plasmid, particularly a lentiviral vector genome, particularly the HIV-1 vector genome disclosed below. The nucleic acid molecule can also be modified, particularly to make it more stable, such as for use as RNA. In another embodiment, the nucleic acid is a mammalian codon-optimized sequence, particularly a human codon-optimized sequence, for expression in mammalian, particularly human cells. Examples of codon-optimized nucleic acids are provided in the exemplary constructs of the transgene.

[0085] Accordingly, the present invention discloses a recombinant lentiviral vector genome comprising at least one recombinant polynucleotide of the present invention encoding a fusion polypeptide of the present invention, wherein the fusion polypeptide is expressed as a multi-domain recombinant protein, the multi-domain recombinant protein comprising a number of antigen domains, the antigen domains comprising MHC class I T cell epitopes of one or more viruses selected from DENV, ZIKV, and YFV.

[0086] The fusion polypeptide is encoded by a recombinant polynucleotide as defined herein, the recombinant polynucleotide being inserted into the backbone of a lentiviral transfer vector to provide a vector genome comprising the recombinant polynucleotide of the present invention, thereby enabling the preparation of lentiviral vector particles expressing one or more fusion polypeptides carrying MHC class I T cell epitopes for eliciting an immune response, particularly a protective immunogenic response or advantageously sterile protection, against one or more viruses from which the epitopes are derived.

[0087] The "vector genome" of the carrier particles disclosed herein is a recombinant nucleic acid, which further contains a polynucleotide or a target transgene as the inserted sequence, and the polynucleotide or the related transgene encodes a fusion polypeptide according to the present invention. The fusion polypeptide contains one or more virus-derived antigen polypeptides or immunogenic fragments thereof disclosed herein. In particular, the virus is a flavivirus selected from dengue virus (especially one of the known serotypes DENV-1, DENV-2, DENV-3 and DENV-4), Zika virus (ZIKV) or yellow fever virus (YFV). The lentivirus-based sequence and polynucleotide / transgene of the vector genome are carried by a plasmid vector, thereby generating a "transfer vector" (also called a "sequence vector") to prepare a lentivirus vector by transfecting cells. Therefore, these expressions can be used interchangeably in this specification.

[0088] Therefore, the vector genome defined herein contains, in addition to the so-called recombinant polynucleotide encoding the fusion polypeptide of the present invention (the fusion polypeptide contains an antigen polypeptide and is expressed under the control of appropriate regulatory sequences), the sequence of the original lentivirus genome, which is the non-coding region of the genome and is necessary to provide recognition signals for DNA or RNA synthesis and processing (small virus genome). These sequences are particularly cis-acting sequences necessary for encapsidation (ψ), reverse transcription (LTR may have mutated relative to the original LTR), transcription and optionally integration (RRE). In addition, for the specific purpose of the present invention, the sequence contains a functional sequence that facilitates nuclear import in cells and thus facilitates the transgene transfer efficiency in the cell. This element is described as a DNA Flap element, which contains or consists of the so-called central cPPT-CTS nucleotide domain present in the lentivirus genome sequence, especially HIV-1 or certain retroelements (such as the retroelement of yeast).

[0089] The structure and composition of the vector genome for preparing the lentivirus vector of the present invention are based on the principles described in the art and on the examples of such lentivirus vectors mainly disclosed in Zennou et al., 2000; Firat H. et al., 2002; VandenDriessche T. et al., 2002. As will be mentioned herein, constructs of this type have been deposited with the CNCM (Pasteur Institute, France). In this regard, reference is also made to the disclosures of patent applications WO 99 / 55892, WO 01 / 27300 and WO 01 / 27304, including the deposited biological materials.

[0090] According to a particular embodiment of the invention, the vector genome may be a replacement vector, in which all viral protein-coding sequences between two long terminal repeats (LTRs) have been replaced by a recombinant polynucleotide encoding a fusion polypeptide of the invention comprising one or more antigenic polypeptides disclosed herein, and in which the DNA-Flap element has been reinserted together with the desired cis-acting sequences described herein. Other features related to the composition of the vector genome are disclosed in connection with the preparation of the particles.

[0091] In a particular embodiment, the lentiviral vector of the invention may contain, in its genome, by means of the vector genome, one or more than one recombinant polynucleotide encoding at least one fusion polypeptide according to the invention. In particular, the vector genome contains two polynucleotides which are genomically contiguous or separated and which encode different fusion polypeptides of different antigens of the same viral pathogen or different viruses.

[0092] Thus, in a particular embodiment, the invention relates to a recombinant lentiviral vector genome comprising at least one recombinant polynucleotide (in particular 1, 2 or 3 recombinant polynucleotides) disclosed in various embodiments herein and encoding a fusion polypeptide or fusion polypeptides, wherein said fusion polypeptides are as disclosed herein.

[0093] In a more particular embodiment, the recombinant lentiviral vector genome encodes a fusion polypeptide comprising:

[0094] - a polypeptide comprising an MHC class I T cell epitope of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 or a variant thereof that does not contain SEQ ID NO:2 and contains SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34 at the 5' end, wherein optionally the coding sequences of the epitopes in the above SEQ ID Nos. are arranged from the N-terminus to the C-terminus in the above order (from SEQ ID NO:2 to SEQ ID NO:28 or from SEQ ID NO:32 to SEQ ID NO:28 (excluding SEQ ID NO:2) according to the above disclosure) or contain variants thereof as disclosed herein; and / or

[0095] - A polypeptide comprising MHC class I T cell epitopes: SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, wherein optionally, the coding sequences of the epitopes in the above SEQ ID Nos. are arranged from the N-terminus to the C-terminus in the above order (from SEQ ID NO:40 to SEQ ID NO:52 according to the above disclosure) or contain variants thereof disclosed herein; and / or

[0096] - A polypeptide comprising MHC class I T cell epitopes: SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID No.78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID No86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, wherein optionally, the coding sequences of the epitopes in the above SEQ ID Nos. are arranged from the N-terminus to the C-terminus in the above order (from SEQ ID NO:58 to SEQ ID NO:104 according to the above disclosure) or contain variants thereof disclosed herein; and / or

[0097] - A polypeptide comprising MHC class I T cell epitopes: SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, wherein optionally, the coding sequences of the epitopes in the above SEQ ID Nos. are arranged from the N-terminus to the C-terminus according to the above order (from SEQ ID NO:108 to SEQ ID NO:140 according to the above disclosure) or comprise variants thereof disclosed herein.

[0098] In one embodiment of this aspect of the invention, the recombinant lentiviral vector genome comprises at least one recombinant polynucleotide (especially 1, 2 or 3 recombinant polynucleotides) encoding a fusion polypeptide, wherein the polynucleotide comprises the following operably linked nucleotide sequences:

[0099] (i) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 or variants thereof, wherein SEQ ID NO:1 is absent and SEQ ID NO:31 and / or SEQ ID NO:33 and / or SEQ ID NO:35 are included at the 5'-end, wherein optionally, these polynucleotide fragments are provided from 5' to 3' in the fusion polynucleotide according to the order in the above list (from SEQ ID NO:1 to SEQ ID NO:27 or from SEQ ID NO:31 to SEQ ID NO:27 (excluding SEQ ID NO:1) according to the above disclosure); and / or

[0100] (ii) SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, wherein optionally, these polynucleotide fragments are provided 5' to 3' in the fusion polynucleotide in the order in the above list (from SEQ ID NO: 39 to SEQ ID NO: 51 according to the above disclosure); and / or

[0101] (iii) SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID No. 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID No 85, SEQ ID NO: 87, SEQ IDNO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ IDNO: 101, SEQ ID NO: 103, wherein optionally, these polynucleotide fragments are provided 5' to 3' in the fusion polynucleotide in the order in the above list (from SEQ ID NO: 57 to SEQ ID NO: 103 according to the above disclosure); or

[0102] (iv) SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ IDNO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ IDNO: 137, SEQ ID NO: 139, wherein optionally, these polynucleotide fragments are provided 5' to 3' in the recombinant polynucleotide in the order in the above list (from SEQ ID NO: 107 to SEQ ID NO: 139 according to the above disclosure).

[0103] In a particular embodiment, the recombinant polynucleotides of the invention, particularly the recombinant lentiviral vector genome, comprise at least one polynucleotide (particularly 1, 2 or 3 different polynucleotides) encoding a fusion polypeptide selected from the following:

[0104] - the fusion polypeptide of sequence SEQ ID NO:30,

[0105] - the fusion polypeptide of sequence SEQ ID NO:38,

[0106] - the fusion polypeptide of sequence SEQ ID NO:54,

[0107] - the fusion polypeptide of sequence SEQ ID NO:56,

[0108] - the fusion polypeptide of sequence SEQ ID NO:106, and

[0109] - the fusion polypeptide of sequence SEQ ID NO:142.

[0110] In a particular embodiment, the recombinant polynucleotides of the invention, particularly the recombinant lentiviral vector genome, comprise at least one polynucleotide (particularly 1, 2 or 3 different polynucleotides) selected from the following:

[0111] - the recombinant polynucleotide of sequence SEQ ID NO:29,

[0112] - the recombinant polynucleotide of sequence SEQ ID NO:37,

[0113] - the recombinant polynucleotide of sequence SEQ ID NO:53,

[0114] - the recombinant polynucleotide of sequence SEQ ID NO:55,

[0115] - the recombinant polynucleotide of sequence SEQ ID NO:105, and

[0116] - the recombinant polynucleotide of sequence SEQ ID NO:141.

[0117] In another embodiment, the recombinant lentiviral vector genome is provided as an insert in a plasmid: pFlap-β2m-DENV-Ag1-WPREm of SEQ ID NO:151 (CNCM I-5883), or pFlap-β2m-DENV-Ag2-WPREm of SEQ ID NO:152 (CNCM I-5885), or pFlap-β2m-ZIKV-Ag-WPREm of SEQ ID NO:153 (CNCM I-5882), or pFlap-β2m-ZIKV-NS1-WPREm of SEQ ID NO:154 (CNCM I-5887), or pFlap-β2m-YFV-Ag1-WPREm of SEQ ID NO:155 (CNCM I-5884), or pFlap-β2m-YFV-Ag2-WPREm of SEQ ID NO:156 (CNCM I-5886), or pFlap-β2m-DENV-Ag2_ZIKV-Ag-WPREm_(Flavi-2) of SEQ ID NO:157 (CNCM I-5888), or pFlap-β2m-ZIKV-Ag_DENV-Ag2-WPREm_(Flavi-3) of SEQ ID NO:158 (CNCM I-5889), or pFlap-β2m-DENV-Ag2_ZIKV-NS1-WPREm_(Flavi-4) of SEQ ID NO:159 (CNCM I-5890), or pFlap-β2m-ZIKV-NS1-DENV-Ag2-WPREm_(Flavi-5) of SEQ ID NO:160 (CNCM I-5891). The plasmid pFlap-β2m-WPRE for inserting the polynucleotide of the present invention may alternatively be referred to as pFlap-ΔU3-β2m-WPRE.

[0118] In another embodiment, the recombinant lentiviral vector genome is provided as an insert in the following plasmids, which were deposited on September 13, 2022 at the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue du Dr Roux - 75724 Paris Cedex 15 - France): pFlap-β2m-DENV-Ag1-WPREm with the number CNCM I-5883, or pFlap-β2m-DENV-Ag2-WPREm with the number CNCM I-5885, or pFlap-β2m-ZIKV-Ag-WPREm with the number CNCM I-5882, or pFlap-β2m-ZIKV-NS1-WPREm with the number CNCM I-5887, or pFlap-β2m-YFV-Ag1-WPREm with the number CNCM I-5884, or pFlap-β2m-YFV-Ag2-WPREm with the number CNCM I-5886, or pFlap-β2m-DENV-Ag2_ZIKV-Ag-WPREm_(Flavi-2) with the number CNCM I-5888, or pFlap-β2m-ZIKV-Ag_DENV-Ag2-WPREm_(Flavi-3) with the number CNCM I-5889, or pFlap-β2m-DENV-Ag2_ZIKV-NS1-WPREm_(Flavi-4) with the number CNCM I-5890, or pFlap-β2m-ZIKV-NS1-DENV-Ag2-WPREm_(Flavi-5) with the number CNCM I-5891.

[0119] According to one embodiment, the lentiviral vector genome comprises the recombinant polynucleotide disclosed herein, which is cloned under the control of a promoter that functions in mammalian cells, particularly the CMV promoter, the human β-2 microglobulin promoter, the SP1-human β-2 microglobulin promoter of SEQ ID NO: 170, or the composite BCUAG promoter of SEQ ID NO: 172, and wherein the vector optionally comprises a post-transcriptional regulatory element (WPRE) of woodchuck hepatitis virus, particularly the mutant WPRE shown in SEQ ID NO: 173 and / or the KOZAK sequence.

[0120] The present invention also relates to a plasmid vector, which is recombined with a nucleic acid molecule of a recombinant polynucleotide encoding one or more fusion polypeptides comprising MHC class I T cell epitopes, and the MHC class I T cell epitopes are selected to elicit an immune response in the host disclosed herein. Thus, in one embodiment, the plasmid vector is an expression vector.

[0121] In one embodiment, the plasmid vector is a transfer vector, particularly a lentiviral transfer vector, particularly an HIV-1 transfer vector, suitable for providing the genome of the lentiviral vector of the present invention. When expressed in a host, the lentiviral vector expresses one or more fusion polypeptides.

[0122] In a particular embodiment, the nucleic acid molecule containing the genome of the transfer vector is provided in the form of a plasmid, which comprises a lentiviral backbone vector (particularly an HIV-1 backbone vector) recombined with a polynucleotide encoding one or more selected antigens of a pathogen, so that when the vector genome is provided in the lentiviral vector particles for administration to a host, the nucleic acid molecule is expressed as a fusion polypeptide.

[0123] In addition, the recombinant polynucleotide or the vector containing it, particularly the nucleic acid molecule containing the genome of the transfer vector, may contain sequences for controlling transcription and / or for controlling expression, and / or may contain sequences for ligating with different nucleic acids (such as ligating with a plasmid or a vector genome). Thus, the nucleic acid may contain one or more restriction site sequences (such as BamHI (GGATCC) XhoI (CTCGAG)), kozak sequence (GCCACC), stop codon (TAA or TAATGA) sequence, promoter or other sequences disclosed herein and illustrated in the examples.

[0124] Thus, the present invention relates to a DNA plasmid comprising a recombinant lentiviral vector genome as defined herein, particularly wherein the genome is inserted into a vector plasmid, preferably the vector plasmid of nucleotide sequence SEQ ID NO:161, and the fusion polypeptide according to the present invention is inserted between the restriction sites BamHI and XhoI.

[0125] In one embodiment, the plasmid vectors of the present invention are selected from the following: plasmid pFlap-β2m-DENV-Ag1-WPREm of SEQ ID NO: 151, or pFlap-β2m-DENV-Ag2-WPREm of SEQ ID NO: 152, or pFlap-β2m-ZIKV-Ag-WPREm of SEQ ID NO: 153, or pFlap-β2m-ZIKV-NS1-WPREm of SEQ ID NO: 154, or pFlap-β2m-YFV-Ag1-WPREm of SEQ ID NO: 155, or pFlap-β2m-YFV-Ag2-WPREm of SEQ ID NO: 156, or pFlap-β2m-DENV-Ag2_ZIKV-Ag-WPREm_(Flavi-2) of SEQ ID NO: 157, or pFlap-β2m-ZIKV-Ag_DENV-Ag2-WPREm_(Flavi-3) of SEQ ID NO: 158, or pFlap-β2m-DENV-Ag2_ZIKV-NS1-WPREm_(Flavi-4) of SEQ ID NO: 159, or pFlap-β2m-ZIKV-NS1-DENV-Ag2-WPREm_(Flavi-5) of SEQ ID NO: 160.

[0126] In another embodiment, the present invention relates to plasmids deposited at the CNCM: pFlap-β2m-DENV-Ag1-WPREm with the number CNCM I-5883, or pFlap-β2m-DENV-Ag2-WPREm with the number CNCM I-5885, or pFlap-β2m-ZIKV-Ag-WPREm with the number CNCM I-5882, or pFlap-β2m-ZIKV-NS1-WPREm with the number CNCM I-5887, or pFlap-β2m-YFV-Ag1-WPREm with the number CNCM I-5884, or pFlap-β2m-YFV-Ag2-WPREm with the number CNCM I-5886, or pFlap-β2m-DENV-Ag2_ZIKV-Ag-WPREm_(Flavi-2) with the number CNCM I-5888, or pFlap-β2m-ZIKV-Ag_DENV-Ag2-WPREm_(Flavi-3) with the number CNCM I-5889, or pFlap-β2m-DENV-Ag2_ZIKV-NS1-WPREm_(Flavi-4) with the number CNCM I-5890, or pFlap-β2m-ZIKV-NS1-DENV-Ag2-WPREm_(Flavi-5) with the number CNCM I-5891.

[0127] The present invention also relates to a fusion polypeptide as disclosed herein encoded by a recombinant polynucleotide of the present invention, in particular a fusion polypeptide encoded by a nucleic acid molecule as disclosed herein with reference to its SEQ ID No. In one embodiment, the fusion polypeptide is selected from the following:

[0128] - a polypeptide comprising an MHC class I T cell epitope of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 or a variant thereof that does not contain SEQ ID NO:2 and contains SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34 at the 5' end, wherein optionally, the coding sequences of the epitopes in the above SEQ ID No. are arranged from the N-terminus to the C-terminus in the above order or contain variants thereof as disclosed herein; and / or

[0129] - A polypeptide comprising MHC class I T cell epitopes: SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, wherein optionally, the coding sequences of the epitopes in the above SEQ ID No. are arranged from the N-terminus to the C-terminus in the above order or include variants thereof disclosed herein; and / or

[0130] - A polypeptide comprising MHC class I T cell epitopes: SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID No.78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID No86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, wherein optionally, the coding sequences of the epitopes in the above SEQ ID No. are arranged from the N-terminus to the C-terminus in the above order or include variants thereof disclosed herein; and / or

[0131] - A polypeptide comprising MHC class I T cell epitopes: SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122,SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, wherein optionally, the coding sequences of the epitopes in the above SEQ ID No. are arranged from the N-terminus to the C-terminus in the above order or include variants thereof disclosed herein.

[0132] In one embodiment, the present invention relates to a fusion polypeptide selected from the following:

[0133] - The fusion polypeptide of SEQ ID NO:30,

[0134] - The fusion polypeptide of SEQ ID NO:38,

[0135] - The fusion polypeptide of SEQ ID NO:54,

[0136] - The fusion polypeptide of SEQ ID NO:56,

[0137] - The fusion polypeptide of SEQ ID NO:106, and

[0138] - The fusion polypeptide of SEQ ID NO:142.

[0139] According to the present invention, when nucleotide sequences encoding two antigens, epitopes, antigen domain polypeptides or antigen polypeptides are ligated in-frame with each other to form a recombinant polynucleotide or gene encoding a fusion polypeptide, the two antigens, epitopes, antigen domain polypeptides or antigen polypeptides are fused to each other. The fusion between the two polypeptide sequences can be direct or indirect. When the C-terminus of the first polypeptide chain is covalently bonded to the N-terminus of the second polypeptide chain, the two polypeptides are directly fused. In this case, the linker region of the fusion polypeptide consists of the terminal amino acid residues adjacent to the residues being ligated in the polypeptide. Alternatively, the polypeptides are indirectly fused, i.e., a linker or spacer peptide or further polypeptide is present between the two fusion polypeptides to form a linker region, the amino acid residues of which are not initially included in the polypeptides to be fused. The linker region using a linker has been disclosed herein.

[0140] The polypeptide chain providing an MHC class I T cell epitope of each peptide or antigen domain comprises a sequence selected from SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:106 and SEQ ID NO:142 disclosed herein, especially consisting of, or being a variant thereof, said variant comprising an amino acid sequence having at least 85% amino acid sequence identity, preferably 90%, 94%, 95%, more preferably 98% or 99% or 94% to 99% sequence identity with the sequence of the original fusion polypeptide (such as DENV-Ag1, DENV-Ag2, ZIKV-Ag, ZIKV-NS1 or YFV-Ag1 and YFV-Ag2 used as reference sequences). In one embodiment, the polypeptide chain has 1 to 10, especially 1 to 5, more especially 1 to 3 amino acid changes relative to the corresponding reference sequence. As used herein, amino acid changes can consist of amino acid substitutions, additions or deletions. Preferably, the amino acid substitutions are conservative amino acid substitutions.

[0141] In one embodiment of the variant of the fusion polypeptide providing an MHC class I T cell epitope disclosed above, the polypeptide chain of the variant is obtained by substitution of amino acid residues. Preferably, the amino acid substitution is a conservative amino acid substitution.

[0142] According to the invention, the fusion polypeptide carries several polypeptides, which comprise or are an MHC class I T cell epitope or an antigenic domain containing the same or different non-structural antigens of the same virus; or carries several polypeptides, which comprise or are an MHC class I T cell epitope or have different antigens of different viruses (DENV, ZIKV and YFV, especially DENV and ZIKV or DENV and YFV). In a particular embodiment, in addition to the MHC class I T cell epitope or antigenic domain derived from the non-structural protein of the virus, the fusion polypeptide further comprises an MHC class I T cell epitope or antigenic domain derived from the structural protein of the virus.

[0143] Thus, the fusion polypeptide of the invention is a multi-antigen polypeptide.

[0144] "Antigen" or "antigen polypeptide" is defined herein as the wild-type or native antigen of DENV, ZIKV and YFV viruses, or as a fragment of such wild-type or native antigen, or as a mutant polypeptide, or as a synthetic antigen derived from the alignment of the available amino acid sequences or consensus sequences of the native antigens disclosed herein. When a fragment of the wild-type or native antigen or synthetic antigen is expressed by the lentiviral vector of the invention, the fragment of the wild-type or native antigen or synthetic antigen advantageously retains the immunogenic properties of the polypeptide from which it is derived or exhibits improved immunogenic properties; and when expressed in a host, advantageously exhibits immunoprotective properties. Thus, such a fragment or synthetic antigen is an antigen or an immunogenic fragment of an immunogenic antigen. The antigen used to provide the fusion polypeptide of the invention has an amino acid sequence that is sufficient to provide one or advantageously several epitopes, especially T cell epitopes and more especially CD8+ T cell epitopes, and the amino acid sequence retains immunogenicity, especially the protective properties that cause the antigen polypeptide from which it is derived to have protective activity, and / or especially exhibits such protective properties when expressed by the lentiviral vector of the invention.

[0145] In one embodiment, the binding of the antigenic domains in the fusion polypeptide is such that the antigenic domains are arranged from the N-terminus to the C-terminus in the same order as they occur in the antigen from which the antigenic domains are derived. In one embodiment, the binding of the antigenic domains in the fusion polypeptide is such that the arrangement of the antigenic domains from the N-terminus to the C-terminus is changed relative to the order in which they occur in the antigen from which the antigenic domains are derived. Examples of such altered arrangements are illustrated in the disclosed fusion polypeptides.

[0146] In a particular embodiment, more than one recombinant fusion protein is expressed by the lentiviral particles of the lentiviral vector of the present invention. In a particular embodiment, fusion polypeptides of DENV, ZIKV, and YFV, particularly DENV and ZIKV or DENV and YFV, are expressed by the same lentiviral particles of the lentiviral vector of the present invention or by a mixture of particles.

[0147] In a particular embodiment, the fusion polypeptide provides at least 2, particularly at least 3, or at least 4, or at least 5, and particularly 2, 3, 4, or 5, antigenic determinants as determined relative to the native or wild-type antigen of the pathogen, and thus encompasses at least 2, at least 3, or at least 4 antigenic determinants and / or antigenic fragments (antigen domains) or mutant antigens and / or fragments thereof. In a particular embodiment, the antigenic polypeptides contained in the fusion polypeptide comprise or consist of: up to 10 antigen fusions, advantageously up to 25 antigen fragments (such as epitopes encoded by the recombinant polynucleotides of the present invention expressed by the lentiviral vectors disclosed herein) fusions, particularly 7 to 25 antigen fragments or mutant fragments thereof fusions. The inventors have demonstrated that the fusion polypeptides of the present invention are capable of driving the expression of large antigenic polypeptides, such as one or more fusion polypeptides expressed by the lentiviral vectors disclosed herein. In one embodiment, the fusion polypeptide comprises at least 300 amino acids, particularly at least 400 amino acids, more particularly at least 400 or 500 amino acids. In one embodiment, the fusion polypeptide comprises 300 to 1400 amino acids, particularly 300 to 850 amino acids. In one embodiment, one or more fusion polypeptides expressed by the lentiviral vector comprise at least 300 amino acids, more particularly at least 400 or 500 amino acids. In one embodiment, the antigenic polypeptide comprises 300 to 1400 amino acids, particularly 300 to 850 amino acids.

[0148] According to one embodiment, the antigenic polypeptides can be fused via a linker to produce a fusion polypeptide.

[0149] The linker sequence is accordingly used to avoid the formation of new epitopes that would interfere with the specific immune response against the epitopes of the pathogen in the host body. Suitable linkers are selected by those skilled in the art according to well-known techniques and are shown in the examples.

[0150] In one embodiment, one or more antigenic polypeptides are selected and arranged within the fusion polypeptide, and a linker that reduces the appearance of new epitopes between the epitope regions is added or not added within the fusion polypeptide.

[0151] In another aspect of the present invention, the inventors have designed and prepared a lentiviral vector, namely a lentiviral vector particle, which encodes the fusion polypeptide of the present invention, and in which MHC class I T cell epitopes of more than one non-structural protein derived from DENV, ZIKV and / or YFV are fused and thus can be expressed in recombinant lentiviral particles. Accordingly, the present invention provides a novel lentiviral vector expressing the recombinant fusion polypeptide according to any one of the embodiments disclosed herein, which elicits a T cell immunogenicity covering CD8+ T cell immune responses against the fusion polypeptide in a host, or against DENV, ZIKV or YFV viruses responsive to the immune responses elicited by administering the lentiviral vector of the present invention, especially in a mammalian host, particularly in a human host.

[0152] The expression "lentiviral vector" or "lentiviral vector particle" refers to a biological or chemical entity suitable for delivering a recombinant polynucleotide encoding the fusion polypeptide of the present invention to the cells of a host to which such a vector is administered. The viral vectors such as those described herein are lentiviral vectors capable of inducing a human immune response. The present invention particularly relates to the use of HIV vectors, especially the HIV-1 vectors illustrated in the examples. The construction details of HIV-1 vectors are known in the art and are provided below and in the examples.

[0153] According to the present invention, there is provided a lentiviral vector expressing the fusion polypeptide of the present invention, wherein the vector has or contains in its genome (vector genome) a recombinant polynucleotide encoding the fusion polypeptide of the present invention. In one embodiment, the vector has or contains in its genome (vector genome) recombinant polynucleotides encoding a plurality of fusion polypeptides according to the present invention, wherein the fusion polypeptides are derived from in total more than one virus, especially 2 or 3 different viruses of the genus Flavivirus selected from dengue virus, Zika virus (ZIKV) and / or yellow fever virus (YFV). In the case of dengue virus, the 4 known serotypes of the virus can be used to derive the antigenic domains used in the fusion polypeptide. Specific embodiments of the vector genome of the lentiviral vector of the present invention have been disclosed above and in the examples.

[0154] The lentiviral vector of the present invention, particularly preferably an HIV-1-based vector, can be a replication-incompetent pseudotyped lentiviral vector, particularly a replication-incompetent pseudotyped HIV-1 lentiviral vector, wherein the vector contains a genome comprising a mammalian codon-optimized synthetic nucleic acid, particularly a human codon-optimized synthetic nucleic acid, wherein the synthetic nucleic acid encodes at least one fusion polypeptide of the present invention, the fusion polypeptide comprising one or more antigen polypeptides, particularly antigen polypeptides of a defined virus that infects mammals, particularly human hosts, as disclosed herein. The lentiviral vector can be advantageously pseudotyped with a viral envelope protein that is not a lentivirus, particularly not an HIV-1 retrovirus, envelope protein or glycoprotein. The lentiviral vector can be pseudotyped with glycoprotein G (V-SVG) of vesicular stomatitis virus from the Indiana or New Jersey serotype.

[0155] The use of codon-optimized sequences in the genome of the vector particles can particularly improve mRNA stability or reduce secondary structure, thereby particularly enabling strong expression of the antigen polypeptide in the cells of the host to which the vector is administered. In addition, the expressed antigen polypeptide undergoes post-translational modifications suitable for processing the antigen polypeptide in the host cell, and the post-translational modification is particularly carried out by modifying translation modification sites (such as glycosylation sites) in the encoded polypeptide. Codon optimization tools are well known in the art, including algorithms and services provided by, for example, GeneArt (Life technologies-USA) and DNA2.0 (Menlo Park, California-USA). In a particular embodiment, the open reading frame (ORF) sequence encoding the antigen polypeptide is codon-optimized, and the optimization is carried out before introducing the sequence encoding the ORF into the plasmid to be used for preparing the vector genome. In another embodiment, other sequences of the vector genome are also codon-optimized. Codon-optimized nucleic acids of the recombinant polynucleotides of the present invention are provided as examples.

[0156] The active ingredient composed of the viral vector can be an integrative pseudotyped lentiviral vector, particularly a replication-incompetent integrative pseudotyped lentiviral vector, particularly an HIV-1 vector. Such a lentiviral vector can additionally contain a genome comprising a mammalian codon-optimized synthetic nucleic acid, particularly a human codon-optimized nucleic acid, such as the insert contained in recombinant pFLAP of SEQ ID NO:151, SEQ ID NO:152, SEQID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159 or SEQ ID NO:160, wherein the nucleic acid encodes the fusion polypeptide according to the present invention.

[0157] Alternatively, the lentiviral vector and in particular the HIV-1-based vector can be a non-integrating replication-defective pseudotyped lentiviral vector.

[0158] A particular embodiment of the lentiviral vector suitable for implementing the present invention relates to a lentiviral vector whose genome is obtained from the pTRIP vector plasmid or pFLAPΔU3 plasmid known in the art, wherein the nucleic acid encoding the fusion polypeptide has been cloned under the control of a promoter functional in mammalian cells, which promoter is in particular the CMV promoter, the human β2-microglobulin promoter (SEQ ID NO: 170), the SP1-β2m promoter of SEQ ID NO: 171 or the composite "BCUAG" promoter of SEQ ID NO: 172, preferably the SP1-β2m promoter, and wherein the vector optionally comprises a post-transcriptional regulatory element of wild-type or mutant woodchuck hepatitis virus (WPRE - SEQ ID NO: 174). In particular, the WPRE is the mutant WPRE shown in SEQ ID NO: 173.

[0159] pFLAP-β2m-WPREm (SEQ ID NO: 161) is a lentiviral plasmid vector derived from the pFLAPΔU3 plasmid or pFLAP plasmid, which is a lentiviral plasmid vector derived from the pTRIP plasmid. Examples of the pFLAP plasmid of the present invention are pFlap-β2m-DENV-Ag1-WPREm of SEQ ID NO: 151, or pFlap-β2m-DENV-Ag2-WPREm of SEQ ID NO: 152, or pFlap-β2m-ZIKV-Ag-WPREm of SEQ ID NO: 153, or pFlap-β2m-ZIKV-NS1-WPREm of SEQ ID NO: 154, or pFlap-β2m-YFV-Ag1-WPREm of SEQ ID NO: 155, or pFlap-β2m-YFV-Ag2-WPREm of SEQ ID NO: 156, or pFlap-β2m-DENV-Ag2_ZIKV-Ag-WPREm_(Flavi-2) of SEQ ID NO: 157, or pFlap-β2m-ZIKV-Ag_DENV-Ag2-WPREm_(Flavi-3) of SEQ ID NO: 158, or pFlap-β2m-DENV-Ag2_ZIKV-NS1-WPREm_(Flavi-4) of SEQ ID NO: 159, or pFlap-β2m-ZIKV-NS1-DENV-Ag2-WPREm_(Flavi-5) of SEQ ID NO: 160.

[0160] In another embodiment of the present invention, lentiviral vector particles expressing a fusion polypeptide according to the features described herein are pseudotyped with the glycoprotein G (VSV-G) of vesicular stomatitis virus from Indiana or New Jersey serotype.

[0161] Specific features of such lentiviral vectors will be discussed in further detail below.

[0162] The present invention further relates to a host cell comprising the lentiviral vector genome of the present invention or transfected with a DNA plasmid according to the present invention, preferably a mammalian host cell. In particular, the host cell is the HEK-293T cell line or the K562 cell line. The present invention further relates to a culture of said host cells.

[0163] The present invention also relates to a formulation or pharmaceutical composition, particularly a vaccine composition, suitable for administration to a mammalian host, which comprises the recombinant lentiviral vector of the present invention and one or more pharmaceutically acceptable excipients suitable for administration to a host in need thereof, particularly a mammalian host, especially a human host.

[0164] The present invention also relates to a formulation suitable for administration to a mammalian host, particularly a human host, which comprises lentiviral vector particles as defined herein as an active ingredient for protection against viral infection or against viral-induced disorders or diseases, wherein the virus is a flavivirus as disclosed herein or one or at least one of a plurality of dengue viruses selected from the group consisting of, in particular, the known serotypes DENV-1, DENV-2, DENV-3 and DENV-4, especially a viral infection caused by any one or all of DENV-1, DENV-2, DENV-3 or DENV-4, or a flavivirus such as Zika virus (ZIKV) or yellow fever virus (YFV), and one or more excipients suitable for administration to a host in need thereof, particularly a human host.

[0165] The pharmaceutical composition according to the present invention, particularly the vaccine composition or formulation, may further comprise an adjuvant component and / or an immunostimulatory component.

[0166] In particular, the composition or formulation may comprise a Th1-promoting adjuvant such as polyinosinic acid-polycytidylic acid (poly I:C) or a derivative thereof. Derivatives of poly(I:C) refer to mismatched dsRNAs obtained by modifying the specific configuration of poly(I:C) by introducing unpaired bases therein, and include poly(I:CxU), poly(IxU:C) (where x is a value of from 3 to 40 on average), etc. Preferably, the derivative of poly(l:C) is poly(I:C12U) or poly(C:I12U), which is available under the trade name Ampligen TM and can be purchased.

[0167] The composition or formulation may further comprise a Th1 / Th17 promoting adjuvant, such as a cyclic dinucleotide adjuvant. The cyclic nucleotide adjuvant is also referred to as a cyclic dinucleotide adjuvant that activates STING. As used herein, the term "cyclic dinucleotide" ("CDN") refers to a class of molecules that contain 2'-5' and / or 3'-5' phosphodiester linkages between two purine nucleotides. This includes 2'-5'-2',5', 2'-5'-3'5', and 3',5'-3',5' linkages. CDNs are small, ubiquitous second messengers synthesized by bacteria that regulate a variety of different processes and are a relatively new class of adjuvants that have been shown to increase vaccine efficacy. CDNs activate innate immunity by directly binding to the endoplasmic reticulum-resident receptor STING (stimulator of interferon genes), activating the signaling pathway that induces the expression of interferon-β (IFN-β) and nuclear factor-κB (NF-κB)-dependent inflammatory cytokines. Preferably, the CDN is cyclic guanosine-adenosine dinucleotide (cGAMP).

[0168] The use of an adjuvant, particularly a Th1 and / or Th17 promoting adjuvant, and the lentiviral vector of the present invention can elicit the production of Th1 CD8+ T cells.

[0169] In another aspect of the present invention, the active ingredient, particularly the lentiviral vector particle or a composition or formulation comprising the same, is used for providing protective immunity against a viral infection or against a virus-induced disorder or disease, wherein the virus is a flavivirus as disclosed herein in a mammalian host, particularly a human host, or a flavivirus selected from dengue virus (particularly one or at least one of the known serotypes DENV-1, DENV-2, DENV-3, and DENV-4, especially a viral infection caused by any one or all of DENV-1, DENV-2, DENV-3, or DENV-4), Zika virus (ZIKV), or yellow fever virus (YFV), optionally in combination with a suitable delivery vehicle, and optionally in combination with an adjuvant component and / or with an immunostimulatory component (e.g., the adjuvant component and / or immunostimulatory component as defined in the present invention).

[0170] Thus, when administered to a host in need, particularly to a mammalian host, especially to a human host, the active ingredient or composition, particularly the lentiviral vector particle of the present invention, elicits an immune response that encompasses a CD8+ T cell response against the antigenic polypeptide or an immunogenic fragment thereof expressed by the fusion polypeptide. The immune response may encompass activation of naive lymphocytes, generation of an effector T cell response, and generation of an antigen-specific T cell response with immunological memory against one or more antigens of the pathogen.

[0171] One aspect of the present invention relates to the active ingredient of the present invention, in particular lentiviral vector particles, pharmaceutical compositions and / or formulations, which are used for preventing and / or treating viral infections, wherein the virus is a flavivirus disclosed herein in a mammalian host in need thereof or a plurality of flaviviruses selected from dengue virus (in particular, one or at least one of the known serotypes DENV-1, DENV-2, DENV-3 and DENV-4, especially any one or all of DENV-1, DENV-2, DENV-3 or DENV-4 causing viral infections), Zika virus (ZIKV) or yellow fever virus (YFV). The present invention also relates to a method for preventing and / or treating viral infections, wherein the virus is a flavivirus disclosed herein in a mammalian host in need thereof or a plurality of flaviviruses selected from dengue virus (in particular, one of the known serotypes DENV-1, DENV-2, DENV-3 and DENV-4), Zika virus (ZIKV) or yellow fever virus (YFV).

[0172] In a particular embodiment, the active ingredient of the present invention, in particular lentiviral vector particles, pharmaceutical compositions and / or formulations are used for preventing and / or treating infections caused by any one or all of the viruses of DENV-1, DENV-2, DENV-3 or DENV-4. Therefore, the present invention can protect the host to which the active ingredient of the present invention, in particular lentiviral vector particles, pharmaceutical compositions and / or formulations are administered from DENV infections of all serotypes.

[0173] The immune response involves the MHC-I restricted presentation of the antigen polypeptide or its immunogenic fragment contained in the fusion polypeptide of the present invention by antigen-presenting cells, especially dendritic cells, and induces a CD8-mediated immune response.

[0174] The lentiviral vector of the present invention is particularly capable of inducing the generation of pluripotent T cells, including CD8+ T cells that secrete one or more of IFN-γ, TNF-α, IL-2 and the lymphocyte degranulation marker CD107a.

[0175] The immune response can prevent viral infections or can prevent (protect against) the onset or development of pathological conditions caused by infections with dengue virus (in particular, one of the known serotypes DENV-1, DENV-2, DENV-3 and DENV-4, or especially any one or all of the known serotypes DENV-1, DENV-2, DENV-3 and DENV-4), Zika virus (ZIKV) or yellow fever virus (YFV).

[0176] Physiologically acceptable media can be selected according to the administration route of the immunocomposition. In a preferred embodiment, administration can be carried out by injection, especially intramuscularly, intradermally, subcutaneously or by intranasal administration or topical skin application.

[0177] The recombinant lentiviral vector particles of the present invention are used to elicit an immune response against a virus in a host, particularly a mammalian host, especially a human host, wherein the virus is a flavivirus disclosed herein or a plurality of flaviviruses selected from dengue virus (in particular, one of the known serotypes DENV-1, DENV-2, DENV-3, and DENV-4, or especially any one or all of the known serotypes DENV-1, DENV-2, DENV-3, and DENV-4), Zika virus (ZIKV), or yellow fever virus (YFV), and the use involves an immunization pattern comprising administering an effective amount of an active ingredient. In one embodiment, the lentiviral particles that elicit a cellular immune response in the host are administered in a single dose. In one embodiment, the lentiviral particles that elicit a cellular immune response in the host are administered as a prime immunization, and after a period of time, an effective amount of the same active ingredient or another active ingredient is administered, for example, the lentiviral particles are administered to enhance the cellular immune response of the host, and optionally, the administration step is repeated (once or several times) to further enhance it.

[0178] In each administration step of the lentiviral vector particles, especially in a regimen covering multiple administration steps, the pseudotyped envelope protein of the vector particles is preferably different from the pseudotyped envelope protein used in other steps, especially derived from different viruses, especially VSV of different serotypes. In a prime-boost regimen, the combination of compounds administered in each step comprises the lentiviral vector as defined herein.

[0179] The prime immunization and boost steps are separated in time by at least 2 weeks, especially 6 weeks, especially at least 8 weeks.

[0180] In a particular embodiment, the recombinant lentiviral vector particles of the present invention are used to elicit an immune response against a virus that provides an antigen expressed by the particles in a host, particularly a mammalian host, especially a human host, and the use involves an immunization pattern comprising a heterologous prime-boost regimen, wherein the recombinant lentiviral vector particles of the present invention are used for prime immunization or boosting. Details of the administration regimen will be further discussed below.

[0181] The LV particles provide a cellular immune response (T cell immune response), particularly a CD8+ T cell immune response, that is, an adaptive immune response mediated by activated T cells carrying the CD8 receptor.

[0182] In a particularly advantageous embodiment, the immune response conferred by the LV particles is a persistent immune response, that is, the immune response encompasses a memory cell response, and especially a central memory cell response; in a particular embodiment, the immune response can still be detected for at least several months after the last administration step.

[0183] According to the present invention, when lentiviral particles are used in a prime-boost regimen or a multi-step administration regimen, the provided lentiviral vector particles are pseudotyped with a first defined pseudotyping envelope G protein obtained from the Indiana or New Jersey strain of VSV, and subsequently the administered lentiviral vector particles are pseudotyped with a second defined pseudotyping envelope G protein obtained from the New Jersey or Indiana strain of VSV. The order of use of the first and second compounds so described in the prime-boost regimen may alternatively be reversed. Thus, when intended for use in a prime-boost regimen, the lentiviral vector particles contained in the individual active ingredient / compound in the composition or composition of the present invention are different from each other, the difference being at least attributable to the specific pseudotyping envelope protein used to pseudotype the vector particles.

[0184] When using an integrating vector, the dose of the lentiviral vector intended to elicit a cellular immune response used in the administration mode may comprise 10 5 TU to 10 10 TU of recombinant lentiviral particles, in particular 10 5 TU to 10 8 TU. When using a non-integrating competent vector, the dose intended to be administered to the host may comprise 10 8 to 10 10 of each type of recombinant lentiviral vector particle.

[0185] The present invention also relates to a method of providing immunity in a mammalian host, particularly a human host, which comprises the steps of: according to the present invention, administering, as a prime or a boost, the recombinant lentiviral vector particles of the present invention to elicit an immune response, and optionally repeating the administration step one or more times, in particular to enhance the response.

[0186] Optionally, the recombinant lentiviral vector particles may be used in combination with an adjuvant compound suitable for administration to a mammalian, particularly a human host, and / or with an immunostimulatory compound and an appropriate delivery vehicle. Suitable adjuvant and immunostimulatory compounds are described in this specification.

[0187] The recombinant lentiviral vector particles may be administered to the host via different injection routes, including subcutaneous (s.c.), intradermal (i.d.), intramuscular (i.m.) or intravenous (i.v.) injection, or may be administered orally for local administration via the mucosa or skin, in particular intranasal (i.n.) administration or inhalation. The amount (dose) to be administered depends on the subject to be treated, including taking into account the patient's condition, the state of the individual immune system, the administration route and the body size of the host. The suitable dose range may be determined according to the content of the equivalent transduction units of the HIV-1-derived lentiviral vector particles.

[0188] Examples and description of the drawings Preparation and application of lentiviral vector particles, which can be combined individually with the definitions given in this specification. Other examples and features of the present invention will be apparent upon reading the examples and drawings.

[0189] DETAILED DESCRIPTION OF THE LENTIVIRAL VECTOR USED IN THE PRESENT INVENTION

[0190] Accordingly, the present invention relates to a lentiviral vector, which is a recombinant lentiviral particle (i.e., a recombinant vector particle), and can be a replication-defective lentiviral vector, particularly an HIV-1-based replication-defective vector, characterized in that: (i) it is pseudotyped with a defined heterologous viral envelope protein or a viral envelope protein derived from an RNA virus (non-HIV), and (ii) it contains in its genome at least one recombinant polynucleotide encoding a fusion polypeptide of the present invention, the fusion polypeptide comprising at least one antigen polypeptide (or its polypeptide derivative, such as its immunogenic fragment) carrying an epitope of a viral antigen, wherein the virus is a flavivirus disclosed herein or a flavivirus selected from dengue virus (particularly one of the known serotypes DENV-1, DENV-2, DENV-3, and DENV-4, or any one or all of the known serotypes DENV-1, DENV-2, DENV-3, and DENV-4 in particular), Zika virus (ZIKV), or yellow fever virus (YFV), and wherein the epitope encompasses T cell epitopes, particularly CD8+ T cell epitopes.

[0191] According to a specific embodiment of the present invention, the lentiviral vector is designed to be an expression-competent (i.e., integration-competent) or expression-defective (i.e., non-integration-competent) particle. According to a specific embodiment of the present invention, the recombinant lentiviral vector particle is both non-integration-competent and replication-defective.

[0192] The preparation of lentiviral vectors is well known to those skilled in the art and has been widely disclosed in the literature (see Sakuma T. et al. (Biochem. J. (2012) 443, 603-618)). The preparation of such vectors is also illustrated in the examples herein.

[0193] In a specific embodiment of the present invention, one or more polynucleotides (ORFs) encoding the antigen polypeptides of the lentiviral vector have been codon-optimized (CO) for mammals, particularly for humans. Optionally, the lentiviral sequences of the genome of the particle also have a codon-optimized nucleotide sequence for mammals. In a particular aspect of the present invention, codon optimization has been performed for expression in mouse cells. In another embodiment, the sequence of one or more polynucleotides encoding the antigen polypeptides of the lentiviral vector has been codon-optimized (CO) for humans.

[0194] It has been observed that codon-optimized nucleotide sequences, especially when optimized for expression in mammalian and especially human cells, are capable of producing a higher yield of particles in such mammalian or human cells. The production cells are illustrated in the Examples. Thus, when the lentiviral vector particles of the present invention are administered to a mammal, especially to a human host, more particles are produced in the host body, which is beneficial for triggering a strong immune response.

[0195] The recombinant lentiviral vector (i.e., lentiviral vector particle or lentivirus-based vector particle) as defined in the present invention is a pseudotyped lentiviral vector, which consists of vector particles carrying one or more envelope proteins derived from a virus different from a specific lentivirus (especially different from HIV, especially HIV-1), and this envelope protein provides the vector genome of the lentiviral vector particle. Therefore, the envelope protein is one or more "heterologous" viral envelope proteins relative to the vector genome of the particle. In the following pages, "envelope protein" will also be mentioned to cover any type of one or more envelope proteins suitable for implementing the present invention.

[0196] When referring to a "lentiviral" vector (lentivirus-based vector) in the present invention, it especially relates to an HIV-based vector, and especially an HIV-1-based vector.

[0197] The lentiviral vector suitable for implementing the present invention is a so-called replacement vector, which means that the sequence of the original lentivirus encoding the lentiviral protein is substantially absent in the genome of the vector, or when present, is modified, especially mutated, especially truncated, to prevent the expression of biologically active lentiviral proteins, especially in the case of HIV, to prevent the transfer vector providing the genome of the recombinant lentiviral vector particle from expressing functional ENV, GAG, and POL proteins and optionally other structural proteins and / or accessory proteins and / or regulatory proteins of the lentivirus, especially HIV.

[0198] In a particular embodiment, the lentiviral vector is constructed from first-generation vectors, particularly first-generation HIV-based vectors, characterized in that it uses separate plasmids to provide (i) the packaging construct, (ii) the envelope, and (iii) the transfer vector genome of the vector. Alternatively, it can be constructed from second-generation vectors, particularly second-generation HIV-based vectors, which additionally do not contain viral accessory proteins (such as in the case of HIV-1, Vif, Vpu, Vpr, or Nef), and thus include only four of the nine intact HIV genes: gag, pol, tat, and rev. In another embodiment, the vector is constructed from third-generation vectors, particularly third-generation HIV-based vectors, which additionally do not contain the said viral accessory proteins and are also Tat-independent; these third-generation vectors can use 4 plasmids to provide the functional elements of the vector, including, when the vector is based on HIV-1, a plasmid encoding the Rev protein of HIV. Such a vector system contains only three of the nine genes of HIV-1. The structures and designs of the said generations of HIV-based vectors are well known in the art.

[0199] In any of these generations of vectors, additional modifications are made according to the invention by inserting into the vector backbone a polynucleotide encoding the fusion polypeptide described herein to provide an LV vector for targeting and activating APCs (particularly dendritic) to induce a cellular immune response, particularly a CD8+ T cell response.

[0200] Specific features of the lentiviral vectors used in various embodiments of the invention are also disclosed in the examples, and such features can be used alone or in combination to generate the vectors.

[0201] According to one embodiment of the invention, the lentiviral vector particles are pseudotyped with a heterologous viral envelope protein or viral polyprotein from the envelope of an RNA virus that is not a lentivirus whose lentiviral sequence provides the genome of the lentiviral particles.

[0202] As an example of a typing envelope protein for use in preparing lentiviral vectors, the invention relates to the viral transmembrane glycoprotein (the so-called G protein) envelope protein of vesicular stomatitis virus (VSV), which is, for example, selected from the VSV-G protein of the Indiana strain and the VSV-G protein of the New Jersey strain.

[0203] Other examples of VSV-G proteins that can be used to pseudotype the lentiviral vectors of the present invention include VSV-G glycoproteins, which can be selected in particular from species classified in the genus Vesiculovirus: Carajas virus (CJSV), Chandipura virus (CHPV), Cocal virus (COCV), Isfahanvirus (ISFV), Maraba virus (MARAV), Piry virus (PIRYV), Vesicular stomatitis Alagoas virus (VSAV), Vesicular stomatitis Indiana virus (VSIV), Vesicular stomatitis New Jersey virus (VSNJV) and / or strains temporarily classified in the genus Vesiculovirus: Grass carp rhabdovirus, BeAn 157575, Boteke virus (BTKV), Calchaqui virus (CQIV), Eel virus American (EVA), Gray Lodge virus (GLOV), Jurona (JURV), Klamath virus (KLAV), Kwatta virus (KWAV), La Joya virus (LJV), Malpais Spring virus (MSPV), Mount Elgon batvirus (MEBV), Perinet virus (PERV), Pike fry rhabdovirus (PFRV), Porton virus (PORV), Radi virus (RADIV), Spring viremia of carp virus (SVCV), Tupaia virus (TUPV), Ulcerative disease rhabdovirus (UDRV) and Yug Bogdanovac virus (YBV).

[0204] The vesicular stomatitis virus (VSV-G) envelope glycoprotein is a transmembrane protein that functions as the surface coat of wild-type virus particles. It also serves as an envelope protein suitable for engineered lentiviral vectors. Currently, nine virus species are clearly classified as the VSV group, and nineteen rhabdoviruses are provisionally classified in this group, all of which show varying degrees of cross-neutralization. When sequenced, the protein G gene indicates sequence similarity. The VSV-G protein has an N-terminal extracellular domain, a transmembrane region, and a C-terminal cytoplasmic tail region. It is exported to the cell surface via the trans-Golgi network (endoplasmic reticulum and Golgi apparatus).

[0205] Vesicular stomatitis Indiana virus (VSIV) and Vesicular stomatitis New Jersey virus (VSNJV) are preferred strains for pseudotyping the lentiviral vectors of the present invention or for designing recombinant envelope proteins to pseudotype lentiviral vectors. Their VSV-G proteins are publicly available in GenBank, where several strains are shown. For the VSV-G New Jersey strain, particular reference is made to the sequence with accession number V01214. For the VSV-G Indiana strain, reference is made to the sequence with accession number AAA48370.1 corresponding to strain JO2428 in GenBank.

[0206] The viral envelope protein can be taken up by antigen-presenting cells and particularly dendritic cells (including hepatic dendritic cells) by fusion and / or endocytosis. In a particular embodiment, the uptake efficiency can be used as a characteristic for selecting the VSV envelope for pseudotyping. In this regard, the relative transduction titer (DC titer / titer of other transducing cells such as 293T cells) can be considered as a test, and an envelope with a stronger ability to fuse with DCs is preferred.

[0207] Antigen-presenting cells (APCs) and particularly dendritic cells (DCs) are accordingly suitable target cells for pseudotyped lentiviral vectors used as immunocompositions.

[0208] The VSV-G envelope protein is expressed from a polynucleotide containing the coding sequence of said protein, wherein said polynucleotide is inserted into a plasmid (referred to as an envelope expression plasmid or pseudotyping envelope plasmid) for preparing the lentiviral vector particles of the present invention. The polynucleotide encoding the envelope protein is under the control of regulatory sequences for transcription and / or expression of the coding sequence, including optionally present post-transcriptional regulatory elements (PREs), particularly polynucleotides such as the element of the woodchuck hepatitis virus obtainable from Invitrogen (i.e., the WPRE sequence) or a mutant sequence of the WPRE shown in SEQ ID NO:174.

[0209] Accordingly, there is provided a nucleic acid construct comprising an internal promoter suitable for mammalian cells, in particular human cells in vivo, and a nucleic acid encoding an envelope protein under the control of said promoter. A plasmid containing this construct is used to transfect cells suitable for preparing vector particles. The promoter can be selected in particular according to its properties as a constitutive promoter, a tissue-specific promoter or an inducible promoter. Examples of suitable promoters include the promoters of the following genes: the MHC class I promoter disclosed by Jones S. et al. (Jones S. et al. Human Gene Therapy, 20:630-640 (June 2009)), the human β-2 microglobulin gene (β2M promoter), EF1α, human PGK, PPI (preproinsulin), thiodextrin, HLA DR invariant chain (P33), HLA DR α chain, ferritin L chain or ferritin H chain, chymase β4, chymase β10, cystatin ribosomal protein L41, CMVie or a chimeric promoter such as GAG (CMV early enhancer / chicken β-actin), or β-2m-CMV (BCUAG) disclosed herein.

[0210] These promoters can also be used in regulatory expression sequences involved in expressing gag-pol-derived proteins from the encapsidated plasmid, and / or for expressing antigenic polypeptides from the transfer vector.

[0211] Alternatively, when the envelope expression plasmid is intended to be used for expression in a stable packaging cell line, in particular for stable expression (such as continuously expressing viral particles), the internal promoter expressing the envelope protein is advantageously an inducible promoter, such as an inducible promoter disclosed by Cockrell A.S. et al. (Mol. Biotechnol. (2007) 36:184-204). As examples of such promoters, reference is made to the tetracycline and ecdysone inducible promoters. The packaging cell line can be a STAR packaging cell line (refer to Cockrell A.S. et al. (2007), Ikedia Y. et al. (2003) Nature Biotechnol. 21:569-572) or a SODk packaging cell line, such as a cell line derived from SODk0, including SODk1 and SODk3 (refer to Cockrell A.S. et al. (2007), Cockrell A.S. et al. (2006) Molecular Therapy, 14:276-284, Xu K. et al. (2001), Kafri T. et al. (1999) Journal of Virol. 73:576-584).

[0212] According to the present invention, the lentiviral vector is a product recovered from mammalian cells co-transfected with the following substances:

[0213] - A vector plasmid, which contains (i) lentiviral cis - active sequences required for encapsulation, reverse transcription and transcription, especially those of HIV - 1, and further contains a functional lentiviral (especially derived from HIV - 1) DNA flap element, and (ii) at least one polynucleotide encoding the fusion polypeptide of the present invention, which itself contains one or more antigenic polypeptides or their immunogenic fragments capable of generating an immune response against one or more viruses, wherein the virus is a flavivirus disclosed herein under the control of a regulatory expression sequence or one of a plurality of flaviviruses selected from dengue virus (especially one of the known serotypes DENV - 1, DENV - 2, DENV - 3 and DENV - 4), Zika virus (ZIKV) or yellow fever virus (YFV), preferably the human β2 - microglobulin promoter or a modified human β2 - microglobulin promoter, such as the SP1 - β2m promoter, and optionally contains sequences for integration into the genome of the host cell;

[0214] - An expression plasmid encoding a pseudotyped envelope derived from an RNA virus, the expression plasmid containing a polynucleotide encoding one or more envelope proteins for pseudotyping, wherein the envelope pseudotyping protein advantageously comes from VSV and especially VSV - G of the Indiana strain or the New Jersey strain, and

[0215] - A capsid plasmid, which contains a lentivirus suitable for generating integration - competent vector particles, especially the HIV - 1 gag - pol encapsulation sequence, or a modified gag - pol encapsulation sequence suitable for generating integration - defective vector particles.

[0216] Therefore, the present invention also relates to the lentiviral vector particles described above, which are products recovered from a stable cell line transfected with the following:

[0217] - A vector plasmid, which contains (i) lentiviral cis - active sequences required for encapsulation, reverse transcription and transcription, especially those of HIV - 1, and further contains a functional lentivirus, especially the HIV - 1 DNA flap element and optionally contains cis - active sequences required for integration, and the vector plasmid further contains (ii) a recombinant polynucleotide, especially a recombinant polynucleotide with mouse or human codon - optimized sequences, which encodes the fusion polypeptide of the present invention and contains one or more antigenic polypeptides or their immunogenic fragments of one or more viruses disclosed herein under the control of a regulatory expression sequence (especially a promoter);

[0218] - A VSV - G envelope expression plasmid, which contains a polynucleotide encoding the VSV - G envelope protein, especially the VSV - G envelope protein of the Indiana strain or the New Jersey strain, wherein the polynucleotide is under the control of a regulatory expression sequence, especially an expression sequence containing a promoter, and;

[0219] - A capsidated plasmid, wherein the capsidated plasmid contains a lentivirus suitable for generating integration-competent vector particles, in particular an HIV-1 gag-pol coding sequence, or a modified gag-pol coding sequence suitable for generating integration-defective vector particles, wherein the gag-pol sequence is from the same lentiviral subfamily as the DNA flap element, and wherein the lentiviral gag-pol or modified gag-pol sequence is under the control of a regulatory expression sequence.

[0220] A stable cell line expressing the vector particles of the present invention is particularly obtained by transfecting the plasmid.

[0221] Thus, the vector plasmid may contain one or several expression cassettes for expressing various fusion polypeptides, or may contain a bicistronic or polycistronic expression cassette, wherein the recombinant polynucleotide encoding the fusion polypeptide containing the antigen polypeptide is optionally separated by an IRES sequence (internal ribosome entry site) of viral origin or by a sequence encoding a 2A peptide as disclosed herein.

[0222] Internal promoters (as transgenes or in expression cassettes) contained in the vector genome and controlling the expression of recombinant polynucleotides encoding viral fusion polypeptides may be selected from the promoters of the following genes: the MHC class I promoters disclosed in Jones S. et al. (2009), such as the human β-2 microglobulin gene (β2M promoter), the SP1-β2m promoter, or EF1a, human PGK, PPI (preproinsulin), thioredoxin, HLA DR invariant chain (P33), HLA DR α chain, ferritin light chain or ferritin heavy chain, chymase β4, chymase β10, or cystatin ribosomal protein L41, CMVie or chimeric promoters, such as GAG (CMV early enhancer / chicken β-actin) or BCUAG.

[0223] Promoters among the above internal promoters may also be selected for expressing envelope proteins and encapsidated (gag-pol-derived) proteins.

[0224] In the preparation of lentiviral vectors based on human lentiviruses, and in particular on HIV-1 viruses, the following specific embodiments may be carried out.

[0225] According to the present invention, the genome of the lentiviral vector is derived from a human lentivirus, in particular from an HIV lentivirus. In particular, the pseudotyped lentiviral vector is an HIV-based vector, such as an HIV-1-based or an HIV-2-based vector, in particular derived from HIV-1M, for example derived from the BRU or LAI isolates. Alternatively, the lentiviral vector providing the essential sequences of the vector genome may be derived from a lentivirus capable of transducing mammalian cells, such as EIAV, CAEV, VISNA, FIV, BIV, SIV, HIV-2, HIV-O.

[0226] As described above, except for the recombinant polynucleotide it finally contains, the vector genome is a replacement vector, in which the nucleic acid between the two long terminal repeats (LTRs) in the original lentiviral genome has been restricted to cis-acting sequences for DNA or RNA synthesis and processing, including those for effectively delivering a transgene to the nucleus of a host, or at least deleted or mutated to essential nucleic acid fragments that would be capable of expressing lentiviral structural proteins including biologically functional GAG polyprotein and possibly POL and ENV proteins.

[0227] In a specific embodiment, the 5' LTR and 3' LTR sequences of a lentivirus are used in the vector genome, but the 3' LTR is at least modified relative to the 3' LTR of the original lentivirus, at least in the U3 region, for example, the enhancer may be deleted or partially deleted (ΔU3). The 5' LTR can also be modified, especially in its promoter region, where, for example, a Tat-independent promoter can replace the U3 endogenous promoter.

[0228] In a specific embodiment, the vector genome contains the coding sequences of one or several (for HIV-1 lentiviral vectors) Vif-, Vpr, Vpu-, and Nef- accessory genes. Alternatively, these sequences can be independently or mutually deleted, or can be non-functional (second-generation lentiviral vectors).

[0229] The vector genome of a lentiviral vector particle contains at least one polynucleotide as an inserted cis-acting fragment, which is included in or contains such a DNA flap element. In a specific embodiment, the DNA flap is inserted upstream of the polynucleotide encoding the fusion polypeptide of the present invention carrying one or more antigen polypeptides, and advantageously (but not necessarily) is located at an approximate central position in the vector genome. The DNA flap applicable to the present invention can be obtained from retroviruses, especially lentiviruses, particularly human lentiviruses, especially HIV-1 retroviruses, or from retrovirus-like organisms such as retrotransposons. Alternatively, it can be obtained from viruses such as CAEV (caprine arthritis encephalitis virus), EIAV (equine infectious anemia virus), VISNA virus, SIV (simian immunodeficiency virus), or FIV (feline immunodeficiency virus). The DNA flap can be prepared synthetically (chemical synthesis) or by amplifying the DNA providing the DNA flap from a suitable source as defined above, such as by polymerase chain reaction (PCR). In a more preferred embodiment, the DNA flap is obtained from an HIV retrovirus, such as HIV-1 or HIV-2 virus, including any isolate of these two types.

[0230] The DNA flap (also referred to as cPPT / CTS) (defined in Zennou V. et al., References 27, 2000, Cell vol 101, 173 - 185 or WO 99 / 55892 and WO 01 / 27304) is a structure located at the center of the genomes of some lentiviruses, particularly HIV, where the DNA flap gives rise to a 3-strand DNA structure that is typically synthesized during HIV reverse transcription in particular and serves as a cis-determinant for nuclear import of the HIV genome. During reverse transcription, the DNA flap enables a central strand displacement event cis-controlled by the central polypurine tract (cPPT) and the central termination sequence (CTS). When inserted into a lentivirus-derived vector, the polynucleotide enabling the generation of the DNA flap during reverse transcription stimulates gene transfer efficiency and restores nuclear import levels to wild-type levels (Zennou et al., Cell, 2000 Cell vol 101, 173 - 185 or WO 99 / 55892 and WO 01 / 27304).

[0231] In the prior art, the sequences of the DNA flap have been disclosed, particularly in the patent applications cited above. These sequences are also disclosed in the sequences of the pTRIP vector described herein. The sequence of the DNA flap is preferably inserted into the vector genome as a fragment optionally having additional flanking sequences, preferably in a position close to the center of the vector genome. Alternatively, the sequence of the DNA flap can be inserted directly upstream of a promoter controlling the expression of a polynucleotide encoding the fusion polypeptide of the present invention. Depending on its origin and preparation, the fragment containing the DNA flap inserted into the vector genome can have a sequence of about 80 bp to about 200 bp.

[0232] According to a particular embodiment, the DNA flap has a nucleotide sequence of about 90 to about 140 nucleotides.

[0233] In HIV-1, the DNA flap is a stable 99-nucleotide-long positive-strand overlap. When used in the genome vector of the lentiviral vector of the present invention, the DNA flap can be inserted as a longer sequence, particularly when the DNA flap is prepared as a PCR fragment. A particularly suitable polynucleotide containing the structure providing the DNA flap is a 124-base pair polymerase chain reaction (PCR) fragment containing the cPPT and CTS regions of HIV-1 DNA.

[0234] It is stipulated that the DNA flap used in the genome vector and the polynucleotide of the encapsidation plasmid encoding the GAG and POL polyproteins should be derived from the same lentiviral subfamily or from the same retrovirus-like organism.

[0235] Preferably, other cis-activating sequences of the genomic vector also originate from the same lentivirus or retrovirus-like organism that provides the DNA flap.

[0236] The vector genome may further comprise one or several unique restriction sites for cloning recombinant polynucleotides.

[0237] In a preferred embodiment, in the said vector genome, the 3' LTR sequence of the lentiviral vector genome lacks at least the activator (enhancer) of the U3 region and may lack the promoter. In another specific embodiment, the 3' LTR region lacks the U3 region (ΔU3). In this regard, reference is made to the descriptions in WO 01 / 27300 and WO 01 / 27304.

[0238] In a specific embodiment, in the vector genome, the U3 region of the 5' LTR is replaced by a non-lentiviral U3 or a promoter suitable for driving tat-independent primary transcription. In this case, the vector is independent of the tat trans-activator (third-generation vector).

[0239] The vector genome also contains the psi (ψ) packaging signal. The packaging signal is derived from the N-terminal fragment of the gag ORF. In a specific embodiment, the sequence of this packaging signal can be modified by one or more frameshift mutations to prevent any interference between the possible transcription / translation of the gag peptide and the transcription / translation of the transgene.

[0240] The vector genome may also optionally contain elements selected from a splice donor site (SD), a splice acceptor site (SA), and / or a Rev response element (RRE).

[0241] According to a specific embodiment, the vector plasmid (or the added genomic vector) contains the following cis-acting sequences for the transgene expression cassette:

[0242] 1. The LTR sequences (long terminal repeats) required for reverse transcription, the sequences required for transcription, and including the sequences optionally present for viral DNA integration. The 3' LTR deletion in the U3 region (at least the promoter) to provide a SIN vector (self-inactivating) without interfering with the functions essential for gene transfer, for two main reasons: first, to avoid the trans-activation of host genes once the DNA is integrated into the genome; second, to allow the self-inactivation of the retroviral cis-sequences after reverse transcription. Optionally, the tat-dependent U3 sequence from the 5' LTR driving genomic transcription is replaced by a non-endogenous promoter sequence. Thus, in the target cell, only the sequences from the internal promoter will be transcribed (transgene).

[0243] 2. The ψ region, which is necessary for viral RNA encapsidation.

[0244] 3. The RRE sequence (Rev response element), which allows viral messenger RNA to be exported from the cell nucleus to the cytosol after binding to the Rev protein.

[0245] 4. The DNA flap element (cPPT / CTS) that promotes nuclear import.

[0246] 5. Optionally present post-transcriptional regulatory elements, in particular elements that improve the expression of the fusion polypeptide and / or the antigen polypeptide in dendritic cells, such as the WPRE cis-acting sequence (woodchuck hepatitis B virus post-response element) are also added to optimize mRNA stability (Zufferey et al., 1999), matrix or scaffold attachment regions (SAR and MAR sequences), such as those of the immunoglobulin κ gene (Park F. et al. Mol Ther 2001; 4:164-173).

[0247] The lentiviral vector of the present invention is non-replicating (replication-defective), i.e., the vector and the lentiviral vector genome are considered suitable for alleviating concerns about replication-competent lentiviruses, in particular the inability to form new particles developed from the infected host cells after administration. This can be achieved in a well-known manner due to the absence of the gag, pol or env genes in the lentiviral genome, or their absence as "functional genes". Therefore, the gag and pol genes are provided only in trans. This can also be achieved by deleting other virus-encoded sequences and / or cis-acting gene elements required for particle formation.

[0248] "Functional" means a gene that is correctly transcribed and / or correctly expressed. Thus, in this embodiment, if sequences containing gag, pol or env are present in the lentiviral vector genome of the present invention, they are not transcribed or incompletely transcribed respectively; the expression "incompletely transcribed" refers to alterations in the transcripts gag, gag-pro or gag-pro-pol, where one or several are not transcribed. Other sequences involved in lentiviral replication may also be mutated in the vector genome to achieve this state. The non-replication of the lentiviral vector should be distinguished from the replication of the lentiviral genome. In fact, as mentioned above, the lentiviral genome may contain an origin of replication that ensures the replication of the lentiviral vector genome, but not necessarily the replication of the vector particles.

[0249] To obtain a lentiviral vector according to the present invention, the vector genome (as a vector plasmid) must be encapsidated by particles or pseudoparticles. Thus, in addition to the envelope protein, lentiviral proteins must be provided in trans with the vector genome to the production system, in particular the vector genome in the production cells, which depends on at least one encapsidation plasmid that carries the gag gene and the pol lentiviral gene or an integrase-incompetent pol gene, and preferably lacks some or all of the coding sequences of the Vif-, Vpr, Vpu, and Nef accessory genes, and optionally lacks Tat (for HIV-1 lentiviral vectors).

[0250] Use another plasmid that carries a polynucleotide encoding an envelope pseudotyping protein selected for pseudotyping lentiviral vector particles.

[0251] In a preferred embodiment, the encapsidation plasmid encodes only the lentiviral proteins necessary for viral particle synthesis. The accessory genes that may cause safety problems present in the plasmid are accordingly removed. Thus, the viral proteins provided in trans for encapsidation are respectively shown by the viral proteins derived from HIV-1:

[0252] 1. GAG proteins for constructing the matrix (MA, with an apparent molecular weight of p17), capsid (CA, p24), and nucleocapsid (NC, p6).

[0253] 2. POL-encoded enzymes: integrase, protease, and reverse transcriptase.

[0254] 3. TAT and REV regulatory proteins, when TAT is necessary for initiating LTR-mediated transcription; if the U3 region of the 5' LTR is replaced by a promoter that drives tat-independent transcription, TAT expression can be omitted. REV can be modified and accordingly used, for example, in a recombinant protein that will be able to recognize the domain that replaces the RRE sequence in the vector genome, or as a fragment that can bind to the RRE sequence via its RBD (RNA binding domain).

[0255] To avoid any encapsidation of the mRNA produced by the genes contained in the encapsidation plasmid in the viral particles, the ψ region is removed from the encapsidation plasmid. A heterologous promoter is inserted into the plasmid to avoid recombination problems, and a polyadenylation tail (poly-A tail) is added at the 3' of the sequence encoding the protein. Suitable promoters have been disclosed above.

[0256] The envelope plasmid encodes the envelope protein for pseudotyping disclosed herein under the control of the internal promoter disclosed herein.

[0257] Any or all of the described plasmids for preparing the lentiviral vector particles of the present invention may be codon-optimized (CO) in the fragment encoding the protein. Codon optimization according to the present invention is preferably carried out to improve the translation of the coding sequences contained in the plasmids in mammalian cells (mouse or especially human cells). According to the present invention, codon optimization is particularly suitable for directly or indirectly improving the preparation of the vector particles, or improving the uptake of the host cells to which they are administered, or improving the transfer efficiency of the polynucleotide encoding the fusion polypeptide (which comprises an antigen polypeptide (transgene)) into the genome of the transduced cells of the host. Codon optimization is illustrated for the coding sequences used in the examples.

[0258] In a particular embodiment of the present invention, the pseudotyped lentiviral vector is also integrase-competent, or alternatively, integrase-competent, so as to be able to integrate the vector genome and the recombinant polynucleotide contained therein into the genome of the transduced cells or into the cells of the host to which it is administered.

[0259] In another particular embodiment of the present invention, the pseudotyped lentiviral vector is also integrase-deficient, or alternatively, integrase-deficient. In this case, the vector genome and thus the recombinant polynucleotide contained therein will not integrate into the genome of the transduced cells or into the cells of the host to which it has been administered.

[0260] Therefore, the recombinant lentiviral vector particles of the present invention may be recombinant integrase-deficient lentiviral vector particles, in particular, wherein the recombinant integrase-deficient lentiviral vector particles are HIV-1-based vector particles and have integrase deficiency for the following reasons: the integrase gene encoded in the lentiviral genome is mutated such that the integrase is not expressed or not functionally expressed, in particular, the integrase gene mutation results in a substitution at amino acid residue 64 of the integrase, in particular, the substitution D64V is made in the catalytic domain of the HIV-1 integrase encoded by Pol.

[0261] The present invention relates to the use of a lentiviral vector in an immunogenic composition, wherein the expressed integrase protein is defective and further comprises at least one polynucleotide encoding at least one fusion polypeptide of the present invention, in particular, comprising at least one antigen polypeptide carrying a viral epitope.

[0262] "Integrase-deficient" means that the integrase, preferably the integrase of lentiviral origin, lacks the ability to integrate the lentiviral genome into the host cell genome, i.e., an integrase protein that has been mutated to specifically alter its integrase activity.

[0263] The non-integrating competent lentiviral vector is obtained by modifying the pol gene encoding integrase, thereby generating a mutant pol gene encoding an integrase-deficient integrase, and the modified pol gene is contained in a packaging plasmid. Such non-integrating competent lentiviral vectors have been described in patent application WO 2006 / 010834. Thus, the integrase ability of the protein is altered, while the correct expression of the packaging plasmid of the GAG, PRO and POL proteins and / or the formation of the capsid and thus the formation of vector particles, as well as other steps of the viral cycle before or after the integration step (such as reverse transcription, nuclear import) remain unchanged. When compared with a lentiviral vector containing the corresponding wild-type integrase, the integration that the integrase should be able to achieve is altered in such a way that the incidence of the integration step is less than 1 / 1000, preferably less than 1 / 10000, and the integrase is said to have the defect.

[0264] In a specific embodiment of the present invention, the defective integrase is caused by a class 1 mutation, preferably an amino acid substitution (one amino acid substitution) or a short deletion that meets the requirements for the expression of the defective integrase. The mutation is made within the pol gene. These vectors may carry a defective integrase with the mutation D64V in the catalytic domain of the enzyme, which specifically blocks the DNA cleavage and ligation reactions in the integration step. The D64V mutation reduces the integration of pseudotyped HIV-1 to at most 1 / 10,000 of the wild type, but retains its ability to transduce non-dividing cells, thus enabling efficient transgene expression.

[0265] Other mutations in the pol gene suitable for affecting the integrase ability of HIV-1 integrase are as follows: H12N, H12C, H16C, H16V, S81R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D116I, D116A, N120G, N120I, N120E, E152G, E152A, D-35-E, K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199C, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264H.

[0266] In a particular embodiment, the mutation in the pol gene is made at any one of the following positions D64, D116 or E152, or at several of these positions located at the catalytic site of the protein. Any substitution at these positions is suitable, including those described above.

[0267] Another proposed substitution is the replacement of the amino acid residues RRK (positions 262 to 264) with the amino acid residue AAH.

[0268] In a particular embodiment of the invention, when the lentiviral vector is non-integration competent, the lentiviral genome further comprises an origin of replication (ori), the sequence of which depends on the nature of the cell in which the lentiviral genome must be expressed. The origin of replication can be of eukaryotic origin, preferably mammalian origin, most preferably human origin. Alternatively, it can be of viral origin, especially from episomal DNA, such as SV40 or RPS. An advantageous embodiment of the invention is the insertion of an origin of replication into the lentiviral genome of the lentiviral vector of the invention. In fact, when the lentiviral genome is not integrated into the cell host genome (due to defective integrase), the lentiviral genome is lost in cells undergoing frequent cell division; this is especially true in immune cells, such as B cells or T cells. The presence of the origin of replication ensures the presence of at least one lentiviral genome in each cell, even after cell division, thus maximizing the efficiency of the immune response.

[0269] The lentiviral vector genome of the lentiviral vector of the invention can in particular be derived from the HIV-1 plasmid pFlap-β2m-WPREm (6155 bp) (SEQ ID NO: 161), which contains the restriction sites BamHI and XhoI for the insertion of a transgene or an expression cassette.

[0270] Vector particles can be produced after transfection of appropriate cells (such as mammalian cells or human cells, such as human embryonic kidney cells shown by 293T cells) with the plasmid or by other processes. In the cells used for expressing lentiviral particles, all or some of the plasmids can be used for stable expression of their encoded polynucleotides, or transient or semi-stable expression of their encoded polynucleotides.

[0271] The concentration of the produced particles can be determined by measuring the P24 (HIV-1 capsid protein) content of the cell supernatant.

[0272] Once administered to a host, the lentiviral vector of the invention infects the host's cells, which may be specific cells, depending on the envelope protein by which it is pseudotyped. The infection results in the release of the lentiviral vector genome into the cytoplasm of the host cell where reverse transcription occurs. Once in the triple helix form (via DNA flap), the lentiviral vector genome enters the nucleus where one or more polynucleotides encoding one or more polypeptides encoding one or more antigens are expressed via the cellular machinery. When transducing non-dividing cells (such as DCs), the expression can be stable. When transducing dividing cells (such as B cells), the expression is transient in the absence of an origin of replication in the lentiviral genome due to nucleic acid dilution and cell division. By providing an origin of replication to ensure proper dissemination of the lentiviral vector genome to daughter cells after cell division, the expression can be more persistent. Stability and / or expression can also be increased by inserting MAR (matrix attachment region) or SAR (scaffold attachment region) elements into the vector genome.

[0273] In fact, these SAR or MAR regions are ATR-rich sequences and are capable of anchoring the lentiviral genome to the matrix of the cell chromosome, thereby regulating the transcription of the polynucleotide encoding the fusion polypeptide of the invention comprising at least one antigenic polypeptide and in particular stimulating the gene expression of the transgene and improving chromatin accessibility.

[0274] If the lentiviral genome is non-integrated, it does not integrate into the host cell genome. However, at least one polypeptide encoded by the transgene is expressed sufficiently and for a long enough time to be processed, associated with MHC molecules and ultimately directed to the cell surface. Depending on the nature of the one or more polynucleotides encoding one or more antigenic polypeptides of a pathogen, at least one polypeptide epitope associated with the MHC molecule triggers a cellular immune response.

[0275] Unless otherwise stated, or unless technically irrelevant, any of the various features, embodiments or examples disclosed in this application regarding the structure or use of lentiviral particles (in particular regarding their envelope proteins or recombinant polynucleotides) can be combined in any possible combination.

[0276] The invention further relates to a combination of compounds for administration to a mammalian host alone, comprising at least:

[0277] (i) the lentiviral vector particles of the invention, pseudotyped with a first defined heterologous viral envelope protein or viral envelope pseudotyping protein; this first pseudotyping protein can be from the New Jersey strain of VSV;

[0278] (ii) The lentiviral vector particles of the present invention provided separately from the lentiviral vector particles in (i), which are pseudotyped with a second defined heterologous viral envelope pseudotyping protein or a viral envelope pseudotyping protein different from the first heterologous viral envelope pseudotyping protein; this second pseudotyping protein can be from the Indiana strain of VSV.

[0279] In another embodiment of the present invention, possibly in combination with alternative forms of the nucleic acids disclosed above, the polynucleotide encoding the fusion polypeptide of the present invention comprising at least one antigenic polypeptide is structurally and / or chemically modified. As an example, the polynucleotide contains a Kozak consensus sequence in its 5' region. Other nucleic acid sequences of non-lentiviral origin that may be present in the vector genome are the IRES sequence (internal ribosome entry site) suitable for initiating polypeptide synthesis, the WPRE sequence or a modified WPRE sequence as a post-transcriptional regulatory element to stabilize the generated RNA, the sequence of a linker or a 2A peptide.

[0280] Other features and characteristics of the present invention, including those to be used in the embodiments described above, will be described in the examples and drawings below and can be used accordingly to characterize the present invention.

[0281] List of SEQ ID No.

[0282] SEQ ID NO:1 and 2 DENV-NS5-5

[0283] SEQ ID NO:3 and 4 DENV-NS3-3A

[0284] SEQ ID NO:5 and 6 DENV-NS4A-2K

[0285] SEQ ID NO:7 and 8 DENV-NS3-1A

[0286] SEQ ID NO:9 and 10 DENV-NS3-3

[0287] SEQ ID NO:11 and 12 DENV-NS3-3B

[0288] SEQ ID NO:13 and 14 DENV-NS5-2

[0289] SEQ ID NO:15 and 16 DENV-NS3-2

[0290] SEQ ID NO:17 and 18 DENV-NS3-1

[0291] SEQ ID NO:19 and 20 DENV-NS5-1

[0292] SEQ ID NO: 21 and 22 DENV-NS4B-2

[0293] SEQ ID NO: 23 and 24 DENV-NS5-3

[0294] SEQ ID NO: 25 and 26 DENV-NS5-4

[0295] SEQ ID NO: 27 and 28 DENV-NS4B-1

[0296] SEQ ID NO: 29 and 30 DENV-Ag1 - Transgenic DENV-Ag1 (Codon Optimized)

[0297] SEQ ID NO: 31 and 32 DENV-NS3-4

[0298] SEQ ID NO: 33 and 34 DENV-NS3-5

[0299] SEQ ID NO: 35 and 36 DENV-NS3-6

[0300] SEQ ID NO: 37 and 38 DENV-Ag2 - Transgenic DENV-Ag2 (Codon Optimized)

[0301] SEQ ID NO: 39 and 40 ZIKV-C-1

[0302] SEQ ID NO: 41 and 42 ZIKV-C-2

[0303] SEQ ID NO: 43 and 44 ZIKV-C-3 / PrM

[0304] SEQ ID NO: 45 and 46 ZIKV-NS4B-1

[0305] SEQ ID NO: 47 and 48 ZIKV-NS4B-2 / NS5-1

[0306] SEQ ID NO: 49 and 50 ZIKV-NS5-2

[0307] SEQ ID NO: 51 and 52 ZIKV-NS5-3

[0308] SEQ ID NO: 53 and 54 ZIKV-Ag - Transgenic ZIKV-Ag (Codon Optimized)

[0309] SEQ ID NO: 55 and 56 ZIKV-NS1 - Transgenic ZIKV-NS1 (Codon Optimized)

[0310] SEQ ID NO: 57 and 58 YFV-NS4B-1

[0311] SEQ ID NO: 59 and 60 YFV-NS5-2

[0312] SEQ ID NO: 61 and 62 YFV-NS5-3

[0313] SEQ ID NO: 63 and 64 YFV-NS4B-3

[0314] SEQ ID NO: 65 and 66 YFV-NS5-4

[0315] SEQ ID NO: 67 and 68 YFV-NS5-5

[0316] SEQ ID NO: 69 and 70 YFV-NS3-3

[0317] SEQ ID NO: 71 and 72 YFV-NS3-4

[0318] SEQ ID NO: 73 and 74 YFV-NS2A-1

[0319] SEQ ID NO: 75 and 76 YFV-NS2A-2

[0320] SEQ ID NO: 77 and 78 YFV-NS2A-3

[0321] SEQ ID NO: 79 and 80 YFV-NS2A-4

[0322] SEQ ID NO: 81 and 82 YFV-NS3-5 / NS4A-1

[0323] SEQ ID NO: 83 and 84 YFV-NS3-2

[0324] SEQ ID NO: 85 and 86 YFV-NS2B-1

[0325] SEQ ID NO: 87 and 88 YFV-NS3-1

[0326] SEQ ID NO: 89 and 90 YFV-NS4A-2

[0327] SEQ ID NO: 91 and 92 YFV-NS2A-5

[0328] SEQ ID NO: 93 and 94 YFV-NS5-6

[0329] SEQ ID NO: 95 and 96 YFV-NS5-7

[0330] SEQ ID NO:97 and 98 YFV-NS4A-3"

[0331] SEQ ID NO:99 and 100 YFV-2K-1

[0332] SEQ ID NO:101 and 102 YFV-NS4B-2

[0333] SEQ ID NO:103 and 104 YFV-NS5-1

[0334] SEQ ID NO:105 and 106 YFV-Ag1

[0335] SEQ ID NO:107 and 108 YFV-C-2

[0336] SEQ ID NO:109 and 110 YFV-C-1

[0337] SEQ ID NO:111 and 112 YFV-PrM-1

[0338] SEQ ID NO:113 and 114 YFV-M-1

[0339] SEQ ID NO:115 and 116 YFV-E-1

[0340] SEQ ID NO:117 and 118 YFV-NS1-2

[0341] SEQ ID NO:119 and 120 YFV-NS1-4

[0342] SEQ ID NO:121 and 122 YFV-NS1-1

[0343] SEQ ID NO:123 and 124 YFV-E-4

[0344] SEQ ID NO:125 and 126 YFV-E-5

[0345] SEQ ID NO:127 and 128 YFV-E-2

[0346] SEQ ID NO:129 and 130 YFV-E-3

[0347] SEQ ID NO:131 and 132 YFV-E-6

[0348] SEQ ID NO:133 and 134 YFV-NS1-5

[0349] SEQ ID NO: 135 and 136 YFV-E-7

[0350] SEQ ID NO: 137 and 138 YFV-NS1-3

[0351] SEQ ID NO: 139 and 140 YFV-NS1-6

[0352] SEQ ID NO: 141 and 142 YFV-Ag2 - Transgenic YFV-Ag2 (codon-optimized)

[0353] SEQ ID NO: 143 and 144 DENV-Ag2_ZIKV-Ag-WPREm_(Flavi-2) - Transgenic DENV-Ag2_ZIKV-Ag-WPREm_(Flavi-2) (codon-optimized)

[0354] SEQ ID NO: 145 and 146 ZIKV-Ag_DENV-Ag2-WPREm_(Flavi-3) - Transgenic ZIKV-Ag_DENV-Ag2-WPREm_(Flavi-3) (codon-optimized)

[0355] SEQ ID NO: 147 and 148 DENV-Ag2_ZIKV-NS1-WPREm_(Flavi-4) - Transgenic DENV-Ag2_ZIKV-NS1-WPREm_(Flavi-4) (codon-optimized)

[0356] SEQ ID NO: 149 and 150 ZIKV-NS1-DENV-Ag2-WPREm_(Flavi-5) - Transgenic ZIKV-NS1-DENV-Ag2-WPREm_(Flavi-5) (codon-optimized)

[0357] SEQ ID NO: 151 pFlap-β2m-DENV-Ag1-WPREm

[0358] SEQ ID NO: 152 pFlap-β2m-DENV-Ag2-WPREm

[0359] SEQ ID NO: 153 pFlap-β2m-ZIKV-Ag-WPREm

[0360] SEQ ID NO: 154 pFlap-β2m-ZIKV-NS1-WPREm

[0361] SEQ ID NO: 155 pFlap-β2m-YFV-Ag1-WPREm

[0362] SEQ ID NO:156 pFlap-β2m-YFV-Ag2-WPREm

[0363] SEQ ID NO:157 pFlap-β2m-DENV-Ag2_ZIKV-Ag-WPREm_(Flavi-2)

[0364] SEQ ID NO:158 pFlap-β2m-ZIKV-Ag_DENV-Ag2-WPREm_(Flavi-3)

[0365] SEQ ID NO:159 pFlap-β2m-DENV-Ag2_ZIKV-NS1-WPREm_(Flavi-4)

[0366] SEQ ID NO:160 pFlap-β2m-ZIKV-NS1-DENV-Ag2-WPREm_(Flavi-5)

[0367] SEQ ID NO:161 Empty vector pFlap-β2m-WPREm

[0368] SEQ ID NO:162 DENV-1 serotype consensus sequence (DENV1_cons) based on sequences representing different lineages (GenBank accession numbers: ADC92350.1, AJQ21317.2, ABG75761.1, AIG59667.1, AAQ19665.2)

[0369] SEQ ID NO:163 DENV-2 serotype consensus sequence (DENV2_cons) based on sequences representing different lineages (GenBank accession numbers: ALI16136.1, AAD18036.1, AUZ41807.1, AHA42535.1, ANT47239.1)

[0370] SEQ ID NO:164 DENV-3 serotype consensus sequence (DENV3_cons) based on sequences representing different lineages (GenBank accession numbers: ACV04798.1, BAE48725.1, AIH13925.1, ALS05358.1, AIO11765.1)

[0371] SEQ ID NO:165 DENV-4 serotype consensus sequence (DENV4_cons) based on sequences representing different lineages (GenBank accession numbers: AVA30162.1, ALI16138.1, AEJ33672.1, ARN79589.1)

[0372] The common sequence of all dengue virus serotypes (DENV1-4_cons) based on the common sequences of each serotype (DENV1_cons, DENV2_cons, DENV3_cons, and DENV4_cons), SEQ ID NO:166

[0373] The common sequence of the Asian lineage of ZIKV (ZIKV-Asian_cons) based on the sequences of the Pf13 / 251013-18 strain (GenBank accession number ARB08102.1) and the BR / AM / 16800005 strain (GenBank accession number AQU12485.1), SEQ ID NO:167

[0374] The common sequence of the African lineage of ZIKV (ZIKV-African_cons) based on the sequences of the SEN / 1984 / 41671-DAK strain (GenBank accession number AMR39836.1) and the MR766-NIID strain (GenBank accession number BAP47441.1), SEQ ID NO:168

[0375] The common sequence of the Asian and African lineages of ZIKV (ZIKV_ALL_cons) based on the sequences of ZIKV-Asian_cons and ZIKV-African_cons, SEQ ID NO:169

[0376] SEQ ID NO:170 human b2m

[0377] SEQ ID NO:171 SP1-human b2m promoter

[0378] SEQ ID NO:172 BCUAG composite promoter

[0379] SEQ ID NO:173 WPREm

[0380] SEQ ID NO:174 WPRE

[0381] SEQ ID NO:175 FLAP forward primer

[0382] SEQ ID NO:176 FLAP reverse primer

[0383] SEQ ID NO:177 GAPDH forward primer

[0384] SEQ ID NO:178 GAPDH reverse primer

[0385] SEQ ID NO:179 DENV Forward Primer

[0386] SEQ ID NO:180 DENV Reverse Primer

[0387] SEQ ID NO:181 DENV Taqman Probe

[0388] SEQ ID NO:182 DENV4 Reverse Primer

[0389] SEQ ID NO:183 DENV4 Taqman Probe Description of the Drawings

[0390] Figure 1 . Genetic diversity of DENV. A phylogenetic tree was constructed based on the complete polyprotein sequences of DENV-1 (84 sequences), DENV-2 (71 sequences), DENV-3 (46 sequences), and DENV-4 (39 sequences) using MEGA 7 software. The strains representing different lineages of each genotype are shown on the right, and the strains were selected for the identification and prediction of MHC class I epitopes. The source of the challenge strain indicates the country where the DENV strain was initially isolated for experimental infection.

[0391] Figure 2 . Selection of the epitope-containing regions for multivalent DENV-Ag (DENV-Ag1). (A) Schematic illustration of the DENV polyprotein. (B) Amino acid identity plot showing the distribution of identical amino acids in the consensus sequences of the four DENV serotypes. The consensus sequences for each genotype are SEQ ID NO:162 for DENV-1 serotype, SEQ ID NO:163 for DENV-2 serotype, SEQ ID NO:164 for DENV-3 serotype, and SEQ ID NO:165 for DENV-4 serotype. The black lines show the regions with an identity score higher than 80%. (C) Distribution of human MHC class I (black) and class II (gray) epitopes marked as positive in various T cell assays in the IEDB database. (D and E) Distribution of human MHC class I epitopes of the four DENV serotypes predicted by the IEDB and netCTLpan prediction servers, respectively. Each point corresponds to the center of the epitope and shows its position along the DENV polyprotein sequence (x-axis). The y-axis indicates the number of times each epitope can be matched to the alignment of the DENV sequences used for prediction. For example, for each epitope predicted in all the sequences used for DENV-1, DENV-2, and DENV-3, y = 5, and for such epitopes of DENV-4, y = 4. Higher values on the y-axis correspond to multi-allelic epitopes (presenting >1 allele) or epitope clusters (those epitopes having the same position on the x-axis). The gray boxes encompass the regions selected to generate the DENV antigen.

[0392] Figure 3. Alignment of the amino acid sequences included in the DENV antigen. The antigen regions were selected from the NS3 (A), NS4A, 2K and NS4B (B), and NS5 (C) proteins. The first sequence aligned (DENV1-4_cons (SEQ ID NO: 166)) shows that the four DENV serotypes have a 75% major consensus sequence, and this first sequence was generated based on the individual consensus sequences of each serotype (DENV1_cons (SEQ ID NO: 162), DENV2_cons (SEQ ID NO: 163), DENV3_cons (SEQ ID NO: 164), and DENV4_cons (SEQ ID NO: 165)).

[0393] Figure 4. Structures of the multivalent DENV antigens DENV-Ag1 and DENV-Ag2. (A) Arrangement of the individual protein fragments of the non-structural proteins from DENV in DENV-Ag1. The amino acid linkers (L1 to L10) that connect different regions and are designed to eliminate non-specific MHC class I epitopes at the junctions are marked. (B) The modified version of the multivalent DENV antigen (DENV-Ag2) was developed by replacing the 26 aa-length N-terminal fragment of DENV-Ag1 (which includes the NS5-5 region and the L1 linker) with a 47 aa-length sequence that includes three additional antigen regions of the NS-3 protein (NS3-4, NS3-5, and NS3-6). (C) The protein sequence of DENV-Ag1.

[0394] Figure 5 . Immunogenicity of integrative (A) or non-integrative (B) lentiviral vectors expressing DENV-Ag1 in A129 mice. (A) T cell responses induced by the integrative vector iLV-DENV-Ag1 pseudotyped with VSV-G of the Indiana (IND) or New Jersey (NJ) serotype 14 days after a single immunization. (B) T cell responses induced by the non-integrative vector LV-DENV-Ag1 after a single immunization protocol (analyzed 14 days after immunization) or a prime-boost protocol (analyzed 6 days after the second immunization). The statistical significance of the total responses was determined by one-way ANOVA test and Tukey correction for multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0395] Figure 6. T cell responses in IFNAR-BL6 mice were analyzed by intracellular cytokine staining after a single immunization with LV-DENV-Ag1 or LV-GFP. (Start) Fourteen days after immunization with a single dose of LV-DENV-Ag1 or LV-GFP, viable CD8+ T lymphocytes in splenocytes isolated from IFNAR-BL6 mice were identified using a gating strategy. (Continued) CD8+ cells expressing the cytokines IFNγ, TNFα, and IL-2 were detected. (End) The response of CD8+ cells expressing IFNγ and the lymphocyte degranulation marker (CD107a) to stimulation with DENV-specific peptides was detected. The last row shows the CD8+ cells expressing IFNγ + / TNFα + 、IFNγ + / IL-2 + double-positive or IFNγ + / TNFα + / IL-2 + triple-positive CD8+ cells. Left panel: Splenocytes from mice immunized with LV-DENV-Ag1 and stimulated with a non-specific peptide (YF-C) not included in DENV-Ag1 (negative control). Middle panel: Splenocytes from mice immunized with LV-GFP and stimulated with DENV-specific peptides (negative control). Right panel: Splenocytes from mice immunized with LV-DENV-Ag1 and stimulated with DENV-specific peptides.

[0396] Figure 7 . Protection of A129 mice from DENV-1 and DENV-2 infection by a single immunization with LV-DENV-Ag1. (A and B) A129 mice (n = 10 / group) were immunized intramuscularly (i.m.) with a single dose of 7.5 × 10 6 TU / mouse of LV-DENV-Ag1 (IND) or LV-GFP (IND) vector and infected intravenously (i.v.) with 1 × 10 7 FFU / mouse of DENV-1 or 5 × 10 5 FFU / mouse of DENV-2 one month after immunization. (C and D) Viremia in the plasma of infected A129 mice was measured by RT-qPCR and expressed as genome equivalents (G.E.) / ml. LOD = limit of detection. Statistical significance of differences between groups was evaluated by unpaired non-parametric Mann-Whitney test (*p < 0.05, **p < 0.01, ***p < 0.001).

[0397] Figure 8. Protection of IFNAR - BL6 mice from DENV - 1 and DENV - 2 infections by single immunization with LV - DENV - Ag1. (A and B) IFNAR - BL6 mice (n = 5 - 6 / group) were immunized i.m. with a single dose of 3×10 8 TU / mouse of LV - DENV - Ag1 (IND) or LV - GFP (IND) vector and infected i.v. with 1×10 7 FFU / mouse of DENV - 1 or 2×10 6 FFU / mouse of DENV - 2 one month after immunization. Mean body weights are shown. (C and D) Viremia in the plasma of infected IFNAR - BL6 mice was measured by RT - qPCR and expressed as genome equivalents (G.E.) / ml. (E and F) Viral loads in the organs of infected mice were collected on day 4 post - infection. LOD = limit of detection. Statistical significance of differences between groups was assessed by unpaired non - parametric Mann - Whitney test (*p < 0.05, **p < 0.01).

[0398] Figure 9 . Protection of IFNAR - BL6 mice from DENV - 3 and DENV - 4 infections by single immunization with LV - DENV - Ag1. (A and B) IFNAR - BL6 mice (n = 6 / group) were immunized i.m. with a single dose of 3×10 8 TU / mouse of LV - DENV - Ag1 (IND) or LV - GFP (IND) vector and infected i.v. with 8×10 6 FFU / mouse of DENV - 3 (2 months after immunization) or 1×10 7 FFU / mouse of DENV - 4 (1 month after immunization). Mean body weights are shown. (C and D) Viremia in the plasma of infected IFNAR - BL6 mice was measured by RT - qPCR and expressed as G.E. / ml. (E) Viral load in the spleens of mice infected with DENV - 3, and (F) viral loads in the organs of mice infected with DENV - 4, expressed as G.E / 1μg of total RNA. Statistical significance of differences between groups was assessed by unpaired non - parametric Mann - Whitney test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001).

[0399] Figure 10 . Protection of A129 mice from DENV - 2 infection by prime - boost immunization with LV - DENV - Ag1. (A) A129 mice were immunized with 1×10 7 TU / mouse of LV - DENV - Ag1 (IND) or LV - GFP (IND) and boosted with 2×108 Mean body weight of A129 mice (n = 12 / group) given a secondary i.m. boost of LV-DENV-Ag1(NJ) or LV-GFP(NJ) at TU / mouse. One month after the boost, both groups were infected i.v. with 1 × 10 7 FFU / mouse of DENV-2. (B) Viremia in the plasma of the mice was measured by RT-qPCR and expressed as G.E. / ml. (C) Viral load in the spleen was expressed as G.E / 1 μg of total RNA. Statistical significance of differences between groups was evaluated by unpaired non-parametric Mann-Whitney test (*p < 0.05).

[0400] Figure 11. Role of CD8+ cells in protection of IFNAR-BL6 mice from DENV-2 infection induced by LV-DENV-Ag1. Mean body weight of mice after prime-boost immunization with LV-DENV-Ag1 or LV-GFP followed by DENV-2 infection one month later. Before infection, six mice per group were pre-injected with anti-isotype control antibody (A), anti-CD4+ (B), or anti-CD8+ (C) antibodies to selectively deplete CD4+ or CD8+ cells of the mice, respectively. (D) Survival rate of mice immunized with LV-DENV-Ag1 or LV-GFP and not depleted of T cells (isotype control Ab) or depleted of CD4+ or CD8+ T cells (anti-CD4+ or anti-CD8+ antibodies, respectively). (E) Viremia in the blood of mice immunized with LV-DENV-Ag1 or LV-GFP and infected with DENV-2, which were injected with anti-isotype antibody (transparent violin), anti-CD4+ antibody (light gray violin), or anti-CD8+ antibody (dark gray violin) before infection.

[0401] Figure 12. Selection of T cell epitope-containing regions of ZIKV and YFV. (A) Schematic illustration of the ZIKV and YFV polyproteins, showing the structural proteins capsid (C), matrix (M), and envelope (E) and the non-structural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. (B and C, top panels) Distribution of human MHC class I (blue / dark gray) and MHC class II (orange / light gray) ZIKV-specific and YFV-specific epitopes, which were referenced as positive in T cell assays in the IEDB database, respectively. (B and C, bottom panels) Distribution of human MHC class I epitopes predicted by IEDB (blue / dark gray) and netCTLpan (orange / light gray) based on the consensus sequences of ZIKV and YFV. Each point shows the position (x-axis) of the center of the epitope in the ZIKV or YFV polyprotein sequence and the number of epitopes located at each position in the sequence (y-axis). Regions selected to be included in each antigen are shaded: ZIKV-Ag (yellow / light gray), YFV-Ag1 (also called YFVN-NS) (green / left), and YFV-Ag2 (also called YFV-S) (purple / middle and right). The y-axis indicates the number of times each epitope can be aligned and matched to two ZIKV sequences representing two ZIKV lineages (African and Asian). For example, y = 2 for each epitope predicted in all ZIKV sequences used. Higher values on the y-axis correspond to multi-allelic epitopes (presenting as >1 allele) or epitope clusters (those epitopes having the same position on the x-axis). The box encompasses the region selected to constitute the ZIKV antigen. (C) Alignment of the amino acid sequences included in the ZIKV antigen selected from the C, PrM, NS4A, NS4B, and NS5 proteins. The first sequence aligned (ZIKV_ALL_cons (SEQ ID NO:169)) shows the consensus sequence of two ZIKV lineages, namely the ZIKV Asian lineage (SEQ ID NO:167:ZIKV-Asian_cons), which is based on the sequences of the Pf13 / 251013-18 strain (GenBank accession number ARB08102.1) and the BR / AM / 16800005 strain (GenBank accession number AQU12485.1), and the ZIKV African lineage (SEQ ID NO:168:ZIKV-African_cons), which is based on the sequences of the SEN / 1984 / 41671-DAK strain (GenBank accession number AMR39836.1) and the MR766-NIID strain (GenBank accession number BAP47441.1), and this consensus sequence was generated based on the individual consensus sequences of each lineage. (D) Arrangement of the protein regions in ZIKV-Ag.

[0402] Figure 13. Protein sequence of ZIKV-Ag. The sequence diagram generated using SnapGene 6.1.1 shows the antigenic regions (gray boxes) and amino acid linkers (black boxes) designed to eliminate non-specific MHC class I epitopes. The hollow colored boxes show the peptide sequences that contain the predicted MHC class I epitopes for H-2b mice used to evaluate the immunogenicity of LV-ZIKV-Ag: peptide pool C (black), PrM (green), NS4B-A (orange), NS4B-B (blue), NS5-A (red), and NS5-B (purple).

[0403] Figure 14 . Immunogenicity of a non-integrating lentiviral vector expressing ZIKV-Ag in A129 mice. A129 mice (n = 5 / group) were immunized with a single dose of 1×10 8 TU / mouse of the LV-ZIKV-Ag or LV-GFP (control) vector, and the T cell response was evaluated by Elispot assay 14 days after immunization. Spleen cells from immunized mice were restimulated with region-specific peptide pools predicted to be immunogenic in A129 mice (Figure 13). The y-axis indicates the number of cells secreting IFNγ in response to this stimulation (per 10 6 total spleen cells). Different shadings indicate responses to epitopes from different antigenic regions.

[0404] Figure 15. T cell responses in IFNAR-BL6 mice were analyzed by intracellular cytokine staining after a single immunization with LV-ZIKV-Ag or LV-GFP. (A) At 14 days after immunization with 3×10 8 TU of LV-ZIKV-Ag or LV-GFP, CD8+ T lymphocytes in spleen cells isolated from IFNAR-BL6 mice were identified using a gating strategy. (B) The response of CD8+ cells expressing the cytokines IFNγ, TNFα, IL-2, and the lymphocyte degranulation marker (CD107a) to stimulation with ZIKV-specific peptides was detected. The last row shows the expression of IFNγ + / TNFα + 、IFNγ + / IL-2 + double-positive or the expression of IFNγ + / TNFα + / IL-2 +Triple-positive CD8+ cells. Left panel: Spleen cells from mice immunized with LV-ZIKV-Ag and stimulated with a non-specific peptide (YF-C) not included in LV-ZIKV-Ag (negative control). Middle panel: Spleen cells from mice immunized with LV-GFP and stimulated with a ZIKV-specific peptide (negative control). Right panel: Spleen cells from mice immunized with LV-ZIKV-Ag and stimulated with a ZIKV-specific peptide.

[0405] Figure 16. T cell responses in IFNAR-BL6 mice were analyzed by intracellular cytokine staining after single immunization with LV-ZIKV-NS1 or LV-GFP. (A) At 14 days after immunization with 3×10 8 TU of LV-ZIKV-NS1 or LV-GFP, CD8+ cytotoxic T lymphocytes in spleen cells isolated from IFNAR-BL6 mice were identified using a gating strategy. (B) Responses of CD8+ cells expressing the cytokines IFNγ, TNFα, IL-2, and the lymphocyte degranulation marker (CD107a) to a single pool of 166 overlapping peptides covering the ZIKV NS1 protein were detected. The last row shows CD8+ cells expressing IFNγ + / TNFα + 、IFNγ + / IL-2 + double-positive or expressing IFNγ + / TNFα + / IL-2 + triple-positive. Left panel: Spleen cells from mice immunized with LV-ZIKV-NS1 and stimulated with a non-specific peptide (YF-C) not included in LV-ZIKV-NS1 (negative control). Middle panel: Spleen cells from mice immunized with LV-GFP and stimulated with a ZIKV-NS1-specific peptide (negative control). Right panel: Spleen cells from mice immunized with LV-ZIKV-NS1 and stimulated with a ZIKV-specific peptide.

[0406] Figure 17 . Single immunization with LV-ZIKV-Ag or LV-ZIKV-NS1 protected A129 mice from ZIKV (strain PF-13) infection. (A) Mean body weight of A129 mice after ZIKV infection. (B) Viremia in the plasma of mice immunized with LV-ZIKV-Ag, LV-ZIKV-NS1, or LV-GFP and infected with ZIKV was measured by RT-qPCR and expressed as genome equivalents (G.E.). (C and D) At the end of infection (day 12 post-infection), viral loads were measured in the brain and spleen organs of infected mice, respectively. Statistical significance of differences between groups was evaluated by unpaired non-parametric Mann-Whitney test (*p < 0.05).

[0407] Figure 18 . Protection of IFNAR-BL6 mice from ZIKV (strain PF-13) infection by single immunization with LV-ZIKV-NS1. (A) Mean body weight of IFNAR-BL6 mice after ZIKV infection. (B) Survival rate of immunized mice after ZIKV infection. (C) Viremia in the blood of mice immunized with LV-ZIKV-NS1 or LV-GFP and infected with ZIKV was measured by PCR and expressed as genomic equivalents (G.E.). (D) Viral load measured in the brains and spleens of surviving mice after infection (day 15 post-infection). Statistical significance of differences between groups was evaluated by unpaired non-parametric Mann-Whitney test (*p < 0.05, **p < 0.01).

[0408] Figure 19. Selection of epitope-containing regions of YFV-Ag1 and YFV-Ag2. (A) Schematic illustration of the YFV polyprotein (top panel), distribution of human MHC class I epitopes that were referenced as positive in various T cell assays in the IEDB database (middle panel), and epitopes predicted by the IEDB and netCTLpan prediction servers (bottom panel). Each point corresponds to the center of an epitope and shows its position along the YFV polyprotein sequence (on the x-axis). The y-axis indicates the number of times each epitope can be matched to the alignment of YFV strains representing three different lineages of YFV. Higher values on the y-axis correspond to multi-allelic epitopes (presenting as >1 allele) or epitope clusters (those epitopes having the same position on the x-axis). (B) Alignment of the protein regions included in the YFV-Ag1 (top panel) and YFV-Ag2 (bottom panel) antigens.

[0409] Figure 20 . Protein sequences of YFV-Ag1 (also known as YFV-NS) and YFV-Ag2 (also known as YFV-S). The sequences of both antigens are identical to the sequences of the corresponding regions (gray boxes) of the YFV live attenuated vaccine strain (17D-204). Non-YFV-specific amino acids (linkers) that connect different regions and eliminate non-specific MHC class I epitopes (black boxes) are shown in the figure.

[0410] Orange (LV-YF-S) and red (LV-YF-NS) boxes show peptide sequences containing MHC class I epitopes of humans and H-2b mice, which are used to evaluate immunogenicity.

[0411] Figure 21. Immunogenicity of non-integrating lentiviral vectors expressing YFV-Ag1 and YFV-Ag2 in A129 mice. T cell responses induced by single immunization with LV-YFV-Ag1, LV-YFV-Ag2 or LV-GFP (control) were evaluated 14 days after immunization by Elispot assay. Spleen cells from immunized mice were harvested and stimulated with pools of antigen-specific peptides reported or predicted to be immunogenic for A129 mice. Pool-1 contains 7 peptides specific for the non-structural protein regions included in YFV-Ag1, and Pool-2 contains 3 peptides specific for the structural protein regions included in YFV-Ag2. The y-axis indicates the number of cells secreting IFNγ in response to this stimulation (per 10 6 total spleen cells). Each combination of vector immunization / peptide pool stimulation analyzed in the assay is marked by a different symbol.

[0412] Figure 22. T cell responses in IFNAR-BL6 mice were analyzed by intracellular cytokine staining after single immunization with LV-YFV-Ag1 or LV-GFP. (A) Fourteen days after immunization with a single dose of each vector, CD8+ cytotoxic T lymphocytes in spleen cells harvested from IFNAR-BL6 mice were identified using a gating strategy. (B) Responses of CD8+ cells expressing the cytokines IFNγ, TNFα, IL-2 and the lymphocyte degranulation marker (CD107a) to stimulation with a single pool (Pool 1) of 7 peptides derived from the YFV non-structural region were detected. The last row shows CD8+ cells expressing IFNγ + / TNFα + 、IFNγ + / IL-2 + double-positive or IFNγ-expressing + / TNFα + / IL-2 + triple-positive. Left panel: Spleen cells from mice immunized with LV-YFV-Ag1 and stimulated with non-specific peptides (YF-C) not included in LV-YFV-Ag1 (negative control). Middle panel: Spleen cells from mice immunized with LV-GFP and stimulated with YFV-specific peptides (negative control). Right panel: Spleen cells from mice immunized with YFV-Ag and stimulated with YFV-specific peptides.

[0413] Figure 23. A129 mice were protected from YFV (strain 17D-204) infection by single immunization with LV-YFV-Ag1, LV-YFV-Ag2, or LV-GFP (control). (A) Mean body weight of A129 mice after YFV infection. (B) Viremia in the blood of mice immunized with LV-ZIKV-Ag, LV-ZIKV-NS1, or LV-GFP and infected with YFV was measured by RT-qPCR and expressed as genome equivalents (G.E.). Weight of the spleen (C) and viral load in the spleen (D) of mice immunized with different vectors and infected with YFV.

[0414] Figure 24 . Schematic illustration of the strategy of modifying the DENV-Ag1 antigen (to generate DENV-Ag2) and using DENV-Ag2, ZIKV-Ag, and ZIKV-NS1 to generate a panel of DENV / ZIKV bivalent antigens.

[0415] Figure 25 . Evaluation of T cell responses induced by lentiviral vectors expressing DENV-specific antigens DENV-Ag1 and DENV-Ag2 and bivalent antigens Flavi-2, Flavi-3, Flavi-4, and Flavi-5. (A) T cell responses induced by single immunization of IFNAR-BL6 mice with individual vectors were evaluated by Elispot assay 14 days after immunization. Spleen cells from immunized mice were harvested and stimulated with a single combinatorial pool of 35 DENV-specific peptides, including all test peptide pools that were previously positive in the A129 mouse LV-DENV-Ag1 Elispot assay. The y-axis indicates the number of cells secreting IFNγ in response to each vector after stimulation with the DENV-specific peptide pool (per 10 6 total spleen cells). (B) T cell responses induced by single immunization of C57BL / 6 (wt) mice with each vector were evaluated by Elispot assay 14 days after immunization. Reactivity against DENV was evaluated by harvesting spleen cells from immunized animals and stimulating the spleen cells with a single pool of 12 DENV-specific peptides (a combinatorial pool of NS4B-1 and NS5-2) that we had previously tested and that showed the highest reactivity against LV-DENV-Ag1 in A129 mice. Reactivity against ZIKV-NS1 was evaluated by stimulating the harvested spleen cells with a single pool of 166 overlapping 15-mers that covered the entire ZIKV NS1 protein. Candidate vectors preselected for further analysis based on Elispot results are circled.

[0416] Figure 26. Protection of IFNAR-BL6 mice from DENV-4 infection by immunization with LV-DENV-Ag1, LV-DENV-Ag2, LV-Flavi-3, LV-Flavi-4 or LV-Flavi-5 vectors. IFNAR-BL6 mice (A) were immunized with each vector at a single dose (3×10 8 TU / mouse) and one month later, viremia in the blood of mice (n = 3) infected with DENV-4 at 1×10 7 FFU / mouse was measured by RT-qPCR and expressed as genome equivalents (G.E.) / ml. (B) At 7 days post-infection, viral load in the spleens of infected mice was measured by RT-qPCR and expressed as the number of viral genome equivalents per 1 μg of total RNA.

[0417] Figure 27 . Protection of IFNAR-BL6 male mice from ZIKV infection by immunization with LV-Flavi-3, LV-Flavi-4 or LV-Flavi-5 bivalent vectors. (A) IFNAR-BL6 male mice were immunized with each vector at a single dose (3×10 8 TU / mouse) and one month later, viremia in the plasma of mice infected with ZIKV (PF-13) at 1×10 3 FFU / mouse was measured by RT-qPCR and expressed as genome equivalents (G.E.) / ml. (B) At 9 days post-infection, viral load in the organs (brain and testis) of infected male mice was measured by RT-qPCR and expressed as the number of viral genome equivalents per 1 μg of total RNA.

[0418] Figure 28 . Protection of IFNAR-BL6 mice from DENV-1, DENV-2, DENV-3 and DENV-4 infections by a single-dose immunization with the LV-Flavi-5 vector. (A, B, C, D: left panels) Mean body weights of IFNAR-BL6 mice immunized with LV-Flavi-5 or LV-GFP (control) and infected with DENV-1, DENV-2, DENV-3 and DENV-4, respectively, one month after immunization. (A, B, C, D: right panels) Viremia in the blood of mice immunized with LV-Flavi-5 and infected with DENV-1, DENV-2, DENV-3 and DENV-4, respectively, one month later was measured by RT-qPCR and expressed as genome equivalents (G.E.) / ml.

[0419] Figure 29. Comparison of the protective effects of LV-Flavi-5 immunization against ZIKV infection in male and female IFNAR-BL6 mice. Mice were immunized with LV-Flavi-5 or LV-GFP (control), and one month after immunization, were infected with 1×10 3 Mean body weights of IFNAR-BL6 female mice (A, left panel) and male mice (B, left panel) infected with ZIKV (PF-13) at 1×10

[0420] Figure 30. Protection of IFNAR-BL6 mice against DENV-2 or ZIKV infection by heterologous prime-boost immunization with LV-Flavi-5 and LV-Flavi-3 vectors. (A) Mean body weights (left panel) and viremia (right panel) of IFNAR-BL6 mice infected with DENV-2 one month after sequential immunization with LV-Flavi-5 and LV-Flavi-3 or twice with LV-GFP (control). (B) Mean body weights (upper left panel), survival rates (upper right panel), viremia measured by RT-qPCR (lower left panel), or viremia measured by virus titration assay (lower right panel) of male IFNAR-BL6 mice infected with ZIKV (PF-13) one month after sequential immunization with LV-Flavi-5 and LV-Flavi-3 or twice with LV-GFP (control).

[0421] Figure 31 . Antigen design rationale for ZIKV and YFV. Phylogenetic trees representing the major gene lineages of ZIKV and YFV were generated based on 17 and 19 complete sequences of each virus, respectively. Consensus sequences representing each lineage were inferred from the sequences, and the consensus sequences were used to identify regions containing known human MHC class I and class II epitopes as well as predicted MHC class I epitopes. Epitope-containing regions were assembled together and optimized as outlined in Materials and Methods.

[0422] Figure 32. Histological analysis of organs of mice inoculated with ZIKV. Brains (A) and spleens (B) of IFNAR-BL6 mice that were non-immunized and non-infected (left column), immunized with LV-ZIK and inoculated with ZIKV (middle column), or immunized with LV-GFP and inoculated with ZIKV (right column) were stained with Hematoxylin-eosin (H&E). Representative images of 3 mice from each experimental group are shown. Red and white arrows indicate the locations of the red pulp and white pulp regions, respectively. Black arrows indicate the perivascular phenomenon observed in the brains of mice immunized with the LV-GFP vector and infected with ZIKV.

[0423] Figure 33 . Histological analysis of organs of mice inoculated with YFV. Brains (A) and spleens (B) of IFNAR-BL6 mice that were non-immunized and non-infected (left column), immunized with LV-YF-NS and inoculated with YFV (middle column), or immunized with LV-GFP and inoculated with YFV (right column) were stained with H&E. Representative images of 3 animals from each group are shown. Red and white arrows indicate the locations of the red pulp and white pulp regions, respectively. Black arrows indicate the perivascular phenomenon observed in the brains of mice immunized with the LV-GFP vector and infected with YFV. Examples

[0424] POLYNUCLEOTIDE AND LENTIVIRAL VECTOR EXPRESSING THE NON - STRUCTURAL ANTIGENS OF DENGUE VIRUS IN THE FORM OF FUSION POLYPEPTIDE

[0425] The following examples relate to the preparation of recombinant polynucleotides and lentiviral vectors expressing dengue virus non-structural antigens in the form of fusion proteins. Similar protocols have been applied to prepare recombinant polynucleotides and lentiviral vectors expressing Zika virus and yellow fever virus non-structural antigens in the form of fusion proteins. However, the design of the YFV fusion polypeptide does not require the design of a common sequence as it is based on the sequence of the 17-204D yellow fever vaccine strain.

[0426] Accordingly, experimental results and illustrative figures of constructs of Zika virus and yellow fever virus and lentiviral vectors expressing them are provided on this basis.

[0427] Materials and Methods

[0428] Design of Dengue Antigen (DENV-Ag1)

[0429] The complete polyprotein sequences of DENV were retrieved from the nucleotide sequence database (NCBI) (16), aligned using ClustalX (17), and used to construct a phylogenetic tree with Mega 7 software (18). A smaller set of sequences selected to represent the genetic variability of DENV included five sequences each of DENV-1 (GenBank accession numbers: ADC92350.1, AJQ21317.2, ABG75761.1, AIG59667.1, AAQ19665.2), DENV-2 (GenBank accession numbers: ALI16136.1, AAD18036.1, AUZ41807.1, AHA42535.1, ANT47239.1), DENV-3 (GenBank accession numbers: ACV04798.1, BAE48725.1, AIH13925.1, ALS05358.1, AIO11765.1), and four sequences of DENV-4 (GenBank accession numbers: AVA30162.1, ALI16138.1, AEJ33672.1, ARN79589.1). The sequences of known and predicted T cell epitopes were aligned with the DENV polyprotein sequences using MAFFT software (19). The alignment was visualized using the BioEdit sequence editor to further assist in the selection of epitope-containing regions (20). The epitope sequences were matched to the alignment of the DENV polyprotein using the Blast search algorithm (NCBI website), and the localization of each epitope in the alignment was determined (16). Using this data, an XY-graph was constructed, where each epitope was represented by a single point showing its position in the alignment (x-axis) and the number of its matches to different DENV sequences (y-axis). Conserved and / or multi-allelic epitopes were identified by higher match scores, and regions containing such epitopes were preferentially included in the DENV antigen. For a set of 27 of the most common human alleles, MHC class I epitope predictions were independently performed for each of the four DENV serotypes using the proteasome cleavage / TAP transport / MHC class I binding combined predictor on the IEDB server (21) (22 - 24). All octameric, nonameric, decameric, and undecameric peptides with a total score greater than zero were retained and pooled in a single peptide pool. The netCTLpan tool (25) was used on the DTU Bioinformatics server to predict nonameric peptides predicted to bind 20 of the most common human alleles, and peptides with a combined rank less than or equal to 1.0 were retained. The distributions of known and predicted T cell epitopes were compared, and conserved regions containing the most epitopes were selected.The 75% majority consensus sequences of each DENV genotype and the major consensus sequence (SEQ ID No. 166) representing all 4 genotypes (which serves as the basis for DENV-Ag1) were created using the ConsensusMaker software tool (26) available from the Los Alamos HIV database website. The consensus sequences corresponding to the selected polyprotein fragments were assembled into a linear polyprotein, and then epitope prediction was repeated to verify that all epitopes expected to be located near the junctions were correctly formed and that non-specific immunodominant epitopes were not artificially generated by joining different regions together. If such epitopes were identified, a de-optimization strategy was employed, inserting a hydrophobic amino acid linker at the junction, followed by multiple rounds of epitope prediction until no such non-specific epitopes were predicted.

[0430] Generation and titration of lentiviral vectors (LVs)

[0431] Commercially (Genescript), the DNA sequence encoding the DENV genomic region assembly (DENV-Ag1) was synthesized with codons optimized for expression in mammalian cells and inserted into the pUC57 subcloning vector. The insert was excised at the BamHI and XhoI restriction sites and recloned into the pFLAPΔU3-β2m-mWPRE vector between the β2m promoter and the mWPRE (mutated woodchuck post-transcriptional regulatory element) sequence, where the atg start codon was mutated to avoid transcription of the downstream truncated "X" protein of woodchuck hepatitis virus to improve vector safety. After recloning, the sequence of the insert was verified by sequencing the regions flanking the restriction sites (Eurofins, Ebersberg, Germany). The plasmid used to generate the vector was purified using the NucleoBond Xtra Maxi EF kit (Macherey Nagel), resuspended in endotoxin-free Tris-EDTA buffer, quantified using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific), aliquoted, and stored at -20 °C. LV was produced in human embryonic kidney HEK293T cells as previously described in detail (27). Briefly, lentiviral particles were produced by transient calcium phosphate triple transfection of HEK293T cells with a transfer vector plasmid (pFLAP-β2m-mWPRE, where the specific antigen was inserted between the β2m and mWPRE elements), an envelope plasmid expressing the G protein of VSV (Indiana (IND) or New Jersey (NJ) serotype), and a packaging plasmid (NDK or NDK-pD64V, for producing integrative-competent or integrative-defective vectors, respectively). The supernatant was collected 48 hours after transfection, clarified by centrifugation at 2500 rpm at 4 °C, and concentrated by ultracentrifugation at 22 000 rpm for 1 hour at 4 °C. The pelleted particles were resuspended in sterile 20 mM PIPES buffer (pH 7.2) supplemented with 2.5% glucose and 75 mM NaCl, aliquoted, and stored at -80 °C. The titer of the lentiviral vector was determined by qPCR on HEK293T cells transduced with the vector, which were treated with aphidicolin to prevent cell division. At the same time, HEK293T cells were transduced with heat-inactivated vector (30 minutes at 70 °C) to control for plasmid contamination in vector preparation. After 48 - 72 hours of transduction, the cells were lysed, genomic DNA was isolated, and the viral titer was determined by qPCR.To determine the titer, fragments of the lentiviral Flap region (forward primer: 5'-TGG AGG AGG AGA TATGAG GG-3' (SEQ ID No. 175); reverse primer: 5'-CTG CTG CAC TAT ACC AGA CA-3' (SEQ ID No. 176)) and fragments of the cellular GAPDH gene (forward primer: 5'-TCT CCT CTG ACT TCA ACA GC-3' (SEQ ID No. 177); reverse primer 5'-CCC TGC ACT TTT TAA GAG CC-3' (SEQ ID No. 178)) were amplified. The lentiviral vector copy number per cell was determined as the ratio of the Flap copy number to the GAPDH copy number, corresponding to the total number of HEK293T cells. Before immunizing the mice, the lentiviral vector was diluted to an appropriate concentration in PBS.

[0432] mouse

[0433] Ifnar1− / − mice (also known as IFNAR-KO) carrying the Ifnar1tm1Agt allele on a 129 (A129) or C57BL / 6J (IFNAR-BL6) genetic background were housed at the Institute Pasteur and maintained in groups under specific pathogen-free conditions. For vaccination experiments, mice at least 6 weeks of age were used. Vaccination was performed by intramuscular injection of a 50 μL volume into the posterior muscle. Dengue virus was inoculated by intravenous (i.v.) injection of a total volume of 150 μL into the tail vein. Zika and YFV were inoculated by intraperitoneal injection of a total volume of 200 μl. Mice were monitored for signs of disease (DENV: lethargy, ruffled fur, hunched posture; ZIKV and YFV: lethargy, ruffled fur, hunched posture, neurological signs (abnormal movements, limb paralysis)), and body weight was recorded daily during observation of weight changes (weekends were excluded in some experiments). A mouse was considered moribund if its body weight loss exceeded 20% of its initial weight, or if it lost 10% of its weight and had neurological signs (i.e., limb paralysis). Blood samples were collected into Microvette 500 K3E tubes containing EDTA (Starstedt) and centrifuged at 5000 g for 10 minutes to separate plasma from blood cells. The clarified plasma samples were kept at -80 °C, followed by RNA extraction and then RT-qPCR analysis with DENV-specific primers. All experiments were conducted in an A3 containment facility at the Institute Pasteur animal facility. Animal experiments were conducted according to European and French guidelines after approval by the Institute Pasteur Safety, Animal Care and Use Committee (agreement issued by the local ethics committee: CETEA no. DAP1800077) and the Ministry of High Education and Research (APAFIS#18428-2019010717408411_v2).

[0434] Propagation and titration of virus stocks

[0435] The dengue virus serotype 1 (DENV-1) virus strain KDH0026A was kindly provided by Dr. Lambrechts (Institute Pasteur, Paris, France). The mouse-adapted virus strain S221 of dengue virus serotype 2 (DENV-2) was kindly provided by Dr. Shresta (La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA). The dengue virus serotype 3 (DENV-3) virus strain PaH881 / 88 and the DENV serotype 4 (DENV-4) virus strain ThD4_0087_77 were isolated in Thailand in 1988 and 1977, respectively. The Zika virus strain H / PF / 2013 (also known as PF13, GenBank: KJ776791) belonging to the Asian genetic lineage of ZIKV was obtained via the DENFREE (FP7 / 2007-2013) consortium. The vaccine strain of YFV (17D-204, Stamaryl) was obtained from a commercial batch of vaccine purchased from the Vaccine Production Center of the Pasteur Institute. All virus stocks were generated in Vero E6 cells grown in T-175 tissue culture flasks with filter cups. Vero E6 cells grown on 24-well plates were titrated. Cells were infected with 300 μl of serial stock dilutions over a 1-hour period with regular shaking, and after removal of the inoculation medium, were overlaid with DMEM containing 1.6% carboxymethylcellulose, 2% FBS, and antibiotics. After 5 days of incubation, the overlaid medium was removed, the cells were fixed with 4% PFA for 30 minutes, and stained with 0.5 μg / ml of mouse anti-DENV antibody (4G2) (produced by the recombinant protein production facility of the Pasteur Institute) to reveal virus plaques, followed by a second staining with goat anti-mouse IgG HRP conjugate (BioRad, France). The HPR signal was revealed using the Vector VIP peroxidase substrate kit (Vector Laboratories, USA) according to the manufacturer's recommendations.

[0436] Immunogenicity of antigen constructs in mice (Elispot)

[0437] Elispot plates pre-coated with anti-mouse IFNγ antibody (Mabtech AB, Nacka Strand, Sweden) were used according to the manufacturer's instructions. Splenocytes from immunized mice were plated in triplicate at 1×10 5Added at [X] cells / well and stimulated with peptide pools at 2 μg / ml of each peptide. Unstimulated splenocytes and splenocytes stimulated with 2.5 μg / ml of concanavalin A were used as negative and positive controls, respectively. After 24 hours of incubation, spots were developed according to the manufacturer's protocol and counted using an AID ELISpot reader system ELR04 (Autoimmune Diagnostika GmbH, Strassberg, Germany). The background signal from wells containing unstimulated cells was subtracted, and the results were expressed as the number of spot-forming cells per million splenocytes.

[0438] Analysis of viremia and viral load in organs (RT-qPCR)

[0439] To analyze viremia, plasma samples were collected from individual animals by submandibular venipuncture and processed as described above. RNA was extracted from 35 μl of plasma using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). To analyze viral load in the peripheral organs, whole organs were collected, weighed and frozen at -80 °C until RNA extraction. During extraction, frozen tissue samples were suspended in 1 ml of TRIzol and homogenized for 30 s at 6.0 m / s in a FastPrep-24 homogenizer (VWR, France). Total RNA was purified according to the TRIzol manufacturer's extraction protocol. RNA concentration was measured by Nanodrop spectrophotometer and the total RNA concentration in all samples was adjusted to 0.1 μg / μl. Ten microliters of each RNA preparation (1 μg total RNA) were used in the RT-qPCR reaction. To analyze DENV, a two-step RT-qPCR reaction (adapted from 28) was performed to measure the viral load in plasma and peripheral organs. RT was performed using Moloney murine leukemia virus (M-MLV) reverse transcriptase and then two identical qPCR reactions (in duplicate) were performed on each sample using the resulting product on a QuantStudio 12K Flex real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). The following primers and probes described in (28) were used to analyze samples from mice infected with DENV-1, DENV-2 and DENV-3: (forward primer: 5'-GARAGACCAGAGATCCTGCTGTCT-3' (SEQ ID No. 179); reverse primer: 5'-ACCATTCCATTTTCTGGCGTT-3' (SEQ ID No. 180); Taqman probe: [5'-FAM]AGCATCATTCCAGGCAC[MGBEQ]-3') (SEQ ID No. 181). Samples from mice infected with DENV-4 were analyzed using the same RT-PCR protocol, but the reverse primer (5'-ACCAATCCATCTCGCGGCGCT-3') (SEQ ID No. 182) and TaqMan probe (5'-[FAM]AACATCAATCCAGGCAC[MGBEQ]-3') (SEQ ID No. 183) were modified to match the sequence of the DENV-4 virus strain used for challenge. The analysis of viremia and organ load of ZIKV was performed using an RT-qPCR protocol similar to that described by Lanciotti et al. (29) for DENV, and the analysis of YFV was performed using the protocol of Bae et al. (30).

[0440] Antibodies and other reagents

[0441] Anti-mouse CD8α (clones 2, 43), anti-mouse CD4 (clone GK1.5), and IgG2b isotype control (LTF-2) rat antibodies (all from InVivoMab) were used for T cell depletion experiments.

[0442] Statistical analysis

[0443] Statistical analysis was performed using statistical tests implemented in GraphPad Prism 9 software. For paired comparisons, unpaired parametric t-tests with or without Welch's corrections (depending on the standard deviation (SD) determined from the data set) or unpaired non-parametric Mann-Whitney tests (for small sample groups) were used. One-way ANOVA tests (ordinary or Welch tests, depending on the SD) or Kruskal-Wallis tests were used for multiple comparisons. Data were considered significant when the p-value was less than 0.05.

[0444] Results

[0445] Design and improvement of DENV T cell antigens

[0446] To design antigens for a multivalent DENV vaccine, a phylogenetic tree was first constructed using 240 complete polyprotein sequences of the four DENV serotypes. Based on this tree, a smaller set of DENV sequences was selected, with each sequence representing each phylogenetic sub-lineage of each serotype ( Figure 1 ).

[0447] The cross-conserved region localization between different serotype DENVs was mapped by plotting alignment scores along the polyprotein length, and the results showed that such regions were mostly located in the non-structural proteins NS3, NS4B, and NS5 ( Figure 2 A and Figure 2 B). Several previous studies have reported that these proteins are targets of cytotoxic T cell responses and can provide cross-protection against DENVs of different serotypes (5 - 7). To identify the T cell epitope-containing regions in DENV sequences, all human MHC class I and MHC class II epitopes labeled as positive in various T cell assays (secretion of IFNγ and / or TNFα and IL2, cytotoxicity) were retrieved from the Immune Epitope Database and Analysis Resource (IEDB) and aligned with a representative set of DENV sequences (21).

[0448] The distribution of known epitopes along the DENV sequence alignment was also plotted by XY graphs ( Figure 2C) Visualization was performed, and the figure can identify epitope-containing regions (epitope clusters) as well as individual epitopes that are highly similar in different sequences (conserved epitopes) and / or individual epitopes that can be presented by multiple HLA alleles (multi-allelic epitopes). According to published studies, most known functional human MHC class I epitopes are located in non-structural proteins NS3, NS4B, and NS5, while MHC class II epitopes are more evenly distributed between structural and non-structural proteins. Although there are thousands of DENV-specific MHC class I epitopes labeled as positive in the IEDB, many of these epitopes were identified in studies conducted in specific geographical regions where the distribution of MHC class I alleles does not necessarily reflect the global population. In addition, many epitopes were identified and characterized in experimental studies conducted in transgenic mice carrying human MHC class I alleles (with strong protective CTL responses against DENV, such as HLA-B*07:02) (31). Therefore, compared with epitopes presented by less protective but more prevalent human alleles globally (such as HLA-A*01:01 and HLA-A*24:02), the representation of epitopes presented by such alleles in the database may be overestimated. In addition, since several serotypes of DENV often co-circulate in many disease-endemic areas, T cell responses induced by a specific DENV serotype may not provide optimal protection against another serotype. The factors mentioned above may lead to bias in the representation of known human T cell epitopes in the database. To compensate for this possible bias and achieve a more balanced representation of epitopes in DENV-based antigens, in silico prediction of MHC class I epitopes was performed on a selected DENV sequence dataset using prediction tools on the Immune Epitope Database (IEDB) and the website of the Technical University of Denmark (DTU Bioinformatics) (21, 25). The predicted epitopes were mapped to the alignment of DENV sequences and visualized by XY-graphs ( Figure 2 D and Figure 2 E). Comparing the distribution of epitopes predicted by two different methods with the distribution of known epitopes can more precisely select regions of DENV-Ag1. Prediction of MHC class II epitopes has not been performed because, as reported, the algorithms used to predict such epitopes lack efficiency and predictive power compared to the algorithms used to predict MHC class I epitopes (32). In addition, studies on DENV in animal models have shown that cytotoxic T cell responses against MHC class I epitopes play a more important role in protecting mice from DENV infection. To incorporate genetic variations presented by 4 DENV genotypes in a single sequence, 75% majority consensus sequences were inferred for each DENV genotype, and then a major consensus sequence was generated based on the 4 individual consensus sequences (Figure 3).

[0449] In general, the sequence of DENV-Ag1 is identical to the major consensus sequence, except at some positions where variability is evenly distributed among different genotypes (e.g., position 1674 in the NS3-1 region, where serine (S) is encoded by DENV-1 and DENV-3 genotypes, and alanine (A) is encoded by DENV-2 and DENV-4 genotypes), or at sites where more significant differences are observed (e.g., position 1928 in the NS3-2 region). In such cases, the amino acid selection is based on the number of known or predicted T cell epitopes it contains; amino acids that are more represented in the dataset are given more weight in the sequence. Although this approach is generally applicable, in several short regions of the alignment, the amino acid variability is too great to be represented by a single consensus sequence. For this reason, three additional short sequences were added to DENV-Ag1: NS3-1A, NS3-3A, and NS3-3B, each of which has a rearranged subregion of the larger sequence and represents the commonality of the remaining genotypes (Figure 3A). The selected regions were joined together and the junctions were optimized to remove any possible non-specific immunodominant epitopes that might occur at the junctions (Figure 4). DENV-Ag2 was formed as a modified version of DENV-Ag1, in which its 26aa-long N-terminal fragment (including the NS5-5 region and the L1 linker) was replaced by a 47aa-long sequence that includes three additional antigenic regions of the NS-3 protein (NS3-4, NS3-5, and NS3-6). This modification includes several MHC class I epitopes that are expected to induce a broader response against DENV compared to the response induced by DENV-Ag1. Prediction of MHC class I epitopes indicates that DENV-Ag2 should contain between 26 (the minimum predicted for HLA-A*01:01) and 55 (the maximum predicted for HLA-A*35:01) human epitopes per allele. Prediction of the expected coverage of DEN-Ag in the population using the Allele Coverage tool (IEDB) indicates that both antigens should induce protection against DENV in 86-100% of individuals from most geographical regions.

[0450] The immunogenicity of DENV-Ag1 was evaluated in A129 mice

[0451] First, we measured the activation of T cell responses induced by the integrative lentiviral vectors (iLV) iLV-DENV-Ag1(IND) and iLV-DENV-Ag1(NJ), which were pseudotyped with the glycoprotein (G) of Indiana (IND) or New Jersey (NJ) serotype vesicular stomatitis virus. 5×10 7Three groups of A129 mice (n = 5) were immunized intramuscularly (i.m.) with iLV-DENV-Ag1(IND), iLV-DENV-Ag1(NJ), or the control vector iLV-GFP(IND) of TU. Spleen cells were extracted 14 days after immunization, and their IFNγ secretion in response to restimulation with a DENV-Ag1-specific peptide pool was analyzed by Elispot. After immunization with two vectors expressing DENV-Ag1, IFNγ secretion by spleen cells was induced upon stimulation with antigen-specific peptides, and there was no significant difference between the two vectors pseudotyped with VSV-IND or VSV-NJ( Figure 5 A).

[0452] To analyze the T cell responses induced by DENV-Ag1 expressed in the context of a non-integrating lentiviral vector (LV), two groups of A129 mice (n = 6) were immunized i.m. with 3 × 10 8 TU / mouse of LV-DENV-Ag1(IND) or LV-GFP(IND). Delivery of the antigen by a single injection of LV (used at a 6-fold higher dose than one of the integrating vectors) has induced a substantial T cell response( Figure 5 B), indicating that non-integrating lentiviral vectors can be successfully used to induce immune responses against DENV-specific antigens and confirming our previously published results (33). To compare the extent of responses induced by a single-dose immunization regimen with a prime-boost regimen, two groups of A129 mice (n = 6) were immunized i.m. with 7.5 × 10 6 TU / mouse of LV-DENV-Ag1(IND) or LV-GFP(IND). Two months later, the mice were re-injected i.m. with 3 × 10 8 TU / mouse of LV-DENV-Ag1(NJ) or LV-GFP(NJ), and T cell activity was analyzed 6 days after the second immunization. Implementation of the prime-boost regimen significantly increased the average T cell response (2.4-fold).

[0453] Intermediates of T cell responses (IFNγ, TNFα, IL2, and the lymphocyte degranulation marker CD107a) were detected by intracellular cytokine staining

[0454] Cytokines released by antigen-experienced cytotoxic T lymphocytes are widely regarded as evidence of their targeted action against specific pathogens. Several previous studies have linked T cell immunity against DENV to the presence of DENV-specific T cells secreting IFNγ, TNFα, and IL2. Multipotent T cells (i.e., cells that secrete two or three cytokines simultaneously in response to DENV) are considered particularly important for the antiviral response. We have investigated whether immunization with the LV-DENV-Ag1 vector induces cytokine production by antigen-specific cells and whether the same T cell population can secrete several cytokines simultaneously. Fourteen days after infection, spleen cells were extracted from several IFNAR-BL6 mice immunized with 3×10 8 TU of the LV-DENV-Ag1 (IND) or LV-GFP (IND) vector, and T cells secreting IFNγ, TNFα, IL2, and the lymphocyte degranulation marker CD107a were analyzed by intracellular cytokine staining (ICS). During this procedure, spleen cells from several mice immunized with the same vector were pooled and stimulated with a pool of 11 DENV peptides for 3 hours, followed by incubation with Brefeldin A / Monensin for 3 hours (Figure 6). Flow cytometric analysis of cytokine-stained cells has indicated that CD8+ T cells respond to antigen exposure / peptide stimulation by secreting IFNγ, TNFα, and IL2. In addition, a subset of cells exposed to LV-DENV-Ag1 / stimulated with DENV peptides co-expressed IFNγ and the lymphocyte degranulation marker CD107a, indicating that such cells have targeted cytotoxicity and are capable of mediating the lysis of virus-infected cells. Multipotent CD8+ T cells co-expressing three cytokines (IFNγ, TNFα, and IL2) were also detected in spleen cells exposed to LV-DENV-Ag1 and stimulated with DENV peptides, but not in spleen cells exposed to an irrelevant antigen (LV-GFP) or in spleen cells stimulated with a non-specific peptide (YF-C).

[0455] Single-dose immunization protocol: protection of A129 mice against DENV-1 and DENV-2 infection

[0456] A group of A129 mice (n = 10) was immunized intramuscularly with 7.5×10 6 TU / mouse of LV-DENV-Ag1 (IND), and another group was immunized with the same dose of LV-GFP (IND). Twenty-seven days after immunization, each group was further divided into two equal subgroups and infected with DENV-1 (1×10 7 FFU / mouse) or DENV-2 (5×10 5FFU / mouse). The body weights of the infected mice were measured daily, and blood samples were collected from several mice of the sub - groups on days 1, 2, 3, 4, and 7 to 8 post - infection (p.i.) to monitor the level of viremia in the plasma. The mice gained weight on days 7 to 8 p.i., indicating their recovery from the infection, at which time the mice were sacrificed. DENV - 1 did not produce any symptoms in A129 mice, while the mice infected with DENV - 2 developed obvious ruffled fur on days 1 - 2 p.i., and it gradually became less obvious during the recovery phase (around days 5 - 6, when the mice started to regain weight). On days 3 - 4 post - infection, the average body weight of the mice immunized with LV - DENV - Ag1(IND) and infected with DENV - 1 or DENV - 2 was significantly higher than that of the mice immunized with LV - GFP(IND) ( Figure 7 A and Figure 7 B).

[0457] Although viremia was detected in all mouse groups, the levels of DENV - 1 and DENV - 2 viremia measured in the mice immunized with the DENV - specific vector were on average 20 to 30 - fold lower than those in the mice immunized with the control vector from day 1 to day 4 post - infection( Figure 7 C and Figure 7 D). In addition, compared with the control, viremia subsided earlier in the group immunized with LV - DEN - Ag1(IND): DENV - 1 could not be detected in the plasma from day 6 p.i., and DENV - 2 could not be detected after day 3 p.i. In contrast, both viruses could be detected in the plasma of the mice immunized with LV - GFP(IND) for up to 7 to 8 days post - infection. These results indicate that immunization of A129 mice with a single dose of LV - DENV - Ag1(IND) induces partial protection against DENV - 1 and DENV - 2 infections, significantly reducing the level of viremia and shortening its duration.

[0458] Single - dose immunization protocol: Protection of IFNAR - BL6 mice against DENV - 1, DENV - 2, DENV - 3, and DENV - 4 infections

[0459] The immunization / protection study was also conducted in IFNAR - KO mice (IFNAR - BL6) derived from C57BL / 6. Compared with the mice of the A129 lineage, IFNAR - KO mice have previously been shown to be more sensitive to experimental infections with various flaviviruses (34, and our unpublished data). To test protection against DENV - 1 and DENV - 2, 3×10 8Two groups of IFNAR - BL6 mice were immunized with LV - DENV - Ag1(IND) or LV - GFP(IND) at 10 TU / mouse. One month later, half of the immunized mice in each group were infected with DENV - 1(1×10 7 FFU / mouse), and the other half were infected with DENV - 2(2×10 6 FFU / mouse). No obvious symptoms were detected in any of the infected mice, except for weight loss observed in all groups in the first two days after infection( Figure 8 A and Figure 8 B). All mice immunized with LV - DENV - Ag1(IND) regained weight from day 3 to day 4 after infection (significantly different from control mice), while weight recovery in mice immunized with LV - GFP(IND) was delayed, generally occurring from day 4 to day 7 - 8 after infection. In addition, similar to the results obtained in A129 mice, viremia of DENV - 1 and DENV - 2 in IFNAR - BL6 mice immunized with LV - DENV - Ag1(IND) also significantly decreased starting from day 1 and day 2 after infection respectively( Figure 8 C and Figure 8 D). The level of viremia in mice immunized with the DENV - Ag1 - expressing vector also decreased more rapidly: starting from day 5 p.i., DENV - 2 could not be detected in the plasma of such mice, and the level of DENV - 1 measured on day 7 after infection was significantly lower than the corresponding viremia level of control mice. In mice immunized with LV - DENV - Ag1(IND) against DENV - 1( Figure 8 E) and DENV - 2( Figure 8 F), the viral load detected in three different tissues (spleen, liver and small intestine) on day 4 after infection was also significantly reduced. In summary, the immunization / protection experiments conducted in two lineages of IFNAR - KO mice produced similar results: faster weight recovery (day 3 - 4 vs day 4 - 8) was observed in all mice immunized with LV - DENV - Ag1(IND), significant reduction of viremia, faster virus clearance and less virus present in peripheral organs.

[0460] To test protection against DENV - 3, two groups of mice (n = 6) were intramuscularly immunized with LV DENV - Ag1(IND) or LV - GFP(IND) at 3×10 8 TU / mouse, and 2 months later (61 days p.i.) were challenged with 8×10 6DENV-3 infection per mouse. Subgroups of mice (n = 3) were monitored for viremia at different days post-infection. Two independent experiments were conducted under the same conditions to demonstrate statistical significance. After verifying that there were no statistically significant differences in the viremia levels and average weight loss in the LV-GFP control in the two experiments, the results were pooled. In addition, spleen samples were collected from the sacrificed animals at 7 days p.i. to determine the viral load in the spleen. This organ was selected for analysis because several studies have shown that the spleen has the highest level of DENV replication in laboratory-infected IFNAR-KO mice (35). Protection against DENV-4 was evaluated in two groups of IFNAR-BL6 mice (n = 6) that were immunized with 3×10 8 TU / mouse of LV-DENV-Ag1 (IND) or LV-GFP (IND) and infected with DENV-4 (1×10 7 FFU / mouse) 1 month later. The body weights of the animals were measured daily, and blood samples were collected at different days post-infection to monitor viremia. Samples of the spleen, liver, and small intestine were collected at 7 days to evaluate the viral load in those organs.

[0461] Similar to the mice infected with DENV-1 and DENV-2, the mice infected with DENV-3 or DENV-4 did not show any symptoms except for weight loss observed during the first 2 days post-infection. The body weights of the mice immunized with LV-DENV-Ag1 (IND) and infected with DENV-3 were significantly higher than those of the control mice on days 3-4 post-infection ( Figure 9 A), and the viremia was significantly reduced starting from day 2 ( Figure 9 C). At 7 days, viremia was still detectable in 4 out of 6 mice immunized with LV-GFP, but no viremia was detected in any of the mice immunized with LV-DE-Ag1 (IND). In addition, the viral load detected in the spleens of the mice immunized with LV-DENV-Ag1 (IND) at 7 days was significantly lower than that of the control mice ( Figure 9 E).

[0462] Significant body weight differences were observed between the various mice infected with DENV-4 and immunized with LV-DENV-Ag1 (IND) and LV-GFP (IND) at 4 and 7 days post-infection ( Figure 9 B). The viremia in the mice immunized with LV-DENV-Ag1 (IND) was also significantly reduced on days 3 and 4 post-infection ( Figure 9D). Similar to the results obtained with DENV-3 infection, at the end of the experiment, viral RNA was detectable in only 2 out of 6 animals in the group immunized with LV-DENV-Ag1(IND), while viral RNA was detected in all animals in the group immunized with LV-GFP(IND). A significantly reduced viral load was detected in the spleens of mice infected with DENV-4 ( Figure 9 F).

[0463] Prime-boost vaccination regimen: protection of A129 mice against DENV-2 infection

[0464] To evaluate the efficacy of a prime-boost vaccination regimen to protect IFNAR-KO mice against DENV infection, two groups of A129 mice (n = 12) were immunized i.m. with 1×10 7 TU / mouse of LV-DENV-Ag1(IND) or LV-GFP(IND). Fifty-nine days after immunization, two groups of mice were boosted with an LV expressing the same antigen but pseudotyped with VSV-G (New Jersey (NJ)) of different serotypes at 2×10 8 TU / mouse to avoid potential anti-vector immune responses. Twenty-eight days after the second immunization, all mice were inoculated with 1×10 7 FFU / mouse of DENV-2. The weights of the animals were measured daily and blood samples were collected from subgroups of mice at different days post-infection ( Figure 10 ). The animals were sacrificed on day 9 post-infection, i.e., after two consecutive days of weight gain. Similar to the previous experiment analyzing the protection of A129 mice against DENV-2 by single immunization with LV-DENV-Ag1(IND), all infected mice started to lose weight in the first two days post-infection ( Figure 10 A) and developed a ruffled coat.

[0465] However, compared to the mice immunized with LV-GFP(IND) / LV-GFP(NJ), the mice immunized with LV-DENV-Ag1(IND) and LV-DENV-Ag1(NJ) started to regain weight earlier, and a significant difference in weight was observed between the groups on days 2-4 post-infection ( Figure 10 A). The appearance of a ruffled coat in infected animals is usually associated with weight loss and becomes less obvious once the mice start to regain weight. Analysis of viremia by RT-qPCR showed that the viral load in the sera of mice immunized with an LV expressing DENV-Ag1 was reduced by approximately 10-fold on days 1-2 p.i. Figure 10B). Similar to the results of the single-dose immunization experiment, compared with viremia in control mice, DENV-2 viremia in mice immunized with LV-DENV-Ag1(IND) also decreased more rapidly and became undetectable since day 5 p.i. In the group immunized with LV-DEN-Ag1(IND) / LV-DENV-Ag1(NJ), the viral load in the spleen (measured at day 9 p.i.) also decreased significantly ( Figure 10 C). Overall, the results of this experiment were similar to those of the single-dose immunization protocol ( Figure 7 B and Figure 7 D), and indicated that prime-boost immunization did not provide a significant advantage in protecting A129 mice from DENV-2 infection.

[0466] Protection mechanism: Depletion of CD8+ T cells leads to reduced protection

[0467] To analyze the role of different T lymphocyte populations (CD8+ and CD4+) in DENV-specific vector-induced protection against DENV infection, IFNAR-BL6 mice immunized with LV-DENV-Ag1(IND) or LV-GFP(IND) were selectively depleted of CD8+ or CD4+ cells before infection with DENV-2. First, two groups of mice were immunized intramuscularly with 3×10 8 TU / mouse of the LV-DENV-Ag1(IND) or LV-GFP(IND) vector, and boosted 40 days later by injection of the same vector at the same dose. Four days after boosting, each group was further divided into 3 subgroups, which were injected intraperitoneally with 250 μg / mouse of anti-mouse CD8α antibody, anti-mouse CD4 antibody, or IgG2b isotype control antibody. Three days later, i.e., one day before infection, the same antibodies were injected a second time. The next day, the mice were infected intravenously with 1×10 7 FFU / mouse of DENV-2. Infection was monitored for seven days, and measurements of animal weights were taken at days 1, 2, 3, 4, 6, and 7 after infection. Blood samples were collected from the mouse subgroups at the same days for monitoring viremia. The efficacy of CD8+ and CD4+ T cell depletion was verified by cytometry of spleen cells extracted from euthanized animals at day 7. The kinetics of DENV-2 infection in mice pre-injected with IgG2b isotype control antibody were the same as those of mice not previously injected with antibody (Figure 11A and Figure 8B). All mice infected with DENV-2 lost weight 2 days before infection, followed by a recovery phase. The recovery phase started earlier (day 2 p.i.) in mice immunized with the LV-DENV-Ag1(IND) vector compared to mice immunized with the LV-GFP(IND) vector (day 4 p.i.). Viremia in animals immunized with the DENV-specific vector was lower at all time points than that observed in mice immunized with the GFP-containing vector, and a significant decrease in viremia levels was observed at day 4 p.i. (Figure 11E). Depletion of CD4+ T cells in mice immunized with the LV-DENV-Ag1(IND) or LV-GFP(IND) vector did not significantly alter the course of infection: the kinetics of weight loss and recovery and the viremia levels in CD4+ T cell-depleted mice were very similar to those seen in the corresponding non-depleted mice (Figure 11A, Figure 11B, and Figure 11E). In contrast, depletion of CD8+ cells from the groups immunized with LV-DENV-Ag1(IND) or LV-GFP(IND) significantly affected the course of infection. These two groups showed a slower rate of weight recovery and a longer duration of viremia compared to the non-depleted or CD4+ depleted groups (Figure 11C and Figure 11E).

[0468] Clearly, the rapid reduction of viremia observed in the group immunized with LV-DENV-Ag1(IND) and injected with isotype control or anti-CD4 antibody on day 4 did not occur in the CD8+ cell-depleted group (Figure 11E). Deaths were only observed in the group immunized with the LV-GFP(IND) vector: 2 out of 6 mice injected with isotype antibody (33%), 1 out of 6 mice injected with anti-CD4 antibody (17%), and 4 out of 6 mice injected with anti-CD8 antibody (67%) died from the infection. Although the levels of viremia observed in the three groups of mice immunized with LV-GFP(IND) were similar on days 1-3 p.i., viremia in the CD8+ depleted group was prolonged from day 4 post-infection compared to the other two groups (Figure 11D). In addition, compared to the other two groups, the CD8+ depleted group had the highest DENV-2 load measured in the spleen on day 7, and this group also showed the slowest body weight recovery and the lowest survival rate. These results indicate that in IFNAR-KO mice, CD8+ T cell responses play an important role in protection against DENV challenge induced by LV-DENV-Ag1, helping to control the infection at two distinct stages: in the initial phase, DENV-Ag1-stimulated CD8+ cells are responsible for the initial reduction of viremia and faster virus clearance. Thus, significant differences in viremia (days 1-4 p.i.) and body weight (days 3-4 p.i.) were observed between the groups immunized with LV-DENV-Ag1 and LV-GFP, regardless of whether CD4+ cells were depleted. In contrast, the differences in viremia and body weight between the corresponding groups with CD8+ cell depletion were smaller and not significant. Naïve CD8+ T cells (not specific for DENV at the initial stage of infection) appear to be crucial for controlling the infection at the later stage (days 5-7) of infection, as CD8+ T cell depletion prolonged viremia and delayed body weight recovery in the groups immunized with LV-DENV-Ag1 and LV-GFP, and increased the mortality of mice immunized with LV-GFP.

[0469] POLYNUCLEOTIDE AND LENTIVIRAL VECTOR EXPRESSING THE NON - STRUCTURAL ANTIGENS OF ZIK VIRUS AND YF VIRUS IN THE FORM OF FUSION POLYPEPTIDE

[0470] Materials and Methods

[0471] Antigen Design

[0472] To design ZIKV and YFV T cell antigens, the complete nucleotide sequences of 17 ZIKV strains (GenBank accession numbers: KU955595.1, LC002520.1, KF268948.1, OL414716.1, HQ234500.1, KX377336.1, OK054351.1, MH119185.1, OQ661918.1, KY241712.1, ON209935.1, KY766069.1, KY014295.2, MF438286.1, KU922960.1, KU820897.5, KU509998.3) and 19 YFV strains (GenBank accession numbers: JN620362.1, KF769015.1, DQ235229.1, MF004383.1, MW960207.1, JX898878.1, MF405338.1, HM582851.1, JF912187.1, JF912190.1, MF004382.1, JF912181.1, MW158361.1, U54798.1, KU978763.1, AY968064.1, KY861728.1, AF094612.1, KU921608.1) were retrieved from the NCBI sequence database (66). These sequences represent different gene lineages (64, 65) of each virus, were aligned, and a phylogenetic tree was constructed using Mega 7 (67). The consensus sequences of the major phylogenetic groups of ZIKV (African and Asian lineages) and YFV (South American, West African, and Southeast African lineages) were inferred from the corresponding amino acid sequences using the Consensus Maker software tool (68) to limit sequence diversity and identify conserved regions. MHC class I epitopes were predicted from the consensus sequences using the proteasome cleavage / TAP transport / MHC class I combinatorial predictor tool (69, 70) in the Immune Epitope Database (71) and the netCTLpan predictor (72) on the DTU Bioinformatics Server website (73). All 9-mer and 10-mer peptides that could be presented by 27 of the most common human leukocyte antigen (HLA) alleles (74) were identified using the IEDB predictor, and those with a total positive score and a cut-off binding affinity IC50 ≤ 500 nM were selected. All 9-mer peptides for the same set of HLA alleles were predicted using netCTLpan, and 100 epitopes with the best combined prediction scores were retained for each HLA allele. The epitopes predicted by these two methods were aligned with the consensus sequences of ZIKV and YFV using Blast (66) and plotted along the sequence length to identify regions containing the highest number of predicted MHC class I epitopes.The final selection of the antigenic regions maximally includes characterized human epitopes (retrieved from the IEDB database) and epitope-containing regions identified by prediction. The antigenic regions are first joined together to form a single polypeptide, and then the prediction of T cell epitopes is repeated to verify that the individual regions joined together do not form non-specific epitopes and that all epitopes located near the joining sites are predicted to form correctly. If the joining of the antigen fragments forms a dominant non-specific MHC class I epitope for one of the 27 HLA alleles, such an epitope is eliminated by inserting a hydrophobic amino acid linker at the joining site. Multiple rounds of epitope prediction are then performed to confirm that all non-specific epitopes have been eliminated.

[0473] Generation of lentiviral vectors

[0474] The sequences encoding the ZIKV and YFV multi-antigens (LV-ZIK, LV-YF-S and LV-YF-NS) were codon-optimized for expression in mammalian cells and synthesized by GeneCust (France). Each antigen-encoding sequence was inserted between the β2-microglobulin (β2m) promoter and the previously mutated woodchuck transcriptional regulatory element (mWPRE) in the pFLAPΔU3-β2m-WPRE vector to improve vector safety. Plasmids for the production of non-integrating LVs, including the transfer vector plasmid containing the antigen, the packaging plasmid NDK encoding a mutated version of the integrase protein (D64V), and the envelope plasmid encoding the VSV viral G glycoprotein, were purified using the NucleoBond Xtra Maxi EF Kit (Macherey Nagel), aliquoted and stored at -80 °C. As previously described, LVs were produced in HEK-293T cells and the LV titre was determined by qPCR on HEK293T cells transduced with the LV, which were treated with aphidicolin to prevent cell division (63, 38).

[0475] Mice

[0476] Interferon-γ receptor knockout mice (IFNAR-KO) aged 6 to 16 weeks, carrying the Ifnar1 tm1Agt allele on a 129 (A129) or C57BL / 6J (IFNAR-BL6) genetic background were used in the experiments. Both mouse lineages belong to H-2 bMHC haplotypes and thus have similar antigen presentation and T cell responses. Preliminary evaluations of the immunogenicity and protective efficacy against LV-ZIK, LV-YF-S, and LV-YF-NS were conducted in A129 mice because this lineage represents one of the established Zika virus (ZIKV) infection models (33-34). However, our comparison of YFV infection in A129 and IFNAR-BL6 mice (not published) and published data (63, 34) indicate that IFNAR-BL6 lineage mice are more susceptible to ZIKV and YFV viruses. Therefore, immunogenicity and protection studies were also conducted in this mouse lineage. Mice were housed and maintained under specific pathogen-free conditions in the animal facilities of the Pasteur Institute, and all experiments involving ZIKV and YFV infections were conducted in the A3 animal facilities. Animal experiments were conducted according to French and European guidelines after approval by the Safety, Animal Care and Use Committee of the Pasteur Institute (CETEA no. DAP1800077) and the Ministry of Higher Education and Research (APAFIS#18428-2019010717408411_v2).

[0477] Immunization and challenge with ZIKV and YFV

[0478] All lentiviral vectors at 1 - 3 × 10 8 TU / mouse (depending on the experiment) were used to immunize A129 and IFNAR-BL6 mice, which was achieved by intramuscular (i.m.) injection of a total volume of 50 μL of LV in the posterior muscle. Mice were challenged intraperitoneally with a total volume of 300 μl of ZIKV and YFV, and the challenge dose (specified herein) depended on the efficiency of virus propagation in VeroE6 cultures. The infective dose was first verified in IFNAR-KO mice in a preliminary experiment. The disease signs of the mice, such as lethargy, ruffled fur, hunched posture, and neurological symptoms (partial paralysis, exhaustion, spasms, gait instability, and / or falls) were monitored and their body weights were regularly recorded. If a mouse lost more than 20% of its initial body weight, or if it lost 10% accompanied by neurological symptoms (i.e., abnormal movement and / or limb paralysis), the mouse was euthanized.

[0479] Propagation and titration of virus stocks

[0480] The Asian strain Zika PF-13 (strain H / PF / 2013; GenBank: KJ776791) was obtained via the DENFREE (FP7 / 2007 - 2013) consortium. The vaccine strain of YFV (17D - 204, Stamaryl) was obtained from a commercial batch of vaccine purchased from the Vaccine Production Center of the Pasteur Institute. All virus stocks for infection were generated essentially as previously described (63) and titrated in Vero E6 cells. Plaques generated by ZIKV and YFV were visualized by staining with a Gram Crystal Violet solution (BD) (1:1) diluted in H2O for 15 minutes, counted, and used to calculate the infectious virus titer, expressed as the number of plaques per milliliter of virus stock.

[0481] ELISpot assay

[0482] The ELISPOT procedure was generally performed according to the protocol supplied with the ELISPOT kit for IFNγ detection ((Mabtech AB, Nacka Strand, Sweden), except that 96-well PVDF culture plates (Millipore, Sigma) were activated by incubation with 35% ethanol, washed, and coated by incubation overnight with 100 μl / well of 5 μg / ml rat anti-mouse IFNy antibody (clone AN18, BD Pharmingen). Spleen cells from immunized mice were added at 1 × 10 5 cells / well in triplicate and stimulated with an antigen-specific peptide pool with a peptide content of 2 μg / ml for 18 hours. Negative controls (unstimulated spleen cells) and positive controls (spleen cells stimulated with 2.5 μg / ml concanavalin A) were also analyzed in triplicate for each animal. After incubation, spots were developed according to the Mabtech AB Elispot kit protocol and counted on an AID ELISpot Reader System ELR04 (Autoimmune Diagnostika GmbH, Strassberg, Germany). The mean background signal of the negative control was subtracted from the mean positive signal recorded for each spleen cell / peptide pool combination, and the results were expressed as the number of spot-forming cells per million spleen cells.

[0483] Flow cytometry

[0484] Analysis of intracellular cytokine secretion was performed according to the protocol described previously (76). Briefly, spleen cells obtained by homogenization through a 100 μm nylon filter (Cell Strainer, BD Bioscience) were at 4 × 10 6Cells were seeded at

[0485] Viremia and viral load in peripheral organs

[0486] Mouse blood samples were collected by direct bleeding from the submandibular vein into EDTA-containing Microvette 500 K3E tubes (Starstedt) at different time points during infection. Plasma samples clarified from blood cells by centrifugation and organ samples collected from euthanized animals were stored at -80 °C until RNA extraction for determination of viremia and viral load in the organs. RNA extraction and RT-qPCR analysis were performed as previously described (63). Analysis of viremia and organ load of ZIKV was performed using the two-step RT-qPCR protocol of Lanciotti et al. (29). The YFV load in the sera and peripheral organs of A129 mice was analyzed using the RT-PCR protocol of Bae et al. (30), and the YFV load in the sera and organs of IFNAR-BL6 mice was analyzed using the protocol of Fischer et al. (78). Standards for viral RNA quantification were obtained by in vitro transcription of cloned virus-specific DNA fragments amplified from ZIKV stock (primers ZIKV-F-CGGGATCCCGAGCCAAAAAGTCATATACTTG (SEQ ID NO:213) and ZIKV-R-ACCGCTCGAGTCAGTTTCATGTCCTGTGTCATT (SEQ ID NO:214)) and YFV (YFV-F-AACCCACACATGCAGGACAA (SEQ ID NO:215) and YFV-R-GTTGCAGGTCAGCATCCACA (SEQ ID NO:216)). RT reactions were performed using Moloney murine leukemia virus reverse transcriptase (M-MLV) and virus-specific primers. The resulting cDNA was subjected to duplicate qPCR analysis on a QuantStudio 12K Flex real-time PCR system (Applied Biosystems, Carlsbad, CA, USA), and the amount of viral RNA was determined according to the standard curve reproduced for each RT-PCR run.

[0487] Immunohistochemistry

[0488] Mouse brain and spleen samples were fixed in formalin for 72 h and embedded in paraffin. Paraffin sections (5-μm thick) were stained with hematoxylin and eosin (H&E). Slides were scanned using an AxioScan Z1 (Zeiss) system, and images were analyzed using Zen 2.6 software.

[0489] Statistical analysis

[0490] Statistical analysis was performed as publicly described above for the DENV experiments.

[0491] Design and improvement of ZIKV T cell antigens

[0492] To generate multi-antigens that can induce T cell responses against ZIKV and YFV, first, phylogenetic trees were inferred based on strains ([ Figure 31 ) representing the major gene lineages of each virus.

[0493] For ZIKV, the phylogenetic tree was based on representative strains of the African genotype, which originated from Senegal, Guinea, and Nigeria (West Africa), Uganda, and the Central African Republic (East Africa) (80), and the Asian genotype, including strains from: Malaysia and India (ZB.1.0 lineage, Southeast Asia / South Asia), Thailand (ZB.1.1 lineage, Southeast Asia / South Asia), Singapore and Cambodia (ZB.1.2 lineage, Southeast Asia / South Asia), French Polynesia and Haiti (ZB.2.0 lineage, Polynesia, Caribbean, South America), Mexico and Colombia (ZB.2.1 lineage, Central America), USA and Cuba (ZB.2.2 lineage, North America) (64).

[0494] The phylogenetic tree based on YFV strains included representative strains of the South American, West African, and Southeast African genotype lineages. To find T cell epitopes that can be presented by HLA alleles and are shared by different strains of ZIKV and YFV, consensus amino acid sequences were inferred for the gene lineages of each virus. The genetic diversity of ZIKV is represented by the consensus sequences of its two major genotypes (Asian and African), while the diversity of YFV is outlined by three consensus sequences, each representing one lineage of the virus.

[0495] Sequences of 33 human MHC class I and 227 MHC class II ZIKV-specific epitopes and 122 and 407 YFV-specific epitopes labeled as positive in T cell assays in the IEDB database (71) were downloaded and aligned with the ZIKV consensus sequences of each virus for matching to identify known T cell immunogenic regions (Figure 12). To ensure that potential immunogenic regions were not missed due to current insufficient knowledge of ZIKV and YFV immunogenicity and to better delimit immunogenic regions, bioinformatics tools provided by the IEDB and DTU technical websites (71, 73) were used to predict MHC class I epitopes presented by 27 HLA alleles (74) shared by 97% of the global population. The distribution of the predicted MHC class I epitopes was compared with the distribution of the validated epitopes, and several regions with the highest epitope density were selected to generate multi-antigens specific for each virus (Figure 13 and Figure 13). The consensus sequences of two ZIKV genotypes representing lineages of ZIKV in Asia and Africa had 97% similarity, and thus, the consensus sequence of the Asian genotype, which is more widespread, more diverse globally, and has also led to multiple major outbreaks of Zika disease, was selected as the main consensus sequence, resulting in the basis for the ZIKV-specific antigen (also known as "ZIK"). Two aa residues (E143K and P147A) in the ZIKV alignment (Figure 13) were converted to the consensus sequence of the African lineage because this sequence predicted more human MHC class I epitopes presented by a wider range of HLA alleles compared to the corresponding sequences of the Asian lineage. The first antigen (ZIK-Ag) was based on the ZIKV conserved region containing known and predicted T cell epitope clusters and was designed and optimized using a method similar to that described above for DEN-Ag design (Figure 12). It included regions of the C, PrM, NS4B, and NS5 proteins (Figure 12). Regions from the structural proteins capsid (C) and pre-membrane (PrM) were included because these regions contain MHC class I epitope clusters (known or predicted), and the sequence homology levels between these regions and the corresponding DENV regions were low enough to avoid cross-reactive antibody responses that could lead to ADE (Figure 13). The second antigen represented the full sequence of the ZIKV-NS1 protein, to which a 20-aa length signal peptide derived from the E protein coding region was added to ensure proper processing and targeting of NS1 intracellularly.

[0496] Immunogenicity of a non-integrating lentiviral vector expressing ZIKV-Ag in A129 mice

[0497] To analyze the T cell responses induced by ZIKV-Ag expressed from non-integrating LV, a single dose of 3×10 8Two groups of mice (six mice per group) were immunized with LV-ZIKV-Ag(IND) or LV-GFP(IND) at 1 TU / mouse. Fourteen days after immunization, spleen cells from the immunized mice were collected and IFNγ production was analyzed by ELISpot assay using region-specific peptide pools representing MHC class I epitopes of human and A129 mice (H-2b mice) ( Figure 14 and Table 1). Tests have shown that LV-ZIKV-Ag induces a T cell response in A129 mice, with the highest reactivity observed against NS5A, followed by the NS5B and PrM regions of the antigen.

[0498] The immunogenicity of LV-ZIKV-Ag and ZIKV-ZIKV-NS1 was also evaluated in IFNAR-BL6 mice by intracellular cytokine staining (Figure 15 and Figure 16, respectively). Fourteen days after infection, spleen cells from IFNAR-BL6 mice immunized with 3×10 8 TU of the LV-ZIKV-Ag, LV-ZIKV-NS1 or LV-GFP vectors were extracted and the secretion of IFNγ, TNFα, IL2 and the lymphocyte degranulation marker CD107a was analyzed. Flow cytometric analysis of cytokine-stained cells has shown that CD8+ T cells respond to antigen exposure / peptide stimulation by secreting IFNγ, TNFα and IL2. A subset of cells from mice immunized with the LV expressing two ZIKV antigens co-express IFNγ and CD107a. Similar to what was shown for the LV expressing DENV-Ag1, CD8+ T cells co-expressing three effector cytokines (IFNγ, TNFα and IL2) were also detected among spleen cells exposed to the two ZIKV antigens and restimulated with ZIKV-specific peptides (but IL2 was not detected in the CD8+ T cells of mice immunized with ZIKV-Ag), but not in spleen cells exposed to an irrelevant antigen (LV-GFP) or in spleen cells stimulated with a non-specific peptide (YF-C).

[0499] Table 1

[0500]

[0501]

[0502] LV-ZIKV-Ag and LV-ZIKV-NS1 Protect A129 and IFNAR-BL6 Mice from ZIKV Infection

[0503] In the first experiment, A129 mice were immunized with LV-ZIK-Ag (1×10 8 TU / mouse), LV-ZIK-NS1 (0.75×10 8TU / mouse) and LV-GFP (1×10 8 TU / mouse) were used to immunize three groups of A129 mice, and one month later, they were infected with ZIKV of the "Asian" lineage (strain PF13), which caused large-scale outbreaks of Zika disease in Polynesia and South America. Similar to A129 mice infected with DENV, only some obvious symptoms were observed in the infected animals, such as ruffled fur and temporary weight loss (only observed in mice immunized with the LV-GFP control vector). Compared with mice immunized with the LV-GFP vector, a significant difference in body weight was observed in mice immunized with two ZIKV-specific vectors on days 6 to 12 after infection ( Figure 17 A). Viremia was detected in all infected mouse groups (measured on days 2, 4, and 6 after infection), but on days 2 and 4, viremia in mice immunized with ZIKV-specific vectors was approximately 100-fold lower than that in control mice, and it was undetectable or at the limit of detection on day 6, while it was still detectable in mice immunized with LV-GFP ( Figure 17 B). In addition, the viral load in the peripheral organs (spleen and brain) measured on day 12 after infection was at least 100-fold lower than that in control mice ( Figure 17 C and Figure 17 D).

[0504] This experiment was repeated in IFNAR-BL6 mice, which are more sensitive to ZIKV infection (63, 34) and develop severe diseases usually associated with neurological symptoms and death. Mice immunized with LV-ZIK-NS1 (3×10 8 TU / mouse) were compared with mice immunized with the same dose of the LV-GFP vector, and the mice were inoculated with 1×10 3 PFU / mouse of ZIKV (PF-13) one month later. Immunization with LV-ZIKV-NS1 could completely protect mice from infection-induced symptoms and death: in the group immunized with LV-GFP, neurological symptoms (hind limb weakness and flaccid paralysis) were detected in all mice, and a 70% mortality rate was observed at 9 dpi, while no neurological symptoms and death were observed in mice immunized with LV-ZIK-NS1 ( Figure 18 B). In addition, no significant weight loss was detected in mice immunized with the LV-ZIKV-NS1 vector ( Figure 18 A). Similar to the protection experiment in A129 mice, viremia was detected in both groups, but on days 7, 10, and 15 after infection, a significant reduction in viremia (>100-fold) was observed in the group immunized with the LV-ZIKV-NS1 vector ( Figure 18C). The viral load in the organs (spleen, brain, and testis) of mice immunized with LV-ZIKV-NS1 was also lower than that in LV-GFP-immunized mice that survived ZIKV infection (on day 15 post-infection). Figure 18 D).

[0505] Collectively, these data indicate that, similar to the LV-DEN-Ag1 vector, the ZIKV-specific vectors induce partial protection in mice, resulting in protection from weight loss, lower viremia, and lower viral load in the organs, and (at least established for LV-ZIKV-NS1) protection from the symptoms of ZIKV disease and death.

[0506] At 9 dpi, histopathological analysis of the brain and spleen collected from IFNAR-BL6 mice showed significant differences caused by LV-ZIK vaccination. The brains of control mice injected with LV-GFP showed perivascular cuffing, which was not observed in untreated control mice or ZIKV-challenged mice vaccinated with LV-ZIK. Figure 32 A). The difference between the white pulp and red pulp regions of the spleen of control mice injected with LV-GFP was small compared to the spleens of untreated control mice or ZIKV-challenged mice vaccinated with LV-ZIK. Figure 32 B).

[0507] Thus, LV-ZIK induces significant protection in IFNAR-KO mice, which results in weight loss, lower viremia, lower viral load, and reduced lesions in the organs, as well as protection from the symptoms of ZIKV disease (weakness, hind limb paralysis) and death.

[0508] Design and improvement of YFV T cell antigens

[0509] To develop candidate LV vaccines against YFV, two antigens were designed: the first antigen (YFV-Ag1 or YFV-NS) includes known and predicted T cell epitopes, all of which are from non-structural proteins NS2A, NS3B, NS3, NS4A, NS4B, and NS5, while the second antigen (YFV-Ag2 or YFV-S) includes T cell epitopes located in structural proteins C, M, E, and secreted non-structural protein NS1. Both antigens are based on the sequence of the currently used live attenuated vaccine strain (17D-204) because this strain is suitable for immunization and protection studies in IFNAR-KO mice. The antigen regions selected to constitute YFV-Ag1 and YFV-Ag2 present individual MHC class I epitopes (known and predicted) or clusters of such epitopes (Figure 19A). The antigen regions are arranged in a way that reduces the emergence of new epitopes, and if such epitopes are predicted to form, additional sequences (linkers) are designed to remove such epitopes, using the design procedure for DENV-Ag outlined above (Figure 19B and Figure 20 ).

[0510] Immunogenicity of YFV Antigens in IFNAR(- / -) Mice

[0511] The immunogenicity of LV-YFV-Ag1 and LV-YFV-Ag2 was analyzed in A129 mice. Three groups of mice were immunized with 3×10 8 TU / mouse of LV-YFV-Ag1, LV-YFV-Ag2, or LV-GFP, and 14 days after immunization, splenocytes from immunized mice were restimulated ex vivo with an antigen-specific peptide pool presenting selected MHC class I epitopes, and an ELISpot assay was performed for IFNγ ( Figure 21 ). The analysis showed that lentiviral vectors expressing two YFV-derived antigens are immunogenic and induce specific responses in immunized animals. The immunogenicity of LV-YFV-Ag1 has been additionally verified in IFNAR-BL6 mice using ICS (Figure 22). At 14 days post-infection, 3×10 8Spleen cells from IFNAR - BL6 mice immunized with the LV - YFV - Ag1 or LV - GFP vectors of TU were used, and the secretion of IFNγ, TNFα, IL2, and the lymphocyte degranulation marker CD107a was analyzed. Flow cytometric analysis of cytokine - stained cells has shown that CD8+ T cells respond to antigen exposure / peptide stimulation by secreting IFNγ, TNFα, and IL2, and a subset of polyfunctional CD8+ T cells express all three cytokines simultaneously. Similar to what was observed in LVs expressing DENV and ZIKV antigens, cells co - expressing IFNγ and CD107a were also detected. No cytokine production was detected in spleen cells from mice immunized with LV - GFP and stimulated with YFV - Ag1 - specific peptides, nor in spleen cells immunized with LV - YFV - Ag1 but stimulated with peptides not expressed by the antigen (YF - C), indicating the specificity of cytokine production.

[0512] LV - YFV - Ag1 and LV - YFV - Ag2 protect A129 mice from YFV infection

[0513] To analyze protection against YFV, three groups of A129 mice were immunized with 3×10 8 TU / mouse of the LV - YFV - Ag1, LV - YFV - Ag2, or LV - GFP vectors, and 1 month later, were infected with 6×10 6 PFU / mouse of YFV (strain 17D - 204). No weight loss or other obvious symptoms were noted in the infected animals ( Figure 23 A). Viremia was analyzed on days 2, 3, and 4 post - infection (dpi), but was detectable only in mice immunized with the control vector LV - GFP ( Figure 23 B). Analysis of the spleens of infected mice indicated that YFV infection caused splenomegaly (increased spleen size) in all mice immunized with LV - GFP, while mice immunized with the two YFV - specific vectors had spleens of normal size ( Figure 23 C). In addition, although the presence of virus was detectable in all infected animals (in the spleen), the virus was significantly less in the groups of animals immunized with LV - YFV - Ag1 and LV - YFV - Ag2 compared to the control ( Figure 23 D).

[0514] The protective potential of LV - YFV - NS was also evaluated in IFNAR - BL6 mice. Mice (n = 5) were injected i.m. with 3×10 8 TU / mouse of LV - YF - NS or LV - GFP. One month later, they were given 5×10 8Mice were inoculated with YFV (strain 17D-204) at a dose of PFU / mouse. Between 1 and 3 dpi, both groups of mice lost weight. However, between 3 and 7 dpi, the average weight of the mice immunized with LV-YF-NST increased, while the average weight of the mice immunized with LV-GFP continued to decrease ( Figure 23 E). In the mice immunized with LV-YF-NS, viremia measured at 2 dpi and 4 dpi was significantly lower (the geometric means between groups differed by 2.6×10 2 -fold and 5.1×10 3 -fold, respectively), and viremia was undetectable in these mice at 7 dpi ( Figure 23 F). In contrast, viremia was detectable at 7 dpi in all the mice immunized with LV-GFP, and 60% (3 out of 5) of these mice showed signs of severe disease, namely hind limb paralysis, weakness, and exhaustion, and reached the humane endpoint of the experiment ( Figure 23 G). Analysis of the viral load in the brain, spleen, and liver measured at 7 dpi showed that significantly higher loads were present in all organs of the mice immunized with LV-GFP ( Figure 23 H). Histological analysis of the spleen and brain of control mice (inoculated with YFV) injected with LV-GFP showed little difference between the white pulp and red pulp in the spleen ( Figure 33 A) and the perivascular region in the brain ( Figure 33 B), i.e., the pathological changes also observed in the mice injected with LV-GFP (inoculated with ZIKV). In contrast, such changes were not observed in non-immunized and non-infected mice or in YFV-inoculated mice immunized with the LV-YF-NS vector.

[0515] Design of the bivalent DENV / ZIKV T cell antigen

[0516] To facilitate the generation and use of candidate prophylactic lentiviral vaccines against DENV and ZIKV, a set of bivalent antigens was constructed that express the DENV-Ag2 antigen and one of two ZIKV antigens (ZIKV-Ag or ZIKV-NS1) from a single construct. Thus, four antigen constructs ( Figure 24 ) were generated: 1) Flavi-2, which expresses ZIKV-Ag after expressing DENV-Ag2 (); 2) Flavi-3, which expresses DENV-Ag2 after expressing ZIKV-Ag; 3) Flavi-4, which expresses ZIKV-NS1 after expressing DENV-Ag2; and 4) Flavi-5, which expresses DENV-Ag2 after expressing ZIKV-NS1. In all cases, the coding regions of the first and second antigens were separated by the sequence of the self-cleaving polymerase P2A.

[0517] The immunogenicity of the bivalent vectors was evaluated in IFNAR-BL6 mice and wild-type C57BL / 6 mice to confirm that combining DENV and ZIKV antigens in a single construct does not compromise the immunogenicity and protection induced by the individual antigens. After restimulation with DENV-specific and ZIKV-specific peptide pools, each vector at 1 × 10 8 TU / mouse was used to immunize each group of mice (n = 3 - 5) i.m., and T cell response analysis was performed on splenocytes collected 14 days after immunization ( Figure 25 A and Figure 25 B). The results showed that all vectors were immunogenic and their immunogenicity was comparable to or better than that of the vectors expressing the original monovalent antigens.

[0518] Protection of IFNAR-BL6 mice against DENV and ZIKV infections by single-dose immunization with the DENV / ZIKV bivalent vector

[0519] In preliminary experiments, the protective abilities of different "monovalent" and "bivalent" vectors against infection with one DENV serotype (DENV-4) were compared ( Figure 26 ), and the protective abilities of three DENV / ZIKV bivalent vectors (LV-Flavi-3, LV-Flavi-4, and LV-Flavi-5) against ZIKV infection were compared ( Figure 27 ). There were no significant differences in the protection levels of different vectors against DENV or ZIKV, indicating that: 1) co-expression of DENV and ZIKV antigens from the same construct does not compromise protection against DENV; 2) the protection levels against ZIKV induced by different DENV / ZIKV bivalent vectors are similar. Based on the results of these protection experiments and immunogenicity tests described above, the LV-Flavi-5 vector was selected for more detailed analysis of protection against DENV and ZIKV.

[0520] The protection provided by the Flavi-5 bivalent vector against DENV and ZIKV infections with 4 serotypes was re-evaluated in IFNAR-BL6 mice ( Figure 28 ). Each group of mice (n = 6) was immunized with the LV-Flavi-5 vector or the control LV-GFP vector at 3 × 10 8 TU / mouse, and 1 month after immunization, they were challenged with DENV-1 (dose: 1 × 10 7 PFU / mouse), DENV-2 (dose: 2 × 10 6 PFU / mouse), DENV-3 (dose: 8 × 10 6 PFU / mouse), DENV-4 (dose: 1 × 10 7(PFU / mouse) or ZIKV (1×10 3 (PFU / mouse). The body weights of the mice were recorded regularly, and viremia was analyzed in subgroups of infected mice at different days post-infection. Immunization of IFNAR-BL6 with the LV-Flavi-5 vector induced significant protection against all four DENV serotypes, which was very similar to the level of protection achieved previously by immunization with the LV-DEN-Ag1 vector. The LV-Flavi-5 vector also effectively protected female IFNAR-BL6 mice from ZIKV-induced weight loss and death, but male mice, in which ZIKV infection is generally more pathogenic, were not protected, indicating that the bivalent LV-Flavi-5 vector may be less efficient in protecting against ZIKV compared to the protection provided by the monovalent vectors LV-ZIKV-Ag and LV-ZIK-NS1 ( Figure 29 ).

[0521] To improve protection against ZIKV, a heterologous prime-boost strategy was employed, where IFNAR-BL6 mice were first immunized with 3×10 8 TU / mouse of the LV-Flavi-5 vector and boosted 1 month later with the same dose of the LV-Flavi-3 vector. Although both vectors express the same DENV antigen (DENV-Ag2), they express different ZIKV-specific antigens: ZIKV-NS1 and ZIKV-Ag. The protective effect of this prime-boost immunization was analyzed only in male IFNAR-BL6 mice infected with DENV-2 or ZIKV 1 month after the second immunization (to achieve the strongest possible viral challenge conditions). Although the protection against DENV-2 induced by LV-Flavi-5 / LV-Flavi-3 immunization was not significantly different from that induced by single LV-Flavi-5 immunization (Figure 30A), the protection against ZIKV was greatly improved: 70% of male mice immunized with LV-Flavi-5 / LV-Flavi-3 survived upon ZIKV challenge, while all mice immunized with LV-GFP died (Figure 30B). Viremia in infected mice immunized with the LV-Flavi-5 / LV-Flavi-3 vector was significantly reduced (10 to 100-fold) and the amount of infectious virus in the plasma was decreased (150-fold) compared to the control.

[0522] In summary, these data indicate that the "bivalent" DENV / ZIKV vector provides a similar level of protection against DENV 1-4 compared to the "monovalent" DENV vector, but the level of protection against ZIKV may be lower. However, protection against ZIKV can be significantly improved by a sequential immunization strategy using two bivalent vectors (LV-Flavi-5 and LV-Flavi-3) expressing different ZIKV-specific antigens.

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Claims

1. A recombinant polynucleotide comprising at least one polynucleotide encoding a fusion polypeptide, said fusion polypeptide comprising an MHC class I T cell epitope suitable for eliciting a T cell response, wherein said MHC class I T cell epitope is derived from multiple non-structural or structural antigens, wherein said antigen is from at least one flavivirus selected from Dengue virus (DENV), Zika virus (ZIKV), and Yellow fever virus (YFV), and wherein said MHC class I T cell epitopes are assembled together via a linker region that does not contain non-specific neoepitopes.

2. The recombinant polynucleotide according to claim 1, comprising a first polynucleotide encoding a first fusion polypeptide, said first fusion polypeptide comprising an MHC class I T cell epitope, said MHC class I T cell epitope being derived from more than one non-structural DENV protein and forming a DENV-based assembled common antigen of DENV-1, DENV-2, DENV-3, and DENV-4 virus strains.

3. The recombinant polynucleotide according to claim 2, wherein said MHC class I T cell epitope is derived from at least 2 DENV antigens selected from NS3, NS4A, NS4B, and NS5 antigens, and preferably from each of the NS3, NS4A, NS4B, and NS5 antigens.

4. The recombinant polynucleotide according to any one of claims 1 to 3, wherein said linker region comprises a hydrophobic amino acid linker and does not contain a sequence encoding a non-specific immunodominant epitope.

5. The recombinant polynucleotide according to any one of claims 2 to 4, wherein said assembled polynucleotide comprises a polynucleotide encoding an MHC class I T cell epitope of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28 or a variant thereof that does not contain SEQ ID NO:2 and comprises at the 5' end.

6. The recombinant polynucleotide according to claim 5, selected from: a. A recombinant polynucleotide comprising a polynucleotide encoding an MHC class I T cell epitope having the following sequences, which are arranged in the following order from 5' to 3' in said recombinant polynucleotide: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:28, and wherein the linker region between the polynucleotides encoding said MHC class I T cell epitopes does not contain a sequence encoding a non-specific immunodominant epitope, in particular a recombinant polynucleotide in which an amino acid linker sequence is inserted as a linker region between all consecutive said sequences, except between SEQ ID NO:8 and SEQ ID NO:10, between SEQ ID NO:16 and SEQ ID NO:18, and between SEQ ID NO:18 and SEQ ID NO:20; or b. A recombinant polynucleotide comprising a polynucleotide encoding an MHC class I T cell epitope having the following sequences, which are arranged in the following order from 5' to 3' in said recombinant polynucleotide: SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34 and SEQ ID NO:36, and wherein the linker region between the polynucleotides encoding said MHC class I T cell epitopes does not contain a sequence encoding a non-specific immunodominant epitope, in particular a recombinant polynucleotide in which an amino acid linker sequence is inserted as a linker region between all consecutive said sequences, except between SEQ ID NO:8 and SEQ ID NO:10, between SEQ ID NO:16 and SEQ ID NO:18, between SEQ ID NO:18 and SEQ ID NO:20, and between SEQ ID NO:34 and SEQ ID NO:

36.

7. The recombinant polynucleotide according to any one of claims 1 to 6, wherein the MHC class I T cell epitope is derived from at least two ZIKV antigens selected from NS4B and NS5 antigens, and the other MHC class I T cell epitopes are derived from at least two ZIKV antigens selected from C and PrM antigens, wherein the MHC class I T cell epitopes are assembled together via a linker region that does not contain non-specific neo-epitopes, in particular wherein the MHC class I T cell epitopes are SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO:

52.

8. The recombinant polynucleotide according to claim 7, wherein the polynucleotides encoding the MHC class I T cell epitopes are arranged in the recombinant polynucleotide in the following order from 5' to 3': SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO:52, and wherein the linker region between the polynucleotides encoding the MHC class I T cell epitopes does not contain a sequence encoding a non-specific immunodominant epitope, in particular wherein an amino acid linker sequence is inserted as a linker region between all consecutive recombinant polynucleotides of the above sequences, except between SEQ ID NO:44 and SEQ ID NO:46 and between SEQ ID NO:50 and SEQ ID NO:

52.

9. The recombinant polynucleotide according to any one of claims 1 to 6, wherein the MHC class I T cell epitope is derived from at least one YFV antigen selected from NS-1, NS-2A, NS2B, NS-3, NS-4, NS4B and NS5 antigens, and the other MHC class I T cell epitopes are derived from at least two YFV antigens selected from C, M and E antigens, wherein the MHC class I T cell epitopes are assembled together via a linker region that does not contain non-specific neoepitopes, in particular wherein the MHC class I T cell epitopes are SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID No.78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID No 86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102 and SEQ ID NO:104, or are SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138 and SEQ ID NO:

140.

10. The recombinant polynucleotide according to claim 9, selected from: a. A polynucleotide comprising a polynucleotide encoding the MHC class I T cell epitope, which is arranged in the recombinant polynucleotide in the following order from 5' to 3': SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID No.78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ IDNO:100, SEQ ID NO:102 and SEQ ID NO:104, and wherein the linker region between the polynucleotides encoding the MHC class I T cell epitope does not contain a sequence encoding a non-specific immunodominant epitope, in particular a recombinant polynucleotide in which an amino acid linker sequence is inserted as a linker region between all consecutive sequences as above, except between SEQ ID NO:60 and SEQ ID NO:62, between SEQ ID NO:66 and SEQ ID NO:68, between SEQ ID NO:74 and SEQ ID NO:76, between SEQ ID NO:78 and SEQ ID NO:80 and SEQ ID NO:82, and between SEQ ID NO:94 and SEQ ID NO:96; or b. A polynucleotide comprising a polynucleotide encoding the MHC class I T cell epitope, which is arranged in the following order from 5' to 3' in the recombinant polynucleotide: SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138 and SEQ ID NO:140, and wherein the linker region between the polynucleotides encoding the MHC class I T cell epitope does not contain a sequence encoding a non-specific immunodominant epitope, in particular a recombinant polynucleotide in which an amino acid linker sequence is inserted as a linker region between all consecutive sequences as described above, except between SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116 and SEQ ID NO:118 and between SEQ ID NO:124 and SEQ ID NO:

126.

11. The recombinant polynucleotide according to any one of claims 1 to 11, further comprising a sequence encoding a signal peptide at its 5' end.

12. The recombinant polynucleotide according to any one of claims 2 to 6, the sequence of which consists of SEQ ID NO:29 or SEQ ID NO:

37.

13. The recombinant polynucleotide according to claim 12, wherein the sequence of the polynucleotide is modified relative to the sequence of SEQ ID NO or SEQ ID NO:29 or SEQ ID NO:37, wherein the modification consists of one or more point mutations of nucleotides, in particular nucleotide substitutions or deletions, and the modified sequence encodes a fusion polypeptide having at least 90% sequence identity, at least 94% or at least 95% sequence identity or having 94% to 99% sequence identity with the sequence of the original fusion polypeptide.

14. A recombinant polynucleotide according to any one of claims 1 to 13, comprising (i) a first polynucleotide encoding a first fusion polypeptide comprising an MHC class I T cell epitope, said MHC class I T cell epitope being derived from structural and non-structural DENV proteins and forming a DENV-based assembled antigen; and (ii) a second polynucleotide encoding a second fusion polypeptide comprising an MHC class I T cell epitope, said MHC class I T cell epitope being derived from structural and non-structural ZIKV proteins and forming a ZIKV-based assembled antigen; and / or (iii) a third polynucleotide encoding a second fusion polypeptide comprising an MHC class I T cell epitope, said MHC class I T cell epitope being derived from structural and / or non-structural YFV proteins and forming a YFV-based assembled antigen, wherein said first polynucleotide and said second polynucleotide and said third polynucleotide (when present) are operably linked in an expression cassette and separated by a sequence encoding a self-cleaving peptide (such as a 2A self-cleaving peptide), said sequence optionally being linked to a spacer sequence.

15. A recombinant multi-epitope polypeptide, the amino acid sequence of which consists of SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:54 or SEQ ID NO:56, SEQ ID NO:106 and SEQ ID NO:142 or a variant thereof, said variant being obtained by deletion or point mutation of one or more amino acid residues, and wherein the variant sequence has at least 90% sequence identity, at least 94% or at least 95% sequence identity with the sequence of the original fusion polypeptide, or has 94% to 99% sequence identity.

16. A recombinant lentiviral vector genome comprising the recombinant polynucleotide according to any one of claims 1 to 14.

17. The recombinant lentiviral vector genome according to claim 16, wherein the genome is an insert obtained from a pFLAP vector plasmid selected from the following: pFlap-β2m-DENV-Ag1-WPREm of SEQ ID NO: 151, or pFlap-β2m-DENV-Ag2-WPREm of SEQ ID NO: 152, or pFlap-β2m-ZIKV-Ag-WPREm of SEQ ID NO: 153, or pFlap-β2m-ZIKV-NS1-WPREm of SEQ ID NO: 154, or pFlap-β2m-YFV-Ag1-WPREm of SEQ ID NO: 155, or pFlap-β2m-YFV-Ag2-WPREm of SEQ ID NO: 156, or pFlap-β2m-DENV-Ag2_ZIKV-Ag-WPREm_(Flavi-2) of SEQ ID NO: 157, or pFlap-β2m-ZIKV-Ag_DENV-Ag2-WPREm_(Flavi-3) of SEQ ID NO: 158, or pFlap-β2m-DENV-Ag2_ZIKV-NS1-WPREm_(Flavi-4) of SEQ ID NO: 159, or pFlap-β2m-ZIKV-NS1-DENV-Ag2-WPREm_(Flavi-5) of SEQ ID NO:

160.

18. A recombinant lentiviral vector particle comprising the recombinant lentiviral vector genome according to any one of claims 16 to 17.

19. The recombinant lentiviral vector particle according to claim 18, wherein the recombinant lentiviral vector particle is a recombinant replication-defective pseudotyped lentiviral vector particle, particularly a replication-defective pseudotyped HIV-1-based lentiviral vector particle, particularly wherein the HIV-1-based vector particle is pseudotyped with the glycoprotein G (V-SVG) of vesicular stomatitis virus from the Indiana or New Jersey serotype.

20. A host cell, preferably a mammalian host cell, transfected with DNA, particularly a DNA plasmid, comprising the recombinant polynucleotide according to any one of claims 1 to 14 or 16 or 17, particularly wherein the host cell is the HEK-293T cell line or the K562 cell line.

21. A composition, particularly a vaccine composition, suitable for administration to a mammalian host, particularly a human host, the composition comprising the recombinant polynucleotide according to any one of claims 1 to 14 or 16 or 17 or the recombinant lentiviral vector particle according to any one of claims 18 or 19 and one or more pharmaceutically acceptable excipients suitable for administration to a host in need, particularly a human host, and optionally comprising an adjuvant.

22. The composition according to claim 21, which is used to elicit a protective, preferably prophylactic, immune response in a host in need thereof, particularly a human host.

23. The composition according to any one of claims 21 or 22, which is used to elicit a protective, preferably prophylactic, immune response against infection by DENV-1, DENV-2, DENV-3 and DENV-4 viruses or against the onset of diseases caused by DENV-1, DENV-2, DENV-3 and DENV-4 viruses.

24. A multivalent vaccine comprising the composition according to any one of claims 21 to 23, which is used to prevent a disorder or disease caused by infection with a virus selected from DENV, ZIKV and YFV in a human host and / or to prevent the onset of said disorder or disease.

25. The composition according to any one of claims 21 to 23, which is for use in a human host in a multi-dose administration regimen, particularly a prime-boost administration regimen.

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