SELF-AMPLIFIED RNA EXPRESSING THE gB, gBdel, gD AND gE ANTIGENS OF THE INFECTIOUS LARYNGOTRACHEITIS VIRUS (ILT)

Abstract

The present invention relates to self-amplifying RNA (saRNA) vaccines against infectious laryngotracheitis (ILT) that provide safe and effective prophylactic and therapeutic immune responses and improve the symptoms of the disease.In particular, the invention relates to sRNAs encoding at least one laryngotracheitis virus polypeptide, an ILTV polypeptide variant, or an immunogenic fragment or epitope thereof, said sRNA comprising a first nucleic acid sequence including nsP1, nsP2, nsP3, and nsP4 encoding non-structural alphavirus proteins, a second nucleic acid sequence encoding at least one ILTV polypeptide including gB, gBdel, or gD, linked to gE by a nucleic acid sequence encoding at least one protein-binding site or at least one cleavage site for an endogenous or exogenous protease such as a 6k sequence, and also comprising a 5' cap, a 5' UTR upstream of the first nucleic acid sequence, a 26S promoter upstream of the second nucleic acid sequence, a 3' UTR, and a Poly queue A.The sRNA according to the present invention allows, when incorporated into a cell, the expression of at least one ILTV polypeptide, an ILTV polypeptide variant, or an immunogenic fragment or an epitope of one of them.

Description

Title of the invention: SELF-AMPLIFIED RNA EXPRESSING THE gB, gBdel, gD and gE ANTIGENS OF THE INFECTIOUS LARYNGOTRACHEITIS VIRUS (ILT) Field of the invention

[0001] The present invention relates to the fields of immunology, molecular biology and bird care and breeding, and more particularly to self-amplifying RNA (saRNA) vaccines against infectious laryngotracheitis (ILT) which provide safe and effective prophylactic and therapeutic immune responses against ILT and associated comorbidities and which improve the symptoms of the disease. Context of the invention

[0002] Vaccination of commercial poultry flocks is important to reduce mortality and morbidity caused by infectious agents, thereby increasing profitability and meeting applicable regulatory requirements. Commercial farms raising chickens, turkeys, pheasants, guinea fowl, and other poultry are routinely vaccinated to protect them from exposure to pathogens in the environment.

[0003] Infectious laryngotracheitis (ILT), a highly contagious disease of the upper respiratory tract of chickens and other poultry, is an important and widespread disease in poultry. ILT is characterized by conjunctivitis, sinusitis, oculonasal discharge, respiratory distress, bloody mucus, swelling of the orbital sinuses, high morbidity, considerable mortality, and decreased egg production. Historically, ILT has been a problem in the laying hen industry, but in the last two decades, the disease has become more prevalent in broiler production facilities.

[0004] The causative agent of ILT is Gallid herpesvirus 1 (GaHV-1), belonging to the genus Iltovirus and the subfamily Alphaherpesvirinae of the family Herpesviridae. This virus is also known as infectious laryngotracheitis virus (ILTV) and invades the trigeminal ganglion or the trachea, where latency is established. ILTV can survive for days or even months at temperatures between 13 and 23 °C in tracheal exudates and chicken carcasses. ILTV is well established in densely populated poultry production areas worldwide due to its characteristic persistence. The transmission of ILT is illustrated in [Fig. 1].

[0005] ILTV comprises a nucleocapsid surrounded by an irregular envelope (see [Fig. 2]). The envelope contains several glycoproteins that protrude from the surface of the virus. These proteins represent the major antigens of the virus. These glycoproteins mediate the attachment and entry of the virus into a host cell and include gB, gC, gD, gH, gl, gj, gM, and gN; Fuchs W., et al. Molecular biology of avian infectious laryngotracheitis virus. VET. RES. 2007, 38:261-279. doi: 10.1051 / vetres:200657. Another glycoprotein, gG, is not incorporated into the viral envelope but is secreted by infected cells; Kongsuwan K., Johnson MA, and Sheppard M.. 1993. Identification of an infectious laryngotracheitis virus gene encoding an immunogenic protein with a prediction. M(r) of 32 kilodaltons. VIRUS RES 29:125-140. doi:10.1016 / 0168.

[0006] Historically, modified live ILTV vaccines have been used in the field. However, their use presents problems due to the impractical routes of administration and the reversion to virulence of the modified attenuated virus. The reversion to virulence of an attenuated modified viral strain risks infecting unvaccinated birds in a flock, which poses safety and biosecurity problems.

[0007] Nucleic acid-based vectored vaccines expressing isolated ILTV antigens such as gB or UL-32 may be a safer alternative to modified live vaccines because they do not contain live virus. However, subunit vectored vaccines have suffered from a lack of construct stability and insufficient expression of several protective viral antigens. For example, a vectored vaccine typically has a maximum target gene load that limits the number or amount of protective antigens / immunogens it produces. An amount of target DNA exceeding the vector's maximum load will not remain in the vector, and the vector's lack of stability may prevent regulatory approval of a vectored vaccine.

[0008] In view of the limitations described above and other technical problems, the inventors sought to determine whether self-amplified RNA vaccines could offer superior safety and efficacy compared to traditional modified live vaccines and vectored vaccines.

[0009] The inventors sought to develop a non-infectious sRNA vaccine that, once administered, could not spread within the subject or infect other subjects with the VLT. Such an sRNA vaccine could not revert to virulence and could have a safety profile similar to that of an inactivated or subunit vaccine rather than that of a modified live vaccine.

[0010] The inventors also sought to develop a stable sRNA vaccine capable of expressing sufficient quantities of ILTV antigens in a form that produces protective humoral or cellular immune responses, thus overcoming the problems associated with conventional vector vaccines. They also studied ways of administering sRNA, which is generally much larger than non-self-replicating nucleic acid vaccines.

[0011] The inventors have investigated other desirable features for an sRNA vaccine, including how to design and administer an sRNA vaccine that replicates in cells at the administration site but generally activates the immune system, for example by activating the innate immune system, not suppressing immune responses and recruiting immune cells to the sRNA administration site to produce a broader or more robust cellular or humoral response to ILTV antigens than other vaccines.

[0012] The preceding paragraphs have been provided by way of general introduction and are not intended to limit the scope of the following claims. The embodiments described, as well as other advantages, will be better understood by reference to the detailed description that follows, in conjunction with the accompanying drawings. Summary of the invention

[0013] This section gives a brief, non-limiting overview of some aspects of the technology disclosed in this document.

[0014] The present invention relates to self-amplifying RNA (saRNA) encoding at least one polypeptide of infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus 1, GaHV-1), a polypeptide variant of ILTV, or an immunogenic fragment or epitope thereof, characterized in that it comprises, in any order: - a first nucleic acid sequence comprising nsPl, nsP2, nsP3 and nsP4 which code for non-structural alphavirus proteins, and - a second nucleic acid sequence encoding at least one ILTV polypeptide comprising gB, gBdel, gD linked to gE by a nucleic acid sequence encoding at least one protein-splitting site or at least one cleavage site for an endogenous or exogenous protease such as a 6k sequence, and said sRNA comprising a 5' cap, a 5' UTR upstream of the first nucleic acid sequence, a 26S promoter upstream of the second nucleic acid sequence, a 3' UTR and a poly A tail, said sRNA, when incorporated into a cell, allows the expression of at least one ILTV polypeptide, an ILTV polypeptide variant, or an immunogenic fragment or epitope of one of them.

[0015] sRNA constructs. One aspect of this technology is an RNA self-replicating or sRNA that codes for and expresses ILTV antigens / immunogens / epitopes, ILTV antigen / immunogen / epitope variants or fragments, y including epitopes of these (collectively "ILTV antigens or immunogens"). Advantageously, the sRNA is based on, or derived from, a positive-strand viral platform that retains the sequences encoding RNA replication proteins, but which is deleted for the sequences encoding the structural genes of the positive-strand virus. One or more genes of interest are inserted in place of the deleted structural genes. One such platform is derived from the alphavirus and expresses the non-structural alphavirus proteins nspl-nsp4 that are responsible for RNA replication and the sequences encoding genes of interest, such as genes encoding ILTV antigens or immunogens, downstream of the nspl-nsp4 sequences, usually in positions occupied by the structural genes of the alphavirus; see, for example, [Fig. 5].

[0016] In some cases, the sRNA includes additional elements that enhance the expression of ILTV antigens or epitopes or that ensure the trafficking of ILTV antigens once they are expressed.

[0017] These additional elements may encode immunogenic carrier proteins, immunomodulators that enhance the expression of ILTV antigens / immunogens, inhibitors of interferon or RNA encoding such an inhibitor, or other inhibitors of innate immune responses. It may encode signal sequences, head peptides, ER N-terminal signal sequences, or other elements that transport expressed ILTV antigens or epitopes to various parts of a cell such as the ER, the Golgi, the membrane or the cell exterior; see transport sequences described by hypertext transfer protocol secure: / / alg.manifoldapp.org / read / fundamentals-of-cell-biology / section / 925 27ecb-306c-4002-a411-e3c5d5da5e6a (last accessed 1 September 2022, incorporated by reference).

[0018] Another aspect of this technology is an sRNA platform that expresses two, three, four, or more ILTV antigens / immunogens as components of a polyprotein or as separate polypeptides. It is also possible to produce distinct ILTV antigens / immunogens, for example, by interleaving 6K or 6K-like sequences between sequences encoding different ILTV antigens / immunogens, thereby producing distinct ILTV antigen / immunogen polypeptides instead of a polyprotein. An example of a 6K protein is that of the Semliki Forest 6K virus.

[0019] In some embodiments, the sRNA encodes at least two, three, or four ILTV antigens that are expressed as a processed polyprotein to form T and B cell epitopes presentable by MHC class 1 or class 2 molecules. For example, the platform may be designed not to contain cleavage sites between the sequences encoding the three antigens structural at least or to contain translation skip sequences between at least three structural genes and produce proteins that remain in the cytoplasm.

[0020] Compositions. A related aspect of this technology is a composition comprising the self-replicating RNA and a pharmaceutically acceptable carrier. In one case, the composition comprises saRNA and inorganic lipid nanoparticles (LION).

[0021] In addition to the sRNA presented here, a composition may include other active ingredients such as protein-based vaccine components, nucleus-based vaccine components, immunological antigens, or immunogens from other avian diseases. Thus, a composition containing an sRNA encoding ILTV antigens may include other components forming a multivalent vaccine against ILTV and other avian viruses or pathogens. In some cases, a composition will include an sRNA encoding one or more ILTV antigens or immunogens, together with a carrier, adjuvant, or excipient in a form suitable for administration to produce a prophylactic or therapeutic response against ILTV infection. Such a composition may produce or enhance a humoral or cellular response in an ILTV-immunized individual.

[0022] Treatment. Another aspect of this technology is a method for preventing or treating LTI, its symptoms, comorbidities, or sequelae by administering an sRNA platform as described herein, which encodes and expresses one or more ILTV antigens, immunogens, or epitopes. Administration of the vaccine containing the sRNA platform induces innate, humoral, and / or cellular immune responses to the encoded ILTV antigens, including the induction of neutralizing antibodies or antigen-specific T cells that recognize ILTV-infected cells. In a practical embodiment, the sRNA or a composition containing it is administered intramuscularly or subcutaneously.

[0023] Method for manufacturing sRNA. Other aspects of this technology relate to methods for constructing DNA vectors encoding the sRNA presented herein. A DNA vector can be produced synthetically and sRNA generated from this vector, for example, by in vitro transcription of an antigen-coding sequence or ILT immunogen and non-coding alphavirus or regulatory sequences into a synthetic RNA transcript that can be subsequently formulated for delivery. Methods for synthesizing sRNA from a DNA construction template include in vitro RNA transcription, RNA encapsulation, poly(α) encapsulation, and RNA purification. These methods are known, commercially available, and incorporated by reference into the secure hypertext transfer protocol at secure: / / www.neb.com / products / rna-reagents / ma-synthesis / rna-synthesis (last accessed October 21, 2022). Various Techniques for manufacturing ssRNA or other nucleic acid constructs are known. Preferably, ssRNA is produced by in vitro transcription and / or amplification. For example, a linearized DNA template for ssRNA can be transcribed into mRNA using an RNA polymerase, such as bacteriophage T7 or SP6 RNA polymerase. The RNA can be purified by chromatography, filtration, silica gel matrix, or other methods to remove impurities such as dsRNA and proteins. The purified mRNA can be protected by association with cationic nanoparticles, polymers, peptides, or encapsulation in lipid nanoparticles by solvent injection or microfluidic mixing to protect the mRNA from degradation or to facilitate its uptake by target cells. Brief description of the figures

[0024] [FIG1 FIG. 1 shows the ILTV transmission schemes. A more detailed description of ILTV transmission is incorporated by reference to Gowthamen et al, VET. QUART. 2020, 40(1), 140-161.

[0025] [Fig.2] [Fig.2] represents the physical and genetic structure of ILTV. A A more detailed description of the physical and genetic structure of ILTV is incorporated by reference to Gowthamen et al, Infections laryngotracheitis: Etiology, epidemiology, pathobiology, and advances in diagnosis and control - a comprehensive review. Vet. Quart. 2020, 40(1), 140-161; and to Fuchs, et al, Molecular biology of avian infections laryngotracheitis virins, Vet. Res., 2007, 38, 261-279.

[0026] [Fig. 3] Figure 3 describes some identified ILTV genetic products, including gB, gC, gG, gH, gj, and gM. The accession numbers at the bottom of this figure, which describe these genetic products in more detail, are incorporated by reference. A more detailed description of these genetic products is incorporated by reference to Fuchs et al., SNPRA.

[0027] [Fig.4] Fig.4 illustrates the interaction of self-replicating RNA (self-RNA amplifying, sRNA) with a host cell.

[0028] [Fig. 5] [Fig. 5] describes an example of a DNA construct encoding an sRNA containing ILTV genes of interest (gX). This construct includes a 5' nsPl and DLP sequence of a P2A autoprotease as well as sequences encoding the non-structural genes nsPl-nsP4 and gX.

[0029] [Fig.6] [Fig.6] illustrates several different ILTV sRNA constructs. The last gE-VEE 6K-gI construct may include a 6K sequence from VEE or Semliki Forest virus.

[0030] [Fig.7] Fig.7 describes the design and testing of an sRNA vaccine as described In example 1, the vaccine includes between 0.1 pg and 7 pg of each gene of interest listed as gB, gD, gE, gl from ILTV for the intramuscular (IM) route.

[0031] [Fig.8] Fig.8 illustrates protection against LTV as a function of the presence or absence of clinical signs of LTV, including mortality, observed in chickens vaccinated as described in Example 1.

[0032] [Fig.9] Fig.9 presents a system for notating clinical signs ILT specific for the average respiratory severity of clinical signs.

[0033] [Fig. 10] The [Fig. 10] describes the excretion of ILTV in oropharyngeal swabs from vaccinated chickens, tested using qPCR equivalent to an infectious dose of 50% of the egg of the ILTV challenge virus, as described in Example 1. DPC: days after challenge.

[0034] [Fig. 11] Figure 11 shows a table showing the percentage of chickens in each treatment group that tested negative for ILTV by qPCR at 3 or 6 days post-challenge. DPC: days post-challenge. Intramuscular vaccination and booster doses with 0.1 pg to 7 pg of each of the sRNAs encoding gB, gD, gE, and gl eliminated ILTV shedding 6 days post-challenge, and intramuscular vaccination and booster doses with 0.1 pg to 7 pg of each of the sRNAs encoding gD, gE, and gl eliminated ILTV shedding in 95% of chickens 6 days post-challenge. Wingweb vaccination with these same sRNAs prevented 50% of chickens from shedding. The doses described in the figures above or used in the examples are not limiting. Thus, in some embodiments, the amount of sRNA administered is between <0.01, 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 or >5.0 pg or any subrange or intermediate value

[0035] [Fig. 12] Fig. 12 depicts the results of the BioChek ELISA test after primary vaccination (left) and booster vaccination (right). Chickens immunized intramuscularly with sRNAs encoding gB, gD, gE, and gl, or with sRNAs encoding gD, gE, and gl, produced high levels of antibodies compared to groups immunized with other antigens or by other immunization methods. The threshold of 0.5 indicates that the serum samples are positive for ILT-specific antibodies according to the BioChek ELISA kit instructions. BioChek ELISA does not detect gB antibodies. The IDVet gB ELISA kit recognizes gB-specific antibodies and detects seroconversion to gB vaccination.

[0036] [Fig. 13] [Fig. 13] shows the design and testing of an sRNA vaccine as described in Example 2. The vaccine comprises the indicated doses of sRNA encoding various ILTV antigens. Novel sRNA constructs were evaluated, including sRNA comprising gB-del and sRNA comprising gE-VEE 6K-gI. Low and high doses of some sRNA constructs were compared.

[0037] [Fig. 14] Figure 14 illustrates the Western blot probed with pooled serum from chickens vaccinated with gD, gE, and gl from the example to detect gl and gE. Glycoprotein I was linked to glycoprotein E using a 6K linker (V6K) from the Venezuelan equine encephalitis virus or a 6K linker (S6K) of Semlike Forest virus. V6K shows a 110-120 kDa band specific for gl and gE linked together. The broadest band of S6K is absent due to a point mutation.

[0038] [Fig. 15] [Fig. 15] illustrates the clinical signs of ILT, protection, and mortality described in Example 2. Chickens were vaccinated with sRNAs expressing, in group 1, high-dose gD, gE, and gl; in group 2, low-dose gD, gE, and gl; in group 3, high-dose suppressed gB; in group 4, high-dose suppressed gLVEE linker-gE, gD, and gB; in group 5, high-dose gLVEE linker-gE and gD; in group 6, low-dose gLVEE linker-gE and gD; and in group 7, a placebo. Clinical signs of ILT infection and clinical signs of protection were recorded. Mortality rates were also recorded.

[0039] [Fig. 16] [Fig. 16] presents the scoring system for clinical signs of ILT specific to the mean respiratory severity of clinical signs for chickens enrolled in the treatment groups described in [Fig. 15]. A higher clinical score indicates a more severe respiratory clinical sign associated with ILT disease.

[0040] [Fig. 17] Figure 17 shows ILTV shedding on oropharyngeal swabs and ILTV detection by qPCR. The graphs show ILTV shedding in individual chickens and the mean indicated by each black bar. The percentage of immunized chickens not shedding the virus detected by qPCR on oropharyngeal swab samples collected 3 and 6 days post-challenge (PCC) is shown in the table.

[0041] [Fig. 18] Figure 18 illustrates the BioChek ILTV ELISA. Antibody levels after primary infection and after the booster dose were determined by ELISA. A threshold of 0.5 indicates that the serum samples are positive for ILT-specific antibodies according to the BioChek ELISA kit instructions. BioChek ELISA does not detect gB antibodies. The IDVet gB ELISA kit recognizes gB-specific antibodies and detects seroconversion to gB vaccination. Detailed description of the invention

[0042] A more detailed description of the technology disclosed in this document and of the essential and optional elements of the RNA sa and of its methods of use and manufacture is provided below.

[0043] Self-amplified RNA. Self-amplified RNA (saRNA), self-replicated RNA (rRNA), replicon or SRV (synthetic RNA vaccine) as a vaccine and therapeutic modality has been increasingly used for the in vivo production of proteins.

[0044] sRNAs are derived from positive-stranded RNA viruses whose structural proteins have been removed and replaced by heterologous genes of interest; see P. Aliahmad, et al., Next generation self-replicating RNA vectors for vaccines and immunotherapies. CANCER GENE THER (2022). hypertext transfer protocol secure: / / doi.org / 10.1038 / s41417-022-00435-8; Xiong Cet al. Sindbis virus: an efficient, broad host range vector for gene expression in animal cells. SCIENCE. 1989;243:1188-91; Bredenbeek PJ, et al. Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs. J VIROL. 1993;67:6439-46; Liljeström P. & Garoff H. A new generation of animal cell expression vectors based on the Semliki forest virus replicon. BIO / TECHNOL. 1991;9:1356-61; each of these documents is incorporated by reference. Among its other features, the technology disclosed herein involves an RNA sa that codes for one or more ILTV antigens or immunogens.

[0045] Gallid alphaherpes virus 1 (GaHV-1; ILTV), frequently called the virus of the Infectious laryngotracheitis (ILTV) belongs to the type species of the genus Iltovirus and is the causative agent of a highly contagious acute respiratory disease in domestic poultry, known as ILT. It is a virus species in the order Herpesvirales, family Herpesviridae, subfamily Alphaherpesvirinae, and genus Iltovirus; VIRUS TAXONOMY: 2020 RELEASE. INTERNATIONAL COMMITTEE ON VIRUSES TAXONOMY (ILTV). March 2021. Accessed May 10, 2021. Originally identified in chickens in the United States in 1926, this virus causes avian infectious laryngotracheitis (ILT), a potentially fatal and economically damaging disease, widely recognized as one of the most contagious diseases in the poultry industry; Fenner, Frank J., et al. (1993). VETERINARY VIROLOGY (2nd ed.). Academy Press, Inc. ISBN 0-12-253056-X.As used here, this term encompasses all known natural and artificial strains of ILTV, as well as their variants or mutants. Among its other features, the technology disclosed here involves an RNA that codes for one or more ILTV antigens or immunogens that can be used to vaccinate or protect birds against GaHV-1 or ILTV infection and against ILT.

[0046] Infectious laryngotracheitis (ILT) is a disease caused by Gallid alphaherpesvirus 1 (GaHV-1; ILTV). GaHV-1 is excreted in respiratory secretions and transmitted by inhalation of droplets or via fomites. The typical host is Gallus domesticus (domestic chicken). A previously unexposed flock will develop cases two to eight weeks after introduction. The incubation period is two to eight days. Symptoms include coughing, sneezing, head shaking, lethargy, discharge from the eyes and nostrils (sometimes bloody), and difficulty breathing. The name comes from the severe inflammation of the larynx and trachea. A membrane Diphtheria can form in the trachea and cause obstruction. There may be problems with egg laying and the production of abnormal or thin-shelled eggs. Mortality is usually less than 15%. Histopathology, PCR, ELISA, immunofluorescence staining, and viral isolation are all possible diagnostic methods. A vaccine is available, but it is susceptible to undesired recombination and does not prevent latent infections. Live attenuated vaccines against ILTV are highly effective against spreading ILT infection, but reversion to virulence remains problematic. They can be used during an outbreak to reduce morbidity and mortality. A confirmed case usually leads to the establishment of a quarantine zone around the farm. Within this quarantine zone, poultry workers avoid poultry farms to prevent the spread of the virus.Biosecurity measures, including quarantine, isolation, and disinfection, are very important to control the spread of an outbreak.

[0047] ILTV hosts and vaccinated subjects. Includes chickens (e.g., Gallus domesticus), turkeys (Meleagris gallopavo, Meleagris gallopavo domesticus), guinea fowl, pheasants, sparrows (genera Pavo and Afropavo), and other susceptible or carrier birds. Ducks and certain other birds may be carriers. A vaccinated subject may be an avian embryo, a chick, or an adult bird. In chickens, breeders, laying hens, and broilers may be affected. The sa RNA as described herein may be used to vaccinate or treat ILTV hosts, reduce mortality, improve ILTV symptoms, and reduce ILTV transmission.

[0048] ILTV antigens / immunogens include gB, gBdel (gB is deleted), gC, gD, gE, gG, gH, gl, gj, gK, gL, and / or gM, although other ILTV antigens can also be incorporated into the sRNA platforms described herein and used to immunize birds. The inventors considered gB, gD, gl, and gE to be promising candidates for inducing immune responses against ILTV; however, when tested in other types of ILTV vaccines, they proved to lack stability. As described herein, efforts have been made to induce protective immune responses against these antigens using an sRNA platform.

[0049] The RNA disclosed herein can be formulated for administration to domestic birds, gallinules, chickens, and other poultry-like birds such as turkeys and peacocks; or to immunize carriers of ILTV, including those described by, and incorporated by reference to, hypertext transfer protocol secure: / / en.wikipedia.org / wiki / List_of_Galliformes (last accessed 27 July 2022, attached as part of this disclosure). Vaccines formulated for administration to a species, A particular avian strain or breed typically encodes ILTV antigens for viruses capable of infecting that species or strain. In some cases, the ILTV antigens or immunogens disclosed here are derived from the ILTV strains VR2332, HB-13.9, JXwnO6, CHsxl401, or HAGE2018. Other known ILTV immunogens, antigens, or genetic products include those described in [Fig. 3] and by Fuchs et al., supra.

[0050] As mentioned above, ILTV antigens include glycoproteins such as gB, gC, gD, gE, gG, gH, gl, gj, gK, gL, and gM, which are thought to play a crucial role in viral entry and replication; see also Poulsen, DJ, and CL Keeler. 1997. Characterization of the assembly and processing of infectious laryngotracheitis virus glycoprotein B. JOURNAL OF GENERAL VIROLOGY. 78:2945-2951; Pavlova, S., Veits, J., Mettenleiter, TC, 2013. Identification and functional analysis of the infectious laryngotracheitis virus membrane proteins gD, gE, gl, and pUS9. AVIAN DISEASES, 57:416-426; and Bendezu, J., Ruiz SM, Montesinos, R., Guevara, RC, Rojas-Neyra, A., Pauyac-Antezana, K., Fenandez-Diaz, M., 2019. Glycoprotein G production profile during infection with infectious laryngotrachetics (ILTV), PLOS ONE 14(8):e0219475.

[0051] Glycoprotein B is involved in the penetration of ILTV virus into the host cell and is immunogenic for adaptive and cell-mediated immunity (Poulsen and Keeler, Characterization of the assembly and processing of infectious laryngotracheitis virus glycoprotein B. JOURNAL OF GENERAL VIROLOGY, 1997, 78:2945-295).

[0052] Glycoprotein D is responsible for the binding of ILTV to receptors for viral entry into the host cell. A more detailed description of gD, gE, gl, and other ILTV antigens is incorporated by reference to: Pavlova, S., Veits, J., Mettenleiter, TC, 2013. Identification and functional analysis of the membrane proteins gD, gE, gl, and pUS9 of infectious laryngotracheitis virus. AVIAN DISEASES, 57:416-426; and Bendezu, J., et al, 2019. Glycoprotein G production profile during infectious laryngotracheitis virus (ILTV) infection, PLOS ONE 14(8):e0219475.

[0053] Glycoprotein E (gE) and glycoprotein I (gl) are essential for the cell-to-cell propagation of ILTV, which impacts virulence. Therefore, polynucleotides encoding antibodies and specific cellular responses to gE and gl can be incorporated into sa RNA as described herein in order to reduce viral load.

[0054] Glycoprotein G is a virulence factor that acts as an immunomodulator during ILTV infection in chickens (Bendezu et al., 2019, supra). Glycoprotein G (gG) binds to chemokines of the C subfamily, CC and CXC, thereby preventing the interaction between chemokines and their receptors. It also blocks the binding of chemokines to glycosaminoglycans, which is necessary for the in vivo activity of chemokines. The ILTV vCKBP (gG), during the early stages of infection, induces innate immune responses by recruiting specific subsets of immune cells. A saRNA construct described herein can be designed to express a non-functional gG protein that has a reduced capacity to bind cytokines or inhibit innate immune responses.

[0055] Envelope glycoproteins, primarily gC, rather than gB, gD, gH, and gL, are considered to mediate the attachment of ILTV to host cell receptors and contribute to the fusion of the viral envelope with the host cell membrane. A saRNA construct described herein can be designed to induce immune responses against gC.

[0056] Immune responses to glycoprotein C can help to "differentiate infected animals from vaccinated animals" (DIVA). RNA encoding gC can be incorporated into sa RNA (containing other ILTV antigens such as gB, gD, gl, and gE). The sa RNA presented here may also not contain RNA encoding gC. Quantitative PCR can be used to detect copies of ILTV genes on tracheal or oral / pharyngeal swabs, such as genes encoding gC or gB, gD, gl, and gE. These methods are incorporated by reference to Callison, et al., Development and validation of a real-time Taqman PCR assay for the detection and quantification of infectious laryngotracheitis virus in poultry, J. VIROL. METHODS, 2007, 139(1), 31-38.Distinguishing infected animals from unvaccinated animals (DIVA) can be done by measuring immune responses to gC. If gC is absent from an sRNA-based vaccine, the qPCR test can be used to differentiate vaccinated chickens (no gC-positive samples) from ILTV-infected chickens, which would have gC-positive samples.

[0057] In some cases, a sa RNA can express a full-length ILTV antigen as it would appear during a natural infection. In other cases, a fragment of the ILTV antigen can be expressed. An example of a truncated polypeptide antigen is gBdel, which is a shorter fragment of gB. Cloning full-length gB proved difficult, but the inventors discovered that a truncated version of gB exhibited increased stability in a vector system. In gB-del, the transmembrane domain is deleted, so the protein is secreted rather than residing in the cell membrane.

[0058] Platform. This term encompasses a framework into which polynucleotides encoding exogenous antigens can be inserted. It also encompasses a construct or an sa RNA comprising the frame and sequences encoding exogenous antigens, such as an sa RNA that expresses one or more ILTV antigens / immunogens.

[0059] A platform may include viral vector platforms such as harmless viruses designed to encode a polynucleotide encoding one or more ILTV antigens of interest and to transport the polynucleotide into a cell, such as a porcine cell. These viral vectors may be replicative or non-replicative and induce a target cell to produce an ILTV antigen from the polynucleotide carried by the viral vector.

[0060] This term also covers nucleic acid platforms, including messenger RNA encoding one or more target ILTV antigens, which are delivered to a target cell (e.g., a chicken cell), often in a complex with a lipid- or polymer-based nanoparticle. These also induce the target cell to produce an ILTV antigen from the ILTV-encoding polynucleotide in the nucleic acid platform. Typically, such an RNA platform is not infectious, as it may lack the structural protein sequences necessary for the production of an infectious virus. This term also encompasses DNA platforms that carry DNA encoding messenger RNA encoding one or more ILTV antigens and other nucleic acid components for delivery to a target cell where the ILTV antigens are expressed.These DNA platforms may include a plasmid or replicon carrying the polynucleotide(s) encoding an RNA capable of expressing the ILTV antigen(s) in question.

[0061] Alpha virus. Advantageously, an alpha virus platform is used to express ILTV antigens. The genus Alphavirus belongs to the family Togaviridae and contains 28 virus species (Griffin, ENCYLOPEDIA OF VIROLOGY, 2008, 101-107). Alphavirus is a positive-sense RNA virus that is taxonomically distinguished from ILTV, which is a double-stranded DNA virus. Other characteristics of alphaviruses are described and incorporated by reference in Straus, et al. The Alphaviruses: Gene Expression, Replication and Evolution, MICROBIOL. REV, 1994, 491-562; Alphavirus replicon vaccines, Cambridge Press, 2021. ANIMAL HEALTH RESEARCH REVIEWS, Volume 13, Issue 1, June 2012, pp. 1-; https: / / doi.org / 10.1017 / S1466252312000011; Ljungberg, et al, EXPERT REV VACCINES; Self-replicating alphavirus RNA vaccines; 2015, 14(2), 177-94. Ulmer, et al, RNA-based vaccines, VACCINE, 2021, 30(30), 441-8; Strauss & Strauss, The Alphaviruses: Gene Expression, Replication, and Evolution; MICROBIOLOGICAL REVIEWS, 1994, 491-562, all incorporated by reference.

[0062] Alphavirus platform. Alphavirus vectors have demonstrated high levels of transient expression of heterologous genes both in vitro and in vivo and exhibit These are therefore interesting characteristics for vaccine development. The most commonly used vectors are based on three encapsulated single-stranded alphaviruses: Semliki Forest Virus, Sindbis Virus, and Venezuelan Equine Encephalitis Virus. An alphavirus platform advantageously used in many embodiments of the technology disclosed here encodes ILTV antigens instead of the alphavirus structural proteins. However, it contains alphavirus replication sequences, such as nspl-nsp4, which can be used to amplify the RNA encoding ILTV antigens. In some cases, alphavirus vectors are based on platforms of Semliki Forest Virus, Sindbis Virus, or Venezuelan Equine Encephalitis Virus. Alphavirus platforms are described by Lundstrom, K., Alphavirus-based antigen preparation. METHODS MOL. BIOL 2021, 2183-81; Lundstrum, K., Alphavirus-based vaccines, METHODS MOL. BIOL. 2017, 1581, 225-32; or Lundstrum, K., Alphavirus-based vaccines, VIRUSES 2014, 6(6), 2392-415, which are incorporated by reference. .

[0063] In some embodiments, the nucleotides of an RNA platform or saRNA described herein may be modified to stabilize the platform, increase its half-life, enhance its uptake or cellular expression, or reduce undesirable immune responses. For example, uracil in the RNA sequence of the platform may be replaced by l-methyl-3'-pseudouridylyl (the canonical name is Nl-Methylpseudouridine) to reduce host immune responses to the platform after it has been administered. The platform may comprise a modified RNA in which certain nucleosides are replaced by other naturally occurring modified nucleosides or by synthetic nucleoside analogs.

[0064] The compositions and methods disclosed herein may comprise two or more of these platforms, in which each platform encodes ILTV antigens of a different ILTV genotype or strain. Nine ILTV genotypes (LIX) were recognized using the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphisms) method with three viral alleles (gB, gM, and UL47 / gG). A more detailed description of the ILTV genotypes and genotyping is described in Spatz, SJ, et al., MinlON sequencing to genotype US strains of infectious laryngotracheitis virus, AVIAN PATHOLOGY, 2019, 48(3), 255, and is incorporated therein by reference.

[0065] Non-alphavirus platforms. Non-limiting examples of modified non-alphavirus replicons or platforms include modified RNA replicons of viral species belonging to the Togaviridae, Flaviviridae, Orthomyxoviridae, Rhabdoviridae, or Paramyxoviridae families. Accordingly, in some embodiments, the modified non-alphavirus RNA replicon comprises a modified RNA replicon of a negative-strand RNA virus. The appropriate negative-strand RNA virus species These include, but are not limited to, viral species from the families Orthomyxoviridae, Rhabdoviridae, and Paramyxoviridae. In some embodiments, the modified non-alphavirus RNA replicon comprises a modified RNA replicon of a positive-stranded virus species from the family Togaviridae or Flaviviridae. In some embodiments, the modified non-alphavirus RNA replicon comprises a modified RNA replicon of a positive-stranded virus species from the genus Arterivirus of the family Arteriviridae. Suitable arterivirus species include, but are not limited to, species of equine arteritis virus (EAV), porcine reproductive and respiratory syndrome virus (PRRSV), elevated lactate dehydrogenase virus (LDV), simian hemorrhagic fever virus (SHFV), and opossum disease virus (WPDV). Other platforms are described by and incorporated by reference to Kamrud, et al., US2018 / 0171340 Al, which also describes capsid amplifiers and stem-loop structures.

[0066] Expression cassettes. In some embodiments, the sRNA-encoding sequence further comprises one or more expression cassettes, in which each expression cassette includes a promoter operationally linked to a sequence encoding a gene of interest (GOI). In some embodiments, the sRNA comprises at least one, two, three, four, five, or six expression cassettes. In some embodiments, at least one of the expression cassettes is operationally linked downstream of the second nucleic acid sequence encoding at least one non-structural viral protein or a portion thereof.In some embodiments, at least one of the expression cassettes further comprises a third nucleic acid sequence encoding one or more structural elements of a viral capsid activator, the third nucleic acid sequence being operationally linked upstream to the GOI-encoding sequence. In some embodiments, the saRNA further comprises a sequence encoding an autoprotease peptide operationally linked downstream to the third nucleic acid sequence and upstream to the GOI-encoding sequence.

[0067] A platform may comprise or be part of a delivery system containing naked DNA in a buffer with or without adjuvant, DNA coupled to nanoparticles and / or formulated in compounds containing an adjuvant or inserted into live bacterial or viral vectors such as adenovirus, adeno-associated virus (AAV), alphavirus, poxvirus and herpesvirus.

[0068] Other types of platforms include nucleic acid components from a whole virus, a live attenuated whole virus, an inactivated virus (e.g. chemically or by radiation), viral vectors and other nucleic acid-based (RNA or DNA) platforms encoding ILTV antigens or epitopes. Advantageously, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 ILTV antigens are encoded by the platform. In other embodiments, at least 3, 4, 5, or 6 ILTV antigens are encoded by a platform comprising the RNA disclosed herein. Platforms encoding more than one ILTV antigen are designed to allow for more natural processing and association of ILTV antigens with each other, for example, in the form of a complex between different viral antigens that forms virus-neutralizing epitopes not present on the individual proteins composing a complex.

[0069] 5' Cap. The five-prayer cap (5' cap) is a specially modified nucleotide The 5' end of certain primary transcripts, such as precursor messenger RNA. This process, known as mRNA capping, is highly regulated and vital for the creation of a stable and mature messenger RNA. Many preferred platforms, such as those described here, contain a 5' cap. 5' caps are well known. The saRNA platform as described here preferably has a 5' cap.

[0070] 5' UTR. The 5' untranslated region (also called the 5' UTR, leader sequence, The leader RNA (or leader RNA) is the region of a messenger RNA (mRNA) located directly upstream of the start codon. This region is important for regulating transcript translation through various mechanisms. A platform such as the one described here typically contains a 5' UTR, often encoding a leader peptide. In some cases, the 5' UTR, promoters, 3' UTR, or poly-A sequences are derived from alphaviruses or ILTV. The sRNA platform described here preferably includes a 5' UTR.

[0071] Alphavirus 26S promoter. The 26S promoter, which is located between the two ORFs of the alphavirus negative-sense RNA, is recognized by alphavirus non-structural proteins for the transcription of a subgenomic mRNA from which structural proteins, such as exogenous ILTV antigens, are translated. Various alphavirus 26S promoter sequences or their equivalents can be used in the platforms described herein. Each 26S promoter generates subgenomic mRNAs. The number of subgenomic mRNAs produced from a 26S promoter can be approximately 10 times greater than that produced from the full-length replicon RNA. 26S promoters are known, described and incorporated by reference to Vander Veen, et al, Alphavirus replicon vaccines, Cambridge University Press, 2012. An sa RNA may include a single 26S promoter upstream of at least one polynucleotide encoding the ILTV antigen.A 26S promoter of the alphavirus can transcribe subgenomic mRNA encoding ILTV antigens and be used to transcribe multiple polynucleotides encoding ILTV antigens. In other cases, 26S promoters can be paired upstream of polynucleotides encoding the . ILTV antigens. The RNA disclosed here preferably includes a 26S promoter or a functionally equivalent promoter.

[0072] 6K or 6K-type protein. Alphavirus 6K proteins are small (58-61 amino acids), hydrophobic, and associate with membranes. Cleavage of 6K protein residues is carried out by cellular proteases to generate structural proteins. A linker sequence encoding a 6K protein can be interposed between adjacent sequences encoding ILTV antigens. Cleavage of the translated 6K linker sequences produces distinct ILTV antigens. A 6K linker sequence imposed between a first and a second gene of interest can direct a second gene of interest to the ER / Golgi compartments. In some embodiments, such as those comprising an sa RNA encoding two or more ILTV antigens, the sa RNA will include a polynucleotide sequence encoding a 6K linker.

[0073] Ribosome skipping refers to an alternative translation mechanism in which a specific peptide prevents the ribosome from covalently binding a newly inserted amino acid and allows it to proceed with translation. This results in apparent cotranslational cleavage of the polyprotein. This process is induced by a "2A-like" or CHYSEL (cis-acting hydrolase element) sequence, which comprises a non-conserved amino acid sequence with a strong alpha-helical propensity followed by the consensus sequence -D(V / I)ExNPG P, where x = any amino acid. The apparent cleavage occurs between G and P. This class of elements includes polynucleotides encoding so-called self-cleaving peptides or 2A peptides, which are generally 18 to 22 amino acid residues long and can induce ribosome skipping during protein translation. The peptides in this family include P2A, E2A, F2A and T2A.A more detailed description of this process, along with the apparent cleavage sequences of picornaviridae, iflaviradae, tetraviridae, dicistorviridae, and reovriridat and their corresponding UniProt numbers, is available and incorporated by reference to hypertext transfer protocol secure: / / viralzone.expasy.org / 914 (last accessed October 21, 2022). In some embodiments, such as those comprising an saRNA encoding two or more ILTV antigens, the saRNA will include a polynucleotide sequence that causes ribosome blocking or skipping.

[0074] Furin cleavage site. Furin is a protease ubiquitous in mammalian metabolism. Furin cleavage sites are naturally present in many viruses. Polynucleotides encoding other known furin cleavage sites can be inserted between the ILTV antigen-coding sequences in the platforms shown here. Once a polyprotein is expressed, the furin present in the host cell can cleave these sites and produce discrete ILTV antigens. In some embodiments, such as those comprising an sa RNA encoding two or more ILTV antigens, the sa RNA will include a polynucleotide sequence encoding a 6K linker.

[0075] 3' UTR. The untranslated region of the first three letters (3'-UTR) is the section of messenger RNA (mRNA) that immediately follows the translation stop codon. The 3'-UTR often contains regulatory regions that influence gene expression post-transcriptionally. A platform such as the one described herein typically contains a 3'UTR. A 3' or 5' UTR can be derived from ILTV, alphavirus, or other sources. An alphavirus replicon, such as a VEE replicon, typically requires both a 5' and a 3' UTR from alphavirus to replicate, as these sequences are promoters for the production of RNA from the + and - strands driven by the nsPl-4 complex. In many cases, an RNA presented here will contain a 3' UTR or a poly-A sequence.

[0076] Poly-A. Polyadenylation is the addition of a poly(A) tail to an RNA transcript, usually messenger RNA (mRNA). A poly(A) tail is added to an RNA at the end of transcription. On mRNAs, the poly(A) tail protects the mRNA molecule from enzymatic degradation in the cytoplasm and facilitates the termination of transcription, the export of the mRNA from the nucleus, and translation. The poly-A tail may be endogenous to the virus from which the platform is derived, for example, endogenous to the alphavirus, or, in other cases, exogenous. A poly(A) tail is important for nuclear export, translation, and mRNA stability. The polyadenylation sites of many alphaviruses are known.

[0077] The term "antigen," as used here and as commonly used in the field of immunology, refers to an "antibody-generating" molecule or a molecule that induces antigen-specific T cells, i.e., a substance that can trigger an adaptive immune response. An antigen is therefore a molecule that binds to an antigen-specific receptor, whether a T lymphocyte or B lymphocyte receptor. An antigen is generally a polyp, a virus, or a bacterium. An antigen is usually a polypeptide, but it can also be a polysaccharide or a lipid, possibly associated with a protein or polysaccharide carrier molecule. For the purposes of the various aspects and embodiments of the present invention, the antigen is a polypeptide, i.e., a sequence of amino acids.In the case of binding to a T lymphocyte receptor, the antigen is presented to the respective T lymphocyte receptor via an antigen-presenting cell in the form of an antigenic peptide linked to a histocompatibility molecule on the surface of the antigen-presenting cell, the antigenic peptide having been pre-processed by the antigen-presenting cell. Antigen presentation by professional antigen-presenting cells (APCs) is the first step toward initiating an adaptive immune response led by naïve T lymphocytes. Thus, an "antigen" as used here refers to a molecule, such as a protein or polypeptide, comprising one or more epitopes that will stimulate a host's immune system to produce a humoral and / or cellular response specific to the antigen.

[0078] An epitope, also called an antigenic determinant, is the part of an antigen that is recognized by the immune system, in particular by antibodies, B cells, or T cells. Epitopes of protein antigens are divided into two categories: conformational epitopes and linear epitopes. Epitopes of protein antigens are divided into two categories, conformational epitopes and linear epitopes, based on their structure and their interaction with the paratope. Conformational and linear epitopes interact with the paratope according to the three-dimensional conformation adopted by the epitope, which is determined by the surface characteristics of the epitope residues involved and by the shape or tertiary structure of other segments of the antigen. A conformational epitope is formed by the three-dimensional conformation adopted by the interaction of discontinuous amino acid residues.In contrast, a linear epitope is formed by the three-dimensional conformation adopted by the interaction of contiguous amino acid residues. T cell epitopes are presented on the surface of an antigen-presenting cell, where they are bound to major histocompatibility complex (MHC) molecules. In humans, professional antigen-presenting cells are specialized to present MHC class II peptides, while most nucleated somatic cells present MHC class I peptides. T cell epitopes presented by MHC class I molecules are typically peptides between 8 and 11 amino acids in length, while MHC class II molecules present longer peptides, between 13 and 17 amino acids, and non-classical MHC molecules also present non-peptide epitopes, such as glycolipids.

[0079] An epitope that induces a virus neutralization response can induce antibodies that bind to ILTV in a way that inhibits, reduces, or blocks infection or infection progression. A neutralizing antibody or antigen-binding molecule can block interactions with the receptor or bind to a viral capsid in such a way as to prevent genome disincorporation. The expression "neutralizing antibodies" or "antigen-binding neutralizing molecules" also includes antibodies or antigen-binding molecules capable of preventing infection by a pathogen, such as a virus, by facilitating a cytokine response. or by facilitating uptake and elimination by an immune cell. In particular, the term "neutralizing antibodies" includes antibodies (or their fragments or derivatives) capable of inhibiting or blocking infection (or the progression of infection) by a pathogen through antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Only a small subset of the many antibodies that bind to a virus is capable of neutralizing it. Platforms incorporating 6K or 6K-like linkers are used to produce epitopes to which virus-neutralizing antibodies or other responses are directed.

[0080] Antigen processing is an immunological process that prepares antigens for presentation to special immune system cells called T lymphocytes. This process involves two distinct pathways for processing antigens originating from an organism's own proteins or from intracellular pathogens (e.g., viruses), or from phagocytosed pathogens (e.g., bacteria); the subsequent presentation of these antigens onto major histocompatibility complex (MHC) class I or class II molecules depends on the pathway used. The antigen processing pathways are described in [Fig. 4] and include the presentation of peptide fragments via MHC class I or II molecules and the exocytosis of ILTV polypeptides.

[0081] A monovalent vaccine refers to a vaccine designed to immunize against a single ILTV antigen or a single type of ILTV virus, whereas polyvalent or multivalent vaccines are designed to immunize against several strains or genotypes of ILTV or against a mixture of different ILTV antigens. In some cases, a vaccine as described herein may comprise a single platform or a mixture of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more platforms, each encoding antigens from a different strain of ILTV. In other cases, a vaccine may comprise a platform encoding the same antigen from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more ILTV strains.

[0082] Analogues or variants of the polynucleotides or polypeptides described herein may exhibit varying degrees of sequence identity or similarity to said polynucleotides or polypeptides. BLASTN can be used to identify a polynucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99%, or <100% sequence identity with a reference polynucleotide such as a polynucleotide comprising genomic, subgenomic, or ILTV antigen-coding sequences. A modified representative BLASTN parameter to find highly similar sequences uses an expectation threshold of 10 and a word size of 28, maximum matches in the query range of 0, match / mismatch scores of 1 / -2, and a linear gap cost. Less complex regions can be filtered or masked. The default parameters of a standard nucleotide BLAST are described and incorporated by reference to hypertext transfer protocol secure: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome (last accessed June 7, 2022).

[0083] BLASTP can be used to identify an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99%, or <100% sequence identity, or similarity, to a reference amino acid, such as an ILTV amino acid sequence, using a similarity matrix such as BLOSUM45, BLOSUM62, or BLOSUM80, where BLOSUM45 can be used for closely related sequences, BLOSUM62 for intermediate sequences, and BLOSUM80 for sequences further apart. Unless otherwise specified, a similarity score will be based on the use of BLOSUM62. When BLASTP is used, the similarity percentage is based on the BLASTP positive score and the sequence identity percentage is based on the BLASTP identity score.BLASTP "Identities" indicates the number and fraction of total residues in pairs of highly scored sequences that are identical; and BLASTP "Positives" indicates the number and fraction of residues for which alignment scores have positive values ​​and that are similar to each other. Amino acid sequences exhibiting these degrees of identity or similarity, or any intermediate degree of identity or similarity to the amino acid sequences disclosed herein, are considered and covered by this disclosure. The representative BLASTP parameter uses an expectation threshold of 10, a word size of 3, BLOSUM 62 as the matrix, a deviation penalty of 11 (existence) and 1 (extension), and a conditional fit to the composition score matrix. Other default BLASTP parameters are described and incorporated by reference to the disclosure available at: hypertext transfer protocol secure: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome (last accessed June 7, 2022).

[0084] Analogues or variants of a polynucleotide may include those comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or more deletions, substitutions or insertions of nucleotides in a polynucleotide such as those disclosed herein. These analogs may be based on a genomic sequence of the virus, on a sequence of a specific viral gene, or on sequences of other platform elements such as the 3' or 5' UTR, 6k or 6k-like sequences, or any other sequence disclosed herein.

[0085] Polypeptide analogues or variants include those comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or more deletions, substitutions or insertions of amino acid residues in a polypeptide, such as those described herein. These analogs may be based on one or more polypeptides encoded by a viral genome, on a sequence encoded by a specific viral gene, or on sequences encoded by other elements of a 6k platform or 6k-type sequences or any other coding sequence disclosed herein.

[0086] The terms "fragment" or "functional fragment" used herein refer to shorter or truncated segments of a longer nucleic acid or polypeptide sequence, which preferably retain at least one function of the whole nucleic acid or polypeptide, such as an epitope. Functional immunogenic fragments are therefore suitable for vaccination. Typically, a nucleic acid fragment encoding an ILTV antigen will encode at least one of its epitopes, and an ILTV antigen fragment will include at least one epitope of the antigen. Examples of polypeptide fragments include longer ILTV polypeptide fragments having at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, or more contiguous amino acid residues. Examples of polynucleotide fragments include those that encode the peptide fragments described above.

[0087] Pharmaceutically acceptable carriers. An ILTV vaccine generally comprises a platform encoding one or more ILTV antigens and at least one pharmaceutically acceptable carrier, excipient, or diluent. Stabilizers or agents that prevent RNA degradation may be included. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition may be sterile, and the formulation may be suitable for the route of administration. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition may be a liquid solution, suspension, emulsion, tablet, pill, capsule, extended-release formulation, or powder.The composition can be formulated as a suppository, using traditional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Any common pharmaceutical carrier, such as sterile saline or sesame oil, can be used. The medium may also contain conventional pharmaceutical excipients such as, for example, pharmaceutically acceptable salts for adjusting osmotic pressure, buffers, preservatives, and others. Other media that can be used with the compositions and methods are listed below. The carriers provided here are physiological saline and sesame oil. Generally, the nature of the carrier depends on the route of administration. For example, parenteral formulations typically include injectable fluids, which may include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced saline solutions, aqueous dextrose, glycerol, or similar substances as vehicles. For solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional nontoxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, pH buffers, and others, for example, sodium acetate or sorbitan monolaurate. In some embodiments, the carrier may be a particle, a nanoparticle, a liposome, a lipoplex, or a lipid nanoparticle, for example, as described by and incorporated by reference to Kamrud, et al., US 2018 / 0171340 A.

[0088] LION. Inorganic lipid nanoparticles (LION). In some embodiments, the platform is administered in the form of a nanoemulsion particle having a hydrophobic core and comprising a mixture of liquid oil and one or more inorganic solid nanoparticles. The nanoemulsion particle may also be referred to herein as inorganic lipid nanoparticles (LION). The liquid oil is mixed with one or more inorganic nanoparticles to form a hydrophobic core. The liquid oil is generally metabolizable. The suitable liquid oil may be a vegetable oil, an animal oil, or a synthetically prepared oil. In some cases, the liquid oil is fish oil. In other cases, the liquid oil is a natural or synthetic terpenoid.In some cases, the liquid oil is squalene, a triglyceride (such as caprylic / capric triglyceride or myristic acid triglyceride), vitamin E, lauryl polyoxyglyceride, monoacylglycerol, soy lecithin, sunflower oil, soybean oil, olive oil, grapeseed oil, or a combination of these oils. In one case, the liquid oil is squalene, a triglyceride (such as caprylic / capric triglyceride or myristic acid triglyceride), vitamin E, lauryl polyoxyglyceride, monoacylglycerol, soy lecithin, or a combination of these. In one case, the liquid oil is squalene, a triglyceride (such as capric / caprylic triglyceride or myristic acid triglyceride), sunflower oil, soybean oil, olive oil, grapeseed oil, or a combination of these.In some cases, the liquid oil is squalene (of natural or synthetic origin, possibly). in combination with one of the aforementioned liquid oils). Inorganic nanoparticles can be formed from one or more of the same or different metals (all metals, including transition metals), such as metal salts, metal oxides, metal hydroxides, and metal phosphates. Examples include silicon dioxide (SiO2), iron oxides (FeO34, FeO23, FeO, or combinations thereof), aluminum oxide (AlO123), aluminum oxyhydroxide (Al10(OH)), aluminum hydroxyphosphate (Al(OH)x(PO4)4y), calcium phosphate (Ca3(PO4)42), calcium hydroxyapatite (CalO(PO4)6(OH)2), iron gluconate, or iron sulfate. In some cases, the inorganic solid nanoparticle is a metal oxide, such as a transition metal oxide. In one case, the inorganic solid nanoparticle is an iron oxide, for example magnetite (Fe O34), maghemite (γ-).-FeO23), wustite (FeO), hematite (α-FeO23), or combinations thereof. In some cases, the inorganic solid nanoparticle is a metallic hydroxide, such as aluminum hydroxide or aluminum oxyhydroxide. The inorganic solid nanoparticle may contain a reporter element detectable by imaging methods to allow imaging and tracking of the resulting nanoemulsion particles in the body. For example, the inorganic solid nanoparticle may contain a reporter element detectable by magnetic resonance imaging (MRI), such as a paramagnetic, superparamagnetic, ferrimagnetic, or ferromagnetic compound. Iron oxides, iron gluconates, and iron sulfates are examples of inorganic solid nanoparticles detectable by MRI. Solid inorganic nanoparticles typically have an average diameter (number-weighted average diameter) of between about 3 nm and about 50 nm.For example, the inorganic solid nanoparticle can have an average diameter of approximately 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, or 50 nm. The inorganic solid nanoparticle can be surface-modified before being mixed with the liquid oil. For example, if the surface of the inorganic solid nanoparticle is hydrophilic, it can be coated with hydrophobic molecules (or surfactants) to facilitate its miscibility with the liquid oil in the "oil" phase of the nanoemulsion particle.Phosphate-terminated lipids (such as phosphatidylated lipids), phosphorus-terminated surfactants, carboxylate-terminated surfactants, sulfate-terminated surfactants, or amine-terminated surfactants can be used to modify the surface of inorganic solid nanoparticles. Typical phosphate-terminated lipids or phosphorus-terminated surfactants include trioctylphosphine oxide (TOPO). Distearylphosphatidic acid (DSPA) is one example. Typical sulfate-terminated surfactants include, but are not limited to, sodium dodecyl sulfate (SDS). Typical carboxylate-terminated surfactants include oleic acid. Amine-terminated surfactants include oleylamine. In one case, the inorganic solid nanoparticle is a metal oxide such as an iron oxide, and a surfactant, such as oleic acid, oleylamine, SDS, DSPA, or TOPO, is used to coat the inorganic solid nanoparticle before it is mixed with liquid oil to form the hydrophobic core.In one case, the inorganic solid nanoparticle is a metal hydroxide, such as aluminum hydroxide or aluminum oxyhydroxide, and a phosphate-terminated lipid or surfactant, such as oleic acid, oleylamine, SDS, TOPO, or DSPA, is used to coat the inorganic solid nanoparticle before it is mixed with liquid oil to form the hydrophobic core. The lipids used to form the nanoemulsion particles can be cationic lipids, anionic lipids, neutral lipids, or mixtures thereof. In some cases, the lipids used are cationic lipids. For example, positively charged lipids that can interact favorably with negatively charged bioactive agents (such as DNA or RNA) can be used in the nanoemulsion composition. Suitable cationic lipids include 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP); 3.beta.[N(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC Cholesterol); dimethyldioctadecylammonium (DDA); 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP); dipalmitoyl(C16:0)trimethylammonium propane (DPTAP); distearoyltrimethylammonium propane (DSTAP); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA); l,l'-((2-(4-(bis(2-hydroxydodecyl)amino)ethyl) (2-hydroxydodecyl)amino)ethyl)piperazin-l-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200); and their combinations. . A typical cationic lipid is DOTAP. Other examples of suitable lipids include, but are not limited to, phosphatidylcholines (PCs), such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), l-palmitoyl-2-oleoylphosphatidylcholine (POPC), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylcholine (DMPC), etc.; phosphatidylethanolamines (PEs), such as l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), etc.; phosphatidylglycerol (PGs); and PEGylated lipids, including the PEGylated version. of any of the lipids listed above (e.g., DSPE-PEGs, DSPE-PEG). The nanoemulsion particle may also contain one or more surfactants, which may be hydrophobic or hydrophilic. In some cases, the nanoemulsion particle further comprises a hydrophobic surfactant. In some cases, the nanoemulsion particle further comprises a hydrophilic surfactant. In one case, the nanoemulsion particle further comprises both a hydrophobic and a hydrophilic surfactant. Suitable hydrophobic surfactants include those with a hydrophilic-lipophilic balance (HLB) value less than or equal to 10, e.g., less than or equal to 5, between 1 and 5, or between 4 and 5. An example of a hydrophobic surfactant is a sorbitan ester (such as sorbitan monoester or sorbitan trimester).For example, the hydrophobic surfactant may be a sorbitan ester with an HLB value of 1 to 5, or 4 to 5. In some embodiments, the hydrophobic surfactant is a sorbitan monoester or a sorbitan triester. Exemplary sorbitan monoesters include sorbitan monostearate and sorbitan monooleate. Examples of sorbitan triesters include sorbitan tristearate and sorbitan trioleate. Suitable hydrophilic surfactants include polyethylene oxide-based surfactants, for example, a polyoxyethylene sorbitan ester (polysorbate). In some cases, the hydrophilic surfactant is a polysorbate.The exemplary polysorbates are polysorbate 80 (polyoxyethylene sorbitan monooleate or Tween 80), polysorbate 60 (polyoxyethylene sorbitan monostearate or Tween 60), polysorbate 40 (polyoxyethylene sorbitan monopalmitate or Tween 40) and polysorbate 20 (polyoxyethylene sorbitan monolaurate or Tween 20). In one case, the hydrophilic surfactant is polysorbate 80. The nanoemulsion particle can have an oil / surfactant molar ratio ranging from about 0.1:1 to about 20:1, from about 0.5:1 to about 12:1, from about 0.5:1 to about 9:1, from about 0.5:1 to about 5:1, from about 0.5:1 to about 3:1, or from about 0.5:1 to about 1:1. The nanoemulsion particle can have a hydrophilic surfactant / lipid (e.g., cationic lipid) ratio ranging from about 0.1:1 to about 2:1, from about 0.2:1 to about 1.5:1, from about 0.3:1 to about 1:1, from about 0.5:1 to about 1:1, or from about 0.6:1 to about 1:1.The nanoemulsion particle can have a hydrophobic surfactant / lipid (e.g., cationic lipid) ratio ranging from about 0.1:1 to about 5:1, from about 0.2:1 to about 3:1, from about 0.3:1 to about 2:1, from about 0.5:1 to about 2:1, or from about 1:1 to about 2:1. The nanoemulsion particle can comprise from about 0.2% to about 40% w / v of liquid oil, from about 0.001% to about 10% w / v of inorganic solid nanoparticle, from about 0.2% to about 10% w / v of lipid (e.g., cationic lipid), from about 0.25% to about 5% w / v of surfactant. hydrophobic (e.g., sorbitan ester), and about 0.5% to about 10% w / v of hydrophilic surfactant. In some embodiments, the nanoemulsion particle comprises: a hydrophobic core comprising a mixture of: one or more inorganic nanoparticles containing at least one metal oxide nanoparticle optionally coated with a phosphate-terminated lipid, a phosphorus-terminated surfactant, a carboxylate-terminated surfactant, a sulfate-terminated surfactant or an amine-terminated surfactant, and a liquid oil containing natural or synthetic squalene; a cationic lipid comprising DOTAP; a hydrophobic surfactant comprising a sorbitan ester selected from the group consisting of sorbitan monostearate, sorbitan monooleate and sorbitan trioleate; and a hydrophilic surfactant comprising a polysorbate.In one embodiment, the nanoemulsion particle comprises: a hydrophobic core comprising a mixture of: one or more inorganic nanoparticles containing iron oxide nanoparticles, and a liquid oil containing natural or synthetic squalene; the cationic lipid DOTAP; a hydrophobic surfactant comprising sorbitan monostearate; and a hydrophilic surfactant comprising polysorbate 80. In this LION composition, the LION particle may comprise from about 0.2% to about 40% w / v of squalene, from about 0.001% to about 10% w / v of iron oxide nanoparticles, from about 0.2% to about 10% w / v of DOTAP, from about 0.25% to about 5% w / v of sorbitan monostearate and from about 0.5% to about 10% w / v of polysorbate 80.In one embodiment, the LION particle comprises about 2% to about 6% w / v of squalene, about 0.01% to about 1% w / v of iron oxide nanoparticles, about 0.2% to about 1% w / v of DOTAP, about 0.25% to about 1% w / v of sorbitan monostearate, and about 0.5% to about 5% w / v of polysorbate 80.In some embodiments, the nanoemulsion particle comprises: a hydrophobic core comprising a mixture of: one or more inorganic nanoparticles containing at least one metal hydroxide or oxyhydroxide nanoparticle optionally coated with a phosphate-terminated lipid, a phosphorus-terminated surfactant, a carboxylate-terminated surfactant, a sulfate-terminated surfactant or an amine-terminated surfactant, and a liquid oil containing squalene of natural or synthetic origin; a cationic lipid comprising DOTAP; a hydrophobic surfactant comprising a sorbitan ester selected from the group consisting of sorbitan monostearate, sorbitan monooleate and sorbitan trioleate; and a hydrophilic surfactant comprising a polysorbate.In one embodiment, the nanoemulsion particle comprises: a hydrophobic core comprising a mixture of one or more inorganic nanoparticles containing aluminum hydroxide or aluminum oxyhydroxide nanoparticles. possibly coated with TOPO, and a liquid oil containing natural or synthetic squalene; the cationic lipid DOT AP; a hydrophobic surfactant comprising sorbitan monostearate; and a hydrophilic surfactant comprising polysorbate 80. In this LION composition, the LION particle may comprise from about 0.2% to about 40% w / v of squalene, from about 0.001% to about 10% w / v of aluminum hydroxide or aluminum oxyhydroxide nanoparticles, from about 0.2% to about 10% w / v of DOT AP, from about 0.25% to about 5% w / v of sorbitan monostearate and from about 0.5% to about 10% w / v of polysorbate 80.In one case, the LION particle comprises approximately 2% to 6% w / v of squalene, approximately 0.01% to 1% w / v of aluminum hydroxide or aluminum oxyhydroxide nanoparticles, approximately 0.2% to 1% w / v of DOTAP, approximately 0.25% to 1% w / v of sorbitan monostearate, and approximately 0.5% to 5% w / v of polysorbate 80. Nanoparticles and nanoemulsions have been described in the literature, and these terms are used here to refer to particles smaller than 1000 nanometers. The nanoemulsion particle (LION) typically has a mean diameter (hydrodynamic mean diameter z, measured by dynamic light scattering) ranging from approximately 20 nm to approximately 200 nm. In some cases, the average diameter z of the LION particle is between about 20 nm and about 150 nm, between about 20 nm and about 100 nm, between about 20 nm and about 80 nm, between about 20 nm and about 60 nm.In some cases, the mean z-diameter of the LION particle ranges from approximately 40 nm to approximately 200 nm, from approximately 40 nm to approximately 150 nm, from approximately 40 nm to approximately 100 nm, from approximately 40 nm to approximately 90 nm, from approximately 40 nm to approximately 80 nm, or from approximately 40 nm to approximately 60 nm. In one case, the mean z-diameter of the LION particle ranges from approximately 40 nm to approximately 80 nm. In another case, the mean z-diameter of the LION particle ranges from approximately 40 nm to approximately 60 nm. The mean polydispersity index (PDI) of the nanoemulsion particles (LION) can range from approximately 0.1 to approximately 0.5. For example, the average polydispersity index of LION particles can range from about 0.2 to about 0.5, from about 0.1 to about 0.4, from about 0.2 to about 0.4, from about 0.2 to about 0.3, or from about 0.1 to about 0.3.

[0089] Methods of administration. In some cases, the polynucleotides or compositions presented herein are formulated for in vivo administration. For administration to a bird, the composition according to this application may be administered by any enteral or parenteral route, including, but not limited to, intramuscular, skin or wing scarification, intranasal, intradermal, subcutaneous, oral, aerosol, topical, spray-on, gel drop, or any combination thereof.

[0090] In some cases, the sa RNA is formulated for intramuscular, subcutaneous, intra-organ, or intradermal administration. In some cases, the polynucleotide or composition is administered subcutaneously, intravenously, intramuscularly, intra-articularly, intra-synovially, intrasternally, intrathecally, intrahepatically, intrathymically, into a sexual organ, intralesionally, intracranially, intraventricularly, orally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. Other methods of administration, such as those targeting specific tissues or birds at different stages of infection, as described in [Fig. 1], may be used.

[0091] Specific embodiments

[0092] Generic description of sa RNA. One embodiment of this technology relates to self-amplified RNA (sa RNA) that encodes at least one infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus 1, GaHV-1) polypeptide, an ILTV polypeptide variant, or an immunogenic fragment or epitope thereof, comprising in any order (a) a first nucleic acid sequence comprising nsP1, nsP2, nsP3 and nsP4 that encode non-structural proteins of the alphavirus, and (b) a second nucleic acid sequence that encodes at least one ILTV polypeptide, an ILTV polypeptide variant, an immunogenic fragment or an epitope thereof. The sRNA may (c) further comprise a 5' cap, a 5' UTR upstream of the first nucleic acid sequence, a 26S promoter upstream of the second nucleic acid sequence, a 3' UTR and a polyA tail.Generally, RNA incorporated into a target cell expresses at least one ILTV polypeptide, an ILTV polypeptide variant, or an antigenic or immunogenic fragment or epitope of one of them.

[0093] sRNA platform. Preferably, the sRNA is part of an alphavirus platform. The sRNA platform expressing ILTV antigens or immunogens can be selected from the group consisting of Sindbis virus (SINV), Chickungunya virus (CHIKV), Semliki Forest virus (SFV), Ross River virus (RRV), Sagiyama virus (SAGV), Getah virus (GETV), Middleburg virus (MIDV), Bebaru virus (BEBV), O'nyong nyong virus (ONNV), Ndumu (NDUV) and Barmah Forest virus (BFV); or a New World alphavirus selected from the group consisting of Venezuelan equine encephalitis virus (VEEV), Western equine encephalitis virus (WEEV), and Eastern equine encephalitis virus (EEEV). An example of a saRNA platform that is not commercially available is described by Maine et al., 2020.

[0094] Order of RNA elements a. In some preferred embodiments, the sRNA comprises a 5' cap, a 5' UTR upstream of the first sequence The nucleic acid sequence consists of nsPl, nsP2, nsP3, nsP4, the 26S promoter upstream of the second nucleic acid sequence (which encodes one or more ILTV antigens or immunogens), a 3' UTR, a poly A tail, and possibly a terminator. Other functionally or structurally equivalent polynucleotide elements may be substituted for the elements described above, for example, modified nsPl-nsP4 sequences, polynucleotides encoding variants or fragments of ILTV antigens or immunogens, or other promoters or terminators. In some embodiments, the sa RNA further comprises, after the 5' UTR, a polynucleotide encoding nspl, followed by a downstream loop (DLP), followed by an autoprotease, such as P2A; see [Fig. 5], which represents a vector encoding the sa RNA.

[0095] Additional elements of the saRNA. In some embodiments, additional elements may be incorporated into the sRNA. These include elements that produce two or more distinct ILTV antigens or immunogens, such as sequences encoding 6K proteins or ribosome skipping / crystallization sequences, or protease sites. They may also include elements that enhance the transcription of the saRNA or the expression of the ILTV antigen or immunogen, such as DLP loops. They may include polynucleotides encoding immunogenic carrier proteins, adjuvants, or cytokines.

[0096] Protein separation sites. In some embodiments, the saRNA presented herein further comprises a nucleic acid sequence encoding at least one protein separation site, such as a blocking or skipping site of the 6K ribosome. Typically, these sites are placed between nucleic acids encoding different proteins, so that when the protein is expressed from the saRNA, it can be readily expressed or converted into an individual ILTV antigen or immunogen.

[0097] A protein separation site may include at least one cleavage site for an endogenous or exogenous protease, such as a furin cleavage site, which is positioned between segments of the second nucleic acid sequence that encode different ILTV antigens.

[0098] DLP or capsid activator. In some cases, when a DLP or capsid activator is present, the protein-binding sites may be positioned between the viral capsid activator or DLP and the second nucleic acid sequence. In some embodiments, the viral capsid activator or downstream loop ("DLP") is positioned immediately downstream of the 5' UTR or the 26S promoter. In one embodiment, the saRNA further comprises, after a 5' UTR, polynucleotides encoding nspl, followed by a downstream loop (DLP), followed by an autoprotease polynucleotide such as PA2.

[0099] Immunogenic carrier protein. In some embodiments, the saRNA presented herein further comprises a nucleic acid sequence encoding cross-reaction genetically modified material (CRM) of diphtheria toxin, tetanus toxoid (T), meningococcal outer membrane protein complex (OMPC), diphtheria toxoid (D) or H. influenzae protein D (HiD), into which the following nucleic acid sequence is optionally fused.

[0100] Immunomodulatory protein. The sa RNA as disclosed herein may further comprise a nucleic acid sequence encoding at least one immunomodulatory protein, including, but not limited to, the chemokines CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12 or CXCL13; interleukins IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12A, IL-13, IL-14, IL-15, IL-17 or IL-18; cytokines colony-stimulating factor 1 (CSF1), colony-stimulating factor 2 (CSF2) or colony-stimulating factor 3 (CSF3); proteins that are intracellular pattern recognition receptors (PRRs): Sendai virus-derived oligonucleotide that mimics Sendai virus defective interfering particles (DI) (SeVDI) or another PRR-triggering protein; or combinations thereof.

[0101] Protein transport elements. The sa RNAs described herein may further comprise elements that help transport expressed ILTV antigens or immunogens to particular cellular or extracellular compartments, for example, directing an expressed antigen to the endoplasmic reticulum and Golgi apparatus, to lysosomes, to the cell membrane, or to the cell exterior. An sa RNA described above may comprise a polynucleotide encoding a 6k or 6k-type cleavage site or a furin-type cleavage site between polynucleotide sequences encoding ILTV antigens that can be transported to the endoplasmic reticulum and produce one or more glycosylated antigens.

[0102] In some embodiments, the sRNA platforms presented here include polynucleotides encoding head or signal peptides, ILTV a 6k or 6k type cleavage site, a linker encoding a furin cleavage site, or which include P2A or T2A.

[0103] Specific ILTV antigens. In some embodiments, the second nucleic acid sequence of the RNA presented herein encodes at least one ILTV polypeptide encoding gB, gB-del, gC, gD, gE, gG, gH, gl, gj, gK, gL, or gM. The specific ILTV antigenic sequences include those of gB (AGS36506.1), gC (AAA16957.1), gD (AAA98925.1), gE (AGS36509.1), gG (AGC50883.1), gH (AGF43577.1), gl (BAA33003.1; BAA33004.1; BAA33005.1, BAA33006.1, BAA33007.1, and / or BAA33008.1), gJ (AGS36512.1), gK (BAA33003.1; BAA33004.1), BAA33005.1, BAA33006.1, BAA33007.1, and / or BAA33008.1), gL (CAA65897.1, CAA65898.1, CAA65890.1, CAA65891.1, CAA65892.1, CAA65893.1, CAA65894.1, CAA65895.1, CAA65896.1, CAA65899.1, CAA65900.1) and / or gM (ABX59489.1), or any of its isoforms, variants, immunogenic fragments, or epitopes. The sequences described by the accession numbers above are specifically incorporated by reference.

[0104] An example of a gB-del sequence, where gB is wholly or partially deleted, the deletion includes all or part of gB as described by (AGS36506.1) or by patent application WO2005093070.

[0105] Specific ILTV antigen variants. In some embodiments, the second nucleic acid sequence of the RNA shown here codes for a variant of an ILTV polypeptide containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more insertions, substitutions or deletions with respect to the unmodified sequence of the ILTV antigen or immunogen. A variant exhibits at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 99.5, 99.9, or <100% sequence identity or sequence similarity with an ILTV polypeptide, such as gB (AGS36506.1), gC (AAA16957.1), gD (AAA98925.1), gE (AGS36509.1), gG (AGC50883.1), gH (AGF43577.1), gl (BAA33003.1; BAA33004.1; BAA33005.1, BAA33006.1, BAA33007.1, and / or BAA33008.1), gJ (AGS36512.1), gK (BAA33003.1; BAA33004.1), BAA33005.1, BAA33006.1, BAA33007.1, and / or BAA33008.1), gL (CAA65897.1, CAA65898.1, CAA65890.1, CAA65891.1, CAA65892.1, CAA65893.1, CAA65894.1, CAA65895.1, CAA65896.1, CAA65899.1, CAA65900.1) and / or gM (ABX59489.1). .

[0106] Fragments / epitopes. An ILTV antigen or immunogen encoded by the saR RNA presented here may be a functional fragment of, or a variant of, an ILTV antigen or immunogen. In some cases, the saR RNA will encode a polypeptide comprising a cellular epitope of 13 to 17 residues in length or a humoral epitope of 8 to 11 residues in length. An epitope may be linear or conformational.

[0107] DNA construct encoding sa RNA. One embodiment of the invention is a DNA construct that encodes sa RNA as described herein. The DNA construct may be a plasmid or a viral vector. This construct can be transformed within a cell where it can replicate or transcribe sa RNA.

[0108] Single ILTV antigen. In some embodiments, sa RNA will encode a single ILTV antigen or immunogen, such as an antigen or immunogen selected from those encoding gB, gB-del, gC, gD, gE, gG, gH, gl, gj, gK, gL or gM or other ILTV translation products such as those described in [Fig.3].

[0109] Multiple ILTV antigens. In some embodiments, the sa RNA will encode two, three, four, or more ILTV antigens or immunogens, such as two or more selected from those encoding gB, gB-del, gC, gD, gE, gG, gH, gl, gj, gK, gL, or gM, or other ILTV translation products such as those described in [Fig. 3]. The sa RNA sequences encoding two or more antigens or immunogens may express the antigens in a fused form, such as a polyprotein, or as individual antigens, or as mixtures of these options. When an sRNA codes for two or more antigens, the coding sequences may be separated by sequences coding for protein separation sites, such as a sequence coding for a 6K protein, a ribosome blocking site, or a protease cleavage site, such as a site recognized by an endogenous protease.

[0110] Specific sa RNA constructs include, but are not limited to, those described below.

[0111] In some cases, the second nucleic acid sequence codes for gB or for a variant or fragment thereof.

[0112] In some cases, the second nucleic acid sequence codes for gBdel or for a variant or fragment thereof.

[0113] In some cases, the second nucleic acid sequence codes for gD or a variant or fragment thereof.

[0114] In some cases, the second nucleic acid sequence codes for the gE or for a variant or fragment thereof.

[0115] In some cases, the second nucleic acid sequence codes for gl or a variant or fragment thereof.

[0116] In some cases, the second nucleic acid sequence codes for gE-6K-I or for a variant or fragment thereof.

[0117] In some embodiments, the second nucleic acid sequence codes for gB, gBdel, gD or gE, linked by 6K to gl; codes for gB, gBdel or gD, linked by 6K to gE; or codes for gB or gBdel, linked by 6K to gD.

[0118] In some cases, the second nucleic acid sequence codes for at least two different polypeptides chosen from gB, gBdel, gD, gE and / or gl.

[0119] In some cases, the second nucleic acid sequence codes for gl and gE.

[0120] In some cases, the second nucleic acid sequence codes for gB or gBdel, gD, gl and gE.

[0121] In some cases, the second nucleic acid sequence codes for gB or gBdel, gD, gl, gE and gC.

[0122] In certain embodiments, a combination of gB or gB-del, gl and gE (in any order) comprises the following specific order from 5' to 3': gB or gB-del, gl and gE; gB or gB-del, gE and gl; gE, gB or gB-del, and gl; gE and gl, and gB or gB-del; or gl and gE, and gB or gB-del, or gl and gB or gB-del, and gE.

[0123] In other embodiments, a combination of gD, gl and gE (in any order) comprises the following specific order from 5' to 3': gD, gl and gE; gD, gE and gl; gE, gD and gl; gE and gl, and gD; or gl and gE, and gD; or gl and gD, and gE.

[0124] In one case, gl was found to produce the strongest immune response. Thus, a preferred sa RNA may comprise a gene encoding gl alone or in combination with other ITLV genes such as gD.

[0125] Compositions containing sa RNA. Another aspect of this technology relates to a composition comprising sa RNA (or sa RNAs) as disclosed herein in combination with a pharmaceutically acceptable carrier or excipient. Such a carrier or excipient may comprise at least one of the following: lipids, surfactants, polyacids, proteins, polymer particles, self-assembled particles, conjugated phospholipid composite nanoparticles, or inorganic particles (such as silica particles), or combinations thereof.

[0126] In certain embodiments, this composition can advantageously be formulated with one or more sa RNAs and a pharmaceutically acceptable support or excipient comprising lipid inorganic nanoparticles (LION).

[0127] The compositions disclosed herein may be in a form suitable for oral, mucosal or respiratory administration; in a form suitable for intravenous, intramuscular or other parenteral administration; in particulate or nanoparticulate form; in the form of a liposome, lipoplex or lipid nanoparticle (LNP); or in the form of a nanoemulsion comprising a hydrophobic core consisting of a mixture of liquid oil and one or more inorganic nanoparticles, one or more lipids and, optionally, one, two or more surfactants.

[0128] Treatment with saRNA. Another aspect of this technology relates to a method for reducing the severity of ILTV or one or more comorbid conditions in a bird, such as a chicken, comprising administering to a subject in need an sRNA that encodes at least one ILTV antigen or an immunogenic fragment, an epitope or a variant of one of them, as indicated herein; a plasmid or DNA construct encoding the sRNA; a cell containing the DNA construct; or an sRNA composition as indicated herein.

[0129] Reduction of infection severity by ILTV or prophylaxis. In some embodiments of this method, reduction of ILTV severity includes reduction of dyspnea or hemorrhage associated with ILT, at least one symptom of beak blood or nostril depression, and difficulty breathing compared to an unvaccinated control bird (Koski et al. 2015), as shown by the reduction in clinical score in [Fig. 9] and [Fig. 16].

[0130] In some embodiments of this method, the reduction in severity includes the reduction in weight loss of vaccinated birds compared to unvaccinated control birds.

[0131] In other embodiments of this method, the reduction in severity includes the reduction in mortality of vaccinated birds compared to unvaccinated control birds.

[0132] In other embodiments of this method, the reduction of severity includes the reduction of ILTV transmission compared to ILTV transmission from an unvaccinated bird.

[0133] In other embodiments of this method, the reduction of severity includes reducing the number of abnormal or thin-shelled eggs or increasing the number of eggs laid compared to those produced by an unvaccinated bird.

[0134] Avian subjects. In some embodiments, the RNA presented herein is administered to Gallus gallus domesticus (domestic chicken) or other birds that may or may not have been previously exposed to ILTV. The avian subject may be a breeding chicken, a broiler chicken, or a laying hen.

[0135] In some cases, this method involves treating a bird that has not been exposed to ILTV, for example, to induce immunological protection against ILTV.

[0136] In other embodiments, this method consists of treating a bird that has been exposed or is likely to be exposed to ILTV.

[0137] In some embodiments of this method, the poultry carries the ILTV.

[0138] In some cases, sa RNA or sa RNAs are administered intramuscularly or in a wing.

[0139] In some embodiments of this method, the RNA is administered orally, muscularly or respiratoryly.

[0140] In some embodiments of this method, the RNA is administered subcutaneously, intravenously or parenterally.

[0141] In some embodiments of this method, it also involves administering a drug or a biological product that reduces the severity of the ILTV.

[0142] In some embodiments of this method, the administered RNA is administered in a nanoemulsion comprising a hydrophobic core which includes a mixture of liquid oil and one or more inorganic nanoparticles, one or more lipids and, optionally, one or more surfactants.

[0143] In some cases, saRNA may be administered in association with LION.

[0144] In one case, the administered RNA enhances the acquired cellular or humoral immunity of a vaccinated animal compared to a non-immunized control. In another case, the vaccine increases the levels of ILTV-neutralizing antibodies compared to a non-immunized control.

[0145] In yet another case, RNA strengthens cellular immunity against ILTV compared to a non-immunized control.

[0146] In another embodiment, the RNA enhances the innate immunity of a vaccinated animal or does not substantially inhibit the innate immunity of the vaccinated animal compared to that of an unvaccinated control animal. In some embodiments, the methods further comprise administering to the animal at least one agent that enhances innate or adaptive immunity against ILTV, selected from the group consisting of an ILTV protein recognized by the animal's pattern recognition proteins, interferon, or another agent that enhances an innate immune response, for a certain time and under conditions enabling the induction of an innate immune response to the ILTV virus; and then administering at least one platform for a certain time and under conditions enabling the induction of an adaptive immune response to ILTV.In one embodiment, the pattern recognition molecules are selected from the group consisting of RIG (retinoic acid inducible gene)-type receptors (RLRs), Toll-type receptors 3, 7, and 8 (TLR3, TLR7, and TLR8), interferon regulatory factor 3 (IRF-3), IRF7, NF-κB, Nod-type receptors (NLRs), and C-type lectin receptors (CLRs). In another embodiment, the agent at least comprises type 1 interferon (e.g., IFN-α and IFN-γ); a cytokine that induces Thl cell differentiation, including, but not limited to, IL-12 or IFN-γ; a cytokine that induces Th2 cell differentiation, including but not limited to IL-4, IL-5, IL-6 or IL-13; an IL-10 inhibitor, including but not limited to an IL-10 antibody or a Spl inhibitor, including but not limited to mithramycin or the mithramycin analogue MTMox 32E.

[0147] Method for manufacturing sa RNA. Another aspect of this technology relates to a DNA construct encoding sa RNA as described herein or a cell comprising such a DNA construct; see [Fig. 5] which describes a possible construct. Such a DNA construct can be used to transcribe sa RNA as described herein. Another aspect of this technology relates to a method for manufacturing the RNA presented here, comprising the operational insertion into an alphavirus platform of a nucleic acid sequence that expresses at least one ILTV polypeptide or an immunogenic fragment or epitope thereof, or a variant thereof.

[0148] This method may also include the operational insertion of at least one nucleic acid sequence encoding a 5' cap, a 5' UTR upstream of the first nucleic acid sequence, a 26S promoter upstream of the second nucleic acid sequence, a 3' UTR, a poly A tail, a protease, a protease cleavage site, a ribosome blocking site or a protein separation site, a DLP loop, an immunogenic scaffold, an immunomodulatory protein, a trafficking fraction or a cytokine.

[0149] A cell comprising a vector encoding sa RNA as described herein can be cultured in vivo, ex vivo, or in vitro. Preferably, the cell is a chicken cell, but other types of avian or mammalian cells, or other types of non-mammalian cells, including cultured cell lines, can also incorporate such a vector.

[0150] Codon modification. This method may also include replacing at least one degenerate codon in the nucleic acid sequence that codes for the ILTV polypeptide or an immunogenic fragment or variant thereof, with a different codon encoding the same amino acid, which different codon increases GC content, reduces RNA hairpin formation, increases the expression of ILTV polypeptides, polypeptide fragments or variants, or increases RNA stability.

[0151] In one instance, the polynucleotide(s) or other nucleic acid sequence(s) of the sRNA platform or its polynucleotide components are modified depending on codon usage in the host cell or depending on whether their GC content is decreased or increased. Various codon modification functions and methods can be used, including those described in Hanson, G., Coller, J. Codon optimality, bias and usage in translation and mRNA decay and incorporated by reference to these documents. NAT REV MOL CELL BIOL 19, 20-30 (2018). https: / / doi.org / 10.1038 / nrm.2017.91; or https: / / en.wikipedia.org / wiki / Codon_usage_bias#Effect_on_transcription_or_gene_expression (last accessed 7 June 2022) and the references cited therein.

[0152] In another embodiment, the polynucleotide(s) or other nucleic acid sequence(s) of the platform or its polynucleotide components are selected to mitigate the formation of RNA secondary structures that reduce the expression of at least one polynucleotide encoding an ILTV antigen; to provide or enhance secondary structures that stabilize the stability of the encoding mRNAs for ILTV antigens; or to otherwise control the translation and relative abundance of multiple encoded ILTV antigens.

[0153] In another embodiment, the polynucleotide(s) or other nucleic acid sequence(s) of the sRNA platform or its polynucleotide components are selected to increase the expression of at least one polynucleotide encoding an ILTV antigen. For example, codons encoding an ILTV antigen may be selected to increase the relative abundance (or stoichiometric amount) of that antigen compared to another ILTV antigen.

[0154] In some cases, this method makes it possible to produce a platform comprising a self-amplifying RNA designed to resist innate cellular immune responses. Examples

[0155] Example 1: Comparison of an RNAsa vaccine expressing different ILTV immunogenes

[0156] Six groups of twenty chickens (Gallus domesticus) were immunized with various combinations of sa RNA. The chickens were immunized and stimulated intramuscularly (IM) / intramuscularly, or via WingWeb (WW) / intramuscularly. A placebo group received no vaccine. After immunization, a virulent challenge dose (as described in [Fig. 7]) was administered intratracheally, and clinical signs of ILTV (LT) were observed.

[0157] Figure 7 describes the design and testing of an RNA vaccine. The vaccine combinations described in Figure 7 include between 0.1 and 5 pg of each gene of interest listed, for example for the first group "gB, gD, gE, gl".

[0158] Figure 7 also describes the route of administration for each combination of RNA encoding ILTV antigens. For example, the first group was vaccinated and received an IM / IM booster.

[0159] Figure 8 shows the percentage of protection against clinical signs and clinical scores of ILTV observed in vaccinated chickens and the mortality described in Example 1. Route: vaccination / boost. IM: intramuscular; WW: wingweb; SC: subcutaneous.

[0160] ILTV excretion by vaccinated chickens on oropharyngeal swabs was measured using qPCR equivalent to an infectious dose of 50% of the egg ILTV challenge virus; see [Fig.9]. DPC: days post-test.

[0161] Intramuscular vaccination and booster with 0.1–5 pg of each of the sRNAs encoding gB, gD, gE, and gl eliminated ILTV shedding 6 days after the challenge, and intramuscular vaccination and booster with 0.1–5 pg of each of the sRNAs encoding gD, gE, and gl eliminated ILTV shedding in 95% of chickens 6 days after The challenge. Wingweb vaccination with these same sRNAs prevented 50% of the chickens from excreting.

[0162] These results demonstrate the efficacy of the sRNA vaccines described herein in improving the clinical symptoms of LTI, reducing mortality due to LTI and attenuating or eliminating LTI shedding.

[0163] Example!: Comparison of an sRNA vaccine expressing different other ILTV immunogens

[0164] Seven groups of twenty chickens (Gallus domesticus) were immunized with sa RNA platforms encoding the ILTV antigens described in [Fig. 13]. The animals were vaccinated at six weeks of age and received a booster three weeks later, at nine weeks of age.

[0165] All animals underwent a challenge test by intratracheal administration of a virulent ILTV during week 12.

[0166] A Western blot showed that ILTV proteins encoded by RNAsa and recognized by anti-ILTV serum were stably expressed, see [Fig. 17].

[0167] Protection against clinical signs of LTI, severity of clinical signs of LTI and mortality for each group were observed and illustrated by [Fig. 15].

[0168] ILTV excretion three and six days post-challenge was measured and shown in [Fig. 17]. All birds vaccinated with placebo excreted ILTV on the third and sixth days post-challenge. However, 75 to 95% of vaccinated chickens sampled at 6 post-challenge days were ILTV-negative, with the exception of chickens that received the low dose of gD vaccine (gLVEE linker-gE) and chickens that received placebo and were exposed to ILTV.

[0169]

[0170] On the sixth day after the challenge, the IM / IM immunized and reinforced birds with a high dose of gLVEE 6K linker-gE + gD and gB-del were 95% negative with respect to shedding.

[0171] Birds immunized and reinforced by IM / IM with a high dose of gD + gE + gl or with a high dose of gLVEE 6K linker-gE + gD were 90% negative for ILTV excretion on the sixth day after the challenge and those immunized with a low dose of gLVEE 6K linker-gE + gD were 80% negative.

[0172] Immunized and stimulated IM / IM birds with a high dose of gLVEE 6K linker-gE + gD were 90% negative for ILTV shedding on day six after the challenge.

[0173] Birds immunized and reinforced IM / IM with gB-del alone were 75% negative for ILTV shedding.

[0174] Antibodies against ILTV antigens were measured using the BioChek ILT ELISA test which measures the amount of antibodies against ILTV in the serum of chickens. The results are presented in [Fig. 15]. After stimulation, four of the seven groups had anti-ILTV antibody levels above the threshold value, with high-dose IM / IM saRNAs encoding gD, gE and gl showing the highest titers.

[0175] Mortality in each group was determined. Mortality of 25% was observed in the placebo-vaccinated group and mortality of 5% in the group receiving a low dose of gLVEE-6K-linker-gE + gD. None of the birds in the other siRNA-immunized groups died, and all these groups showed at least 90% protection against clinical signs.

[0176] These results demonstrate the efficacy of the sa RNA vaccines described herein in improving the clinical symptoms of LTI, reducing mortality due to LTI and reducing LTI shedding.

[0177] Terminology. The terminology used in this document is solely for the purpose of describing particular embodiments and is not intended to limit the invention.

[0178] Unless expressly stated otherwise, the terms limit the meaning of these terms, unless specifically indicated.

[0179] Although certain aspects of this disclosure have been described in connection with the specific embodiments offered as examples, alternatives, modifications, and variations may be made to the examples. The description and specific examples, while indicating embodiments of the technology, are for illustrative purposes only and are not intended to limit the scope of the technology. Furthermore, the enumeration of several embodiments exhibiting the stated features is not intended to preclude other embodiments with additional features or other embodiments incorporating different combinations of the stated features.Specific examples are provided to illustrate how to manufacture and use the compositions and methods of this technology and, unless explicitly stated otherwise, are not intended to represent any given realizations of this technology that have, or have not, been made or tested.

[0180] In this document, the terms "preferred" and "preferred" refer to embodiments of the technology that offer certain advantages under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, mentioning one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the technology.

[0181] It should be noted that, as used in the specification and the attached claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. See Harari v. Lee, 656 F.3d 1331, 1341, (Fed. Cir. 2011); Baldwin Graphics Sys. v. Siebert, Inc. 512 F.3d 1338, 1342 (Fed. Cir. 2008)); KJC Corp. v. Kinetic Concepts, Inc. 223 F.3d 1351, 1356 (Fed. Cir. 2000).

[0182] In this document, the term "and / or" includes all combinations of one or more of the listed associated elements and may be abbreviated as " / ". A and / or B includes A, B and (A + B).

[0183] As used in the specification and claims, including in the examples, and unless expressly stated otherwise, all numbers may be read as if preceded by the word "substantially," "about," or "approximately," even if the term does not appear expressly. The expression "about" or "approximately" may be used when describing a quantity and / or position to indicate that the described value and / or position lies within a reasonable range of values ​​and / or positions. For example, a numeric value may have a value that is within + / - 0.1% of the stated value (or range of values), + / - 0.2% of the stated value (or range of values), or + / - 0.5% of the stated value (or range of values), + / - 1% of the stated value (or range of values), + / - 2% of the stated value (or range of values), + / - 5% of the stated value (or range of values), + / - 10% of the stated value (or range of values), + / - 15% of the stated value (or range of values), + / - 20% of the stated value (or range of values), etc. Any numeric range mentioned in this document is intended to include all sub-ranges and values ​​it encompasses.

[0184] Any numeric range mentioned in this document is intended to include all subranges and values ​​contained therein. Where a range of values ​​is provided, it is understood that every intermediate value between an upper and lower limit of the range and any other declared or intermediate value within that declared range is included in the disclosure. Where the stated range includes upper and lower limits, ranges excluding either of those limits are also included.

[0185] The disclosure of values ​​and ranges of values ​​for specific parameters (such as temperatures, molecular weights, percentage weights, etc.) is not exclusive of other values ​​and ranges of values ​​useful in this document. It is envisaged that two or more specific exemplified values ​​for a given parameter may define the endpoints of a range of values ​​that may be claimed for the parameter. For example, if parameter X is exemplified here to have the value A and also exemplified to have the value Z, it is envisaged that parameter X may have a range of values ​​from approximately A to approximately Z. Similarly, it is envisaged that the disclosure of two or more ranges of values ​​for a parameter (whether these ranges are nested, overlapping or distinct) encompasses all possible combinations of ranges for the value that could be claimed using the endpoints of the disclosed ranges. For example, if the parameter X is exemplified in this document as having values ​​between 1 and 10, it also describes subranges for the parameter X including 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, 9-10, to name just a few examples. An interval encompasses its endpoints as well as the values ​​located within an endpoint. For example, the interval 0-5 includes 0, >0, 1, 2, 3, 4, <5, and 5.

[0186] In this document, all composition percentages are expressed by weight of the total composition, unless otherwise specified. In this document, the term "include" and its variants are non-limiting, so that the enumeration of elements in a list does not exclude other similar elements that may also be useful in the materials, compositions, devices, and methods of this technology. Similarly, the terms "may" and "can" and their variants are intended not to be limiting, so that a statement that an embodiment may or can include certain elements or features does not exclude other embodiments of the present invention that do not contain those elements or features.

[0187] Although the terms "first" and "second" may be used here to describe various features / elements (including steps), these features / elements should not be limited by these terms unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another feature / element. Thus, a first feature / element examined below could be called a second feature / element, and similarly, a second feature / element examined below could be called a first feature / element, without departing from the teachings of the present invention.

[0188] It is also understood that when a feature or element is said to be "connected," "attached," or "coupled" to another feature or element, it may be directly connected, attached, or coupled to the other feature or element, or intermediate features or elements may be present. Conversely, when a feature or element is said to be "directly connected," "directly attached," or "directly coupled" to another feature or element, there are no intermediate features or elements present. Although described or illustrated with respect to one embodiment, the features and elements so described or illustrated may apply to other embodiments. Those with relevant expertise will also understand that references to a structure or feature arranged "next to" another feature may include parts that overlap or underlie the adjacent feature.

[0189] All publications and patent applications referred to in this specification are herein incorporated by reference in their entirety, to the same extent as if each publication or patent application were specifically and individually indicated as being incorporated by reference, in particular the disclosure appearing in the same sentence, paragraph, page or section of the specification in which the incorporation by reference appears.

[0190] The citation of references in this document does not constitute an acknowledgment of the prior existence of such references or their relevance to the patentability of the technology disclosed herein. Any discussion of the content of the cited references is intended solely to provide a general summary of the claims made by the authors of the references and does not constitute an admission of the accuracy of the content of those references.

[0191] ILTV nucleic acid and polypeptide accession numbers. The sequences of the precursor, transformed, and mature forms of the proteins named below, as well as their variants, modified or glycosylated residues of these proteins, and subcellular locations of these proteins are described and incorporated by reference to the database entries accessed by these accession numbers (last accessed September 1, 2022). The external and cytoplasmic domains are generally separated by the transmembrane domain.

[0192] The section below describes the accession numbers describing the nucleotide (e.g., RNA or DNA) or amino acid (protein) sequences of the ILTV polypeptides. However, the nucleotide and amino acid sequences described here are not limited to these sequences. In some cases, these sequences can be modified to produce variant sequences, such as sequences containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more deletions, substitutions or insertions of nucleotides or amino acid residues, or sequences exhibiting some degree of sequence identity with the sequences described by these accession numbers.Other modifications include glycosylation or other chemicals of amino acid residues, removal of glycosylation from a residue or its replacement with a non-glycosylated amino acid residue, or structural modifications, including multimerization or alternative folding of the protein.

[0193] Amino acid sequences can be deduced from the corresponding RNA or nucleic acid sequences using the genetic code; the sequences of The polynucleotides that encode the amino acid sequences described below can be deduced from the amino acid sequences using the genetic code. All accession numbers described below are incorporated by reference to the GENBANK database, last accessed on September 1, 2022. Information disclosed by GENBANK accession number entries that are not reproduced below is expressly incorporated by reference.

Claims

Demands

1. Self-amplifying RNA (saRNA) encoding at least one infectious laryngotracheitis virus (ILTV; Gallid alphaherpes virus 1, GaHV-1) polypeptide, a polypeptide variant of ILTV, or an immunogenic fragment or epitope thereof, characterized in that it comprises, in any order: - a first nucleic acid sequence comprising nsP1, nsP2, nsP3, and nsP4 encoding non-structural alphavirus proteins, and - a second nucleic acid sequence encoding at least one ILTV polypeptide comprising gB, gBdel, or gD, linked to gE by a nucleic acid sequence encoding at least one protein-binding site or at least one cleavage site for an endogenous or exogenous protease such as a 6k sequence, and said saRNA comprising a 5' cap, a UTR 5' upstream of the first nucleic acid sequence, a 26S promoter upstream of the second nucleic acid sequence, a 3' UTR and a poly A tail,said sRNA, when incorporated into a cell, allows the expression of at least one ILTV polypeptide, an ILTV polypeptide variant, or an immunogenic fragment or epitope of one of them.

2. sRNA according to claim 1, characterized in that the alphavirus is selected from the group consisting of Sindbis virus (SINV), Chickungunya virus (CHIKV), Semliki Forest virus (SFV), Ross River virus (RRV), Sagiyama virus (SAGV), Getah virus (GETV), Middleburg virus (MIDV), Bebaru virus (BEBV), O'nyong nyong virus (ONNV), Ndumu virus (NDUV), and Barmah Forest virus (BFV); or an alphavirus selected from the group consisting of Venezuelan equine encephalitis virus (VEEV), Western equine encephalitis virus (WEEV), and Eastern equine encephalitis virus (EEEV).

3. sRNA according to claim 1 or 2, characterized in that it comprises in the following order the 5' cap, the 5' UTR, nsP1, nsP2, nsP3, nsP4, the 26S promoter, the second nucleic acid sequence, the 3' UTR and the poly A tail, and optionally a terminator.

4. sRNA according to any one of claims 1 to 3, characterized in that it further comprises, after the 5' UTR, a polynucleotide encoding nspl, followed by a downstream loop (DLP), followed by a P2A autoprotease.

5. sRNA according to any one of claims 1 to 4, characterized in that the second nucleic acid sequence encodes at least one ILTV polypeptide comprising gB (AGS36506.1), gD (AAA98925.1) and gE (AGS36509.1) or a variant thereof, or an immunogenic fragment or epitope thereof.

6. sRNA according to any one of claims 1 to 5, characterized in that it further comprises at least one viral capsid activator or downstream loop ("DLP") which optionally is located immediately downstream of the 5' UTR or the 26S promoter.

7. sRNA according to any one of claims 1 to 6, characterized in that it further comprises a nucleic acid sequence encoding a genetically modified cross-reaction (CRM) material of diphtheria toxin, tetanus toxoid (T), meningococcal outer membrane protein complex (OMPC), diphtheria toxoid (D) or H. influenzae protein D (HiD), in which the subsequent nucleic acid sequence is optionally fused.

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