Self-amplifying RNA expressing one or more infectious laryngotracheitis virus (ILT) antigens

Abstract

An alphavirus-based saRNA platform that expresses one or more ILTV antigens. Vaccines incorporating the saRNA and methods for producing the saRNA.

Description

[0001] SELF-AMPLIFYING RNA EXPRESSING ONE OR MORE INFECTIOUS LARYNGOTRACHEITIS VIRUS HLT) ANTIGENS

[0002] Cross-reference to a related application.

[0003] This application claims priority to U.S. Provisional 63 / 508,091, NUCLEIC ACID CONSTRUCT COMPRISING SIGNAL PEPTIDE CLEAVAGE SITES EXPRESSING MULTIPLE ANTIGENS, filed June 14, 2023 which is incorporated by reference for all purposes.

[0004] BACKGROUND OF THE INVENTION

[0005] Field of the Invention. The present invention relates to the fields of immunology, molecular biology, and avian care and husbandry, specifically to self-amplifying RNA (saRNA) vaccines for infectious laryngotracheitis (ILT) which provide a safe and effective prophylactic and therapeutic immune responses against ILT and related comorbidities and which ameliorate disease symptoms.

[0006] Description of Related Art. Vaccination of commercial avian flocks is important to reduce mortality and morbidity caused by infectious agents, thus increasing profitability and meeting applicable regulatory requirements. Commercial chicken, turkey, pheasant, peafowl, and other poultry flocks are routinely vaccinated to protect them against environmental exposure to pathogens.

[0007] A prominent and widespread poultry disease is infectious laryngotracheitis (ILT), a highly contagious upper respiratory tract disease of chickens and other poultry. ILT is characterized by conjunctivitis, sinusitis, oculo-nasal discharge, respiratory distress, bloody mucus, swollen orbital sinuses, high morbidity, considerable mortality, and decreased egg production. Historically, ILT has been a problem of the layer industry, but in the past two decades the disease has become more prevalent in broilers production facilities.

[0008] The causative agent of ILT is Gallid herpesvirus 1 (GaHV-1) belonging to the genus Iltovirus, and subfamily Alphaherpe-svirinae within Herpesviridae family. This virus is also known as infectious laryngotracheitis virus or ILTV which invades the trigeminal ganglion or trachea where latency is established. ILTV can survive for days to months at 13-23 °C in tracheal exudates and chicken carcasses. ILTV is well established in highly dense poultry producing areas of the world due to its characteristic persistence. Tissues infected by ILTV and modes of transmission of ILT are shown in Fig. 1.

[0009] ILTV comprises a nucleocapsid surrounded by an irregular envelope; see Fig. 2. The envelope has several glycoproteins that protrude from the virus’s surface. These proteins represent the main virus antigens. These glycoproteins mediate 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 vims. VET. RES. 2007, 38:261-279. doi: 10.1051 / vetres:200657. Another glycoprotein, gG, is not incorporated into tire viral envelope but it is secreted from infected cells; Kongsuwan K.. Johnson M. A., and Sheppard M.. 1993. Identification of an infectious laryngotracheitis virus gene encoding cm immunogenic protein with a predicted. Mfr) of 32 kilodaltons. VIRUS RES 29: 125-140. doi: 10.1016 / 0168.

[0010] Historically, modified-live ILTV vaccines have been used in the field. However, their use presents problems due to inconvenient routes of administration and reversion of the attenuated modified virus to virulence. Reversion of an attenuated, modified virus strain to virulence risks infecting unvaccinated birds in a flock thus posing concerns about safety and biosecurity.

[0011] Nucleic acid vectored vaccines that express isolated ILTV antigens like gB or UL-32 maybe a safer alternative to modified-live vaccines as they do not contain live virus. However, subunit vectored vaccines have suffered from lack of construct stability and insufficient expression of multiple protective viral antigens. For example, a vectored vaccine generally has a maximum payload of target genes limiting the number or quantities of protective antigens / imniunogens it produces. An amount of target DNA over the maximum for the vector will not remain in the vector and the lack of vector stability can prevent administrative approval of a vectored vaccine.

[0012] Taking into account the limitations described above and other technical problems, the inventors explored whether self-amplifying RNA vaccines could provide superior safety and efficacy compared to traditional modified-live vaccines and vectored vaccines.

[0013] The inventors sought to engineer non-infectious saRNA vaccine which once administered could not spread within the subject or infect other subjects with ILTV. Such a saRNA 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.

[0014] The inventors also sought to engineer a saRNA vaccine that was stable and could express sufficient quantities of ILTV antigens in a form that produces protective humoral or cellular immune responses, thus overcoming problems associated with conventional vectored vaccines. They also considered ways to deliver saRNA, which typically is much larger than non-self replicating nucleic acid vaccines.

[0015] Other desirable features for a saRNA vaccine were considered by the inventors including how to design and administer a saRNA vaccine that replicates within cells at the site of administration but generally activates the immune system, for example, by activating the innate immune system, by not suppressing immune responses, and by recruiting immune cells to the site of saRNA administration to produce a broader or more robust cellular or humoral response to ILTV antigens than other vaccines.

[0016] The foregoing paragraphs have been provided by way of general introduction and are not intended to limit the scope of the following claims. The described embodiments, together with further advantages, wall be best understood by reference to the following detailed description taken in conjunction with the accompanying drawings.

[0017] BRIEF SUMMARY OF THE TECHNOLOGY

[0018] This section provides a brief, non-limited overview of some aspects of the technology disclosed herein.

[0019] The saRNA constructs. One aspect of this technology is a self-replicating RNA or saRNA that encodes and expresses ILTV antigens / immunogens / epitopes, ILTV antigen / immunogen epitope variants, or fragments, including epitopes, thereof (collectively ‘ILTV antigens or immunogens”). Advantageously, the saRNA is based on, or derived from, a positive strand vims platform which retain sequences encoding RNA replication proteins, but which are deleted for sequences encoding 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 alphavirus and expresses alpha virus non-structural proteins nspl-nsp4 which are responsible for RNA replication and sequences encoding genes of interest, such as genes encoding ILTV antigens or immunogens, downstream of the nspl-nsp4 sequences generally in positions occupied by alphavirus structural genes; see for instance, Fig. 5.

[0020] In some embodiments, the saRNA comprises additional elements which enhance the expression of ILTV antigens or epitopes or which traffic ILIA7antigens once expressed. Such additional elements may encode immunogenic carrier proteins, immunomodulators that enhance expression of TLTV antigens / immunogens, interferon inhibitors or RNA encoding such an inhibitor, or other inhibitors of innate immune responses. It may encode signal sequences, leader peptides, ER N-terminal signal sequences, or other elements that traffic expressed ILTV antigens or epitopes to various parts of a cell such as the ER, Golgi, membrane, or outside of a cell: see trafficking sequences described by hypertext transfer protocol secure: / / alg.manifoldapp.org / read / fundamentals-of-cell-biology / section / 92527ecb-306c-4002- a41 l-e3c5d5da5e6a (last accessed September 1, 2022, incorporated by reference).

[0021] Another aspect of this technology is a saRNA platform that expresses two, three, four or more ILTV antigen / immunogens as components of a polyprotein or as separate polypeptides. Alternatively, separate ILTV antigens / immunogens may be produced, for example, by interposing 6K or 6K-like sequences between sequences encoding different ILTV antigens / immunogens, thus producing separate ILTV antigen / immunogen polypeptides instead of a polyprotein. One example of a 6K protein is that of the 6K Semliki Forest Virus.

[0022] In some embodiments, the saRNA encodes at least two, three or four ILTV antigens which are expressed as a polyprotein that are processed to form T and B cell epitopes presentable by MHC class 1 or class 2 molecules. For example, the platform be designed not to contain cleavage sites between sequences encoding the at least three structural antigens or it may be designed to contain translation skipping sequences between the at least three structural genes and produce proteins which remain in the cytoplasm.

[0023] Compositions. A related aspect of this technology is a composition comprising the selfreplicating RNA and a pharmaceutically acceptable carrier. In one embodiment the composition comprises saRNA and Lipid InOrganic Nanoparticles (LION).

[0024] A composition in addition to the saRNA disclosed herein may comprise other active ingredients such as, protein-based vaccine components, nucleic-based vaccine components, or immunological antigens or immunogens of other avian diseases. Thus, a composition containing saRNA encoding ILTV antigens may further comprise other components forming a multivalent vaccine against ILTV and other avian viruses or pathogens. In some embodiments a composition will comprise saRNA encoding one or more ILTV antigens or immunogens, and 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 immunized subject against ILTV. Treatment. Another aspect of this technology is a method for preventing or treating ILT, its symptoms, comorbidities, or sequelae by administering a saRNA platform as disclosed herein which encodes and expresses one or more ILTV antigens, immunogens, or epitopes. Administration of the vaccine containing the saRNA platform induces innate, humoral, and / or cellular immune responses to the encoded ILTV antigens including inducing neutralizing antibodies or antigen-specific T cells that recognize ILTV infected cells. In one convenient embodiment, the saRNA or a composition containing it is administered intramuscularly or subcutaneously.

[0025] Method of making saRNA. Other aspects of this technology involve methods for constructing DNA vectors encoding the saRNA disclosed herein. A DNA vector construct may be produced synthetically and saRNA generated from it, for example, by in vitro transcription of an ILT antigen- or immunogen coding sequence and non-coding alphavirus sequences or regulatory sequences as a synthetic RNA transcript which can be further formulated for delivery. Methods for synthesis of saRNA from a DNA construct template generation inlcude in vitro RNA transcription, RNA capping, poly(a) tailing, and RNA purification are known, commercially available, and incorporated by reference to hypertext transfer protocol secure.7 / www.neb.com / products / rna-reagents / rna-synthesis / ma-synthesis (last accessed October 21, 2022).

[0026] Various techniques for manufacturing saRNA or other nucleic acid constructs are known. Preferably, the saRNA is produced by in vitro transcription and / or amplification. For example, a linearized DNA template for the saRNA may be transcribed into mRNA using a RNA polymerase, such as bacteriophage T7 or SP6 RNA polymerase. RNA may be purified chromatographically, filtration, silica gel matrix or other methods to remove impurities like dsRNA and protein. Purified mRNA may be protected by association with cationic nanoparticles, polymers, peptides or encapsulated in lipid nanoparticles by solvent injection or microfluidic mixing to protect mRNA from degradation or to facilitate uptake by target cells. In vitro transcription is a preferred method.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028] A more complete appreciation of the disclosure and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings below. Fig. 1 shows transmission patterns of TLTV. Further description of TLTV transmission is incorporated by reference to Gowthamen, et al., VET. QUART. 2020, 40(1), 140-161.

[0029] Fig. 2 describes the physical and genetic structure of an ILTV virion. Further description of ILTV physical and genetic structure is incorporated by reference to Gowthamen, et al., Infectious 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 virus, VET. RES., 2007, 38, 261-279.

[0030] Fig. 3 describes some identified gene products of ILTV including gB, gC, gG, gH, gj, and gM. The accession numbers at the bottom of this figure, which further describe these gene products, are incorporated by reference. Further description of these gene products is incorporated by reference to Fuchs, et al., supra. One or more of these genes may be incorporated into constructs as disclosed herein.

[0031] Fig. 4 illustrates interaction of self-replicating RNA (self-amplifying RNA, saRNA) within a host cell. Further description of self-replicating RNA and its interactions with host cells is incorporated by reference to Aliahmad, et al., Next generation self-replicating RNA vectors for vaccines and immunotherapies. CANCER GENE THER. 2022. Hypertext transfer protocol secure.7 / doi.org / 10.1038 / s41417-022-00435-8. One or more of such interactions may be involved in the uptake and expression of ILTV genes in host cells.

[0032] Fig. 5 describes one example of a DNA construct encoding an saRNA containing ILTV genes of interest (gX). This construct comprises a 5’ nsPl and DLP sequence a P2A autoprotease as well as sequences encoding non-structural genes nsPl-nsP4 and gX.

[0033] Fig. 6 describes lengths of several different ILTV saRNA inserts that are incorporated into constructs. The last construct gE-VEE 6K-gI may comprise either a 6K sequence from VEE or from Semliki forest virus; see Example 1.

[0034] Fig. 7 describes saRNA vaccine design and testing as described in Example 1. Vaccine includes between 0.1 pg to 7 pg of each recited gene of interest gB, gD, gE. gl of ILTV for intramuscular (IM) route.

[0035] Fig. 8A discloses protection against LTV based upon the presence or absence of ILT clinical signs, including mortality, observed in vaccinated chickens as described in Example 1.

[0036] Fig 8B discloses an ILT clinical sign scoring system specific for the average of respiratory severity of clinical signs. Fig. 9A describes ILTV shedding to oropharyngeal swabs of vaccinated chickens as tested using qPCR equivalent to 50% egg infectious dose of the ILTV challenge virus as described in Example 1. DPC: days post-challenge.

[0037] Fig 9B describes a table including the percentage of chickens from each treatment group negative for ILTV detected using qPCR at 3 or 6 DPC. DPC: days post-challenge. Intramuscular vaccination and boost with between 0.1 pg to 7 pg each of saRNAs encoding gB, gD, gE and gl eliminated ILTV shedding 6 days post-challenge and intramuscular vaccination and boost with between 0.1 pg to 7 pgeach of saRNAs encoding gD, gE and gl eliminated ILTV shedding in 95% of the chickens 6 days post challenge. Wingweb vaccination with these same saRNAs stopped 50% of chickens from shedding.

[0038] The doses described in the description of the figures above or used in the Examples are non-limiting. Thus, in some embodiments, the amount of saRNA administered ranges 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 intermediate subrange or value.

[0039] Fig. 10 describes results of BioChek ELISA after vaccine priming (left) and boost (right). Chickens immunized intramuscularly with saRNAs encoding gB, gD, gE and gl, or with saRNAs encoding gD, gE and gl produced high levels of antibodies compared to groups immunized with other antigens or by other modes of immunization. The 0.5 thresholds shows serum samples positive for ILT specific antibodies according to the BioChek ELISA kit instructions. BioChek ELISA does not detect gB antibodies. IDVet ELISA gB kit recognizes gB specific antibodies and detection of seroconversion to gB vaccination.

[0040] Fig. 11. saRNA vaccine design and testing as described in Example 2. Vaccine includes the indicated dosages of saRNA encoding various ILTV’ antigens. New saRNA constructs were evaluated including saRNA comprising gB-del and saRNA comprising gE-VEE 6K-gI were tested. High and low doses of some saRNA constructs were compared.

[0041] Fig. 12. Western blot probed with pooled serum collected from chickens vaccinated with gD, gE, and gl from Example to detect gl and gE. Glycoprotein I was linked to glycoprotein E using a Venezuelan equine encephalitis virus 6K ( V6K ) linker or a Semlike Forest virus 6K (S6K) linker. The V6K shows a band 110-I20kDA specific for gl and gE linked together. S6K larger band is missing due to a point mutation.

[0042] Fig. 13 A. ILT clinical signs protection and mortality as described in Example 2. Chickens were vaccinated with saRNAs expressing in group 1 gD, gE and gl high dose; in Group 2 gD, gE and gl low dose, Group 3 gB-deleted high dose; Group 4 gl-VEE linker-gE, gD and gB-deleted high dose; Group 5 gl-VEE linker-gE, and gD high dose; group 6 gl-VEE linker-gE and gD low dose; and group 7 with a placebo. Clinical signs of ILTV infection were recorded along with clinical signs of protection. Mortality rates were recorded.

[0043] Fig 13B discloses the ILT clinical sign scoring system specific for the average of respiratory severity of clinical signs for the chickens enrolled in treatment groups described in Fig. 13 A. Higher clinical score shows a more severe respiratory' clinical sign associated with ILT disease.

[0044] Figs. 14. ILTV shedding on oropharyngeal swabs and qPCR detection of ILTV. The graphs show ILTV shedding from individual chickens and on average indicated by each black bar. The percent of immunized chickens not shedding virus detected using qPCR of oropharyngeal swab samples collected on 3 and 6 days post challenge (DPC) are reported in the table.

[0045] Fig. 15. Biochek ILTV ELISA. Antibody levels after prime and after boost were determined by ELISA. The 0.5 threshold shows serum samples positive for ILT specific antibodies according to the BioChek ELISA kit instructions. BioChek ELISA does not detect gB antibodies. IDVet ELISA gB kit recognizes gB specific antibodies and detection of seroconversion to gB vaccination.

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] A more detailed description of the technology disclosed herein and essential and optional elements of the saRNA and methods of its use and manufacture is provided below.

[0048] Self-amplifying RNA. Self-amplifying (saRNA), self-rep Heating RNA (srRNA), replicon, or SR V (synthetic RNA vaccine) as a vaccine and therapeutic modality has been increasingly used for the in vivo production of proteins. saRNAs are derived from positive-strand RNA viruses where the structural proteins have been removed and replaced with heterologous genes of interest; see P. Aliahmad, et al., Next generation self-replicating RNA vectors for vaccines and imiminotherapies. 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; Liljestrdm 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 which is incorporated by reference. Among its other features, the technology disclosed herein involves saRNA that encodes one or more ILTV antigens or immunogens.

[0049] Gallid alphaherpesvirus 1 (GaHV-1; ILTV). which is frequently referred to as infectious laryngotracheitis virus (ILTV), belongs to the type species of the genus Iltovirus and is the causative agent of a highly contagious, acute, respiratory tract disease of domestic fowl termed ILT. It is a species of virus in the order Herpesvirales, family Herpesviridae, subfamily Alphaherpesvirinae, and genus Iltovirus', VIRUS TAXONOMY: 2020 RELEASE. INTERNATIONAL COMMITTEE ON TAXONOMY OF VIRUSES (ILTV). March 2021. Retrieved 10 May 2021. Originally recognized in chickens in the United States in 1926, this virus causes avian infectious laryngotracheitis (ILT), a potentially fatal, economically deleterious disease, widely recognized as one of the most contagious diseases in the poultry industry; Fenner, Frank J., et al. (1993). VETERINARY VIROLOGY (2nd ed.). Academic Press, Inc. ISBN 0-12-253056-X. As used herein, this term encompasses all known natural and engineered strains of ILTV as well as variants or mutants thereof. Among its other features, the technology disclosed herein involves saRNA that encodes one or more I LTV antigens or immunogens which can be used to vaccinate or protect avians against an infection by GaHV-1 or ILTV and from ILT.

[0050] Infectious laryngotracheitis (ILT) is a disease caused by Gallid alphaherpesvirus 1 (GaHV-1; ILTV). GaHV-1 is shed in respiratory secretions and transmitted by droplet inhalation or via fomites. A typical host is Gallus domesticus (domestic chicken). A previously unexposed flock will develop cases for two to eight weeks following 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 diphtheritic membrane may form in the trachea, causing obstruction. There may be problems in egg laying and the production of abnormal or thin- shelled eggs. Mortality is typically less than 15 percent. Histopathology, PCR, ELISA, immunoflu orescent staining, and viral isolation are all possible methods of diagnosis. A vaccine is available, but it is susceptible to undesired recombination and it does not prevent latent infections. Live attenuated ILTV vaccines are highly effective against a spreading ILT infection but reversion to virulence remains problematic. It can be used during an outbreak to decrease morbidity and deaths. A confirmed case will usually result in the establishment of a quarantine zone around the farm. Inside this quarantine zone, poultry workers will avoid poultry farms to prevent the spread of the virus. Biosecurity measures including quarantine, isolation, and disinfection are very important in controlling the spread of an outbreak.

[0051] ILTV hosts and vaccination subjects. Include chickens (e.g. Gallus domesticus), turkeys (Meleagris gallopavo, Meleagris gallopavo domesticus}, guinea fowl, pheasants, peafowls (genera Pavo and Afropavo} and other susceptible avians or avian carriers. Ducks and some other avians may be carriers. A vaccine subject may be an avian embryo, chick or adult avian. In chickens breeders, layers and broilers can be affected. The saRNA as disclosed herein may be used to vaccinate or treat ILTV hosts, reduce mortality, ameliorate ILT symptoms, and reduce transmission of ILTV.

[0052] ILTV antigens / immunogens include gB, gBdel (gB is deleted), gC, gD, gE, gG, gH, gl, gj, gK, gL and / or gM though other ILTV antigens may also be incorporated into the saRNA platforms disclosed herein and used to immunize avians. The inventors considered that gB, gD, gl and gE were promising candidates for inducing immune responses against ILTV, however, when tested in other types of ILTV vaccines they were found to lack stability. As disclosed herein, efforts were made to induce protective immune responses to these antigens using a saRNA platform.

[0053] The saRNA disclosed herein may be formulated for administration to domesticated avians, galliforms, chickens and other fowl-type birds such as turkeys and peacock; or immunize carriers of ILTV including those described by, and incorporated by reference to, hypertext transfer protocol secure: / 7en.wikipedia.org / wiki / List_of_Galliformes (last accessed July 27, 2022, attached as part of this disclosure ). Vaccines formulated for administration to a particular avian species, strain, or breed typically encode ILTV antigens for viruses capable of infecting that species or strain. In some embodiments the ILTV antigens or immunogens disclosed herein are derived from ILTV strains VR2332, HB-13.9, JXwnO6, CHsxl401, or HAGE2018. Other known ILTV immunogens, antigens or gene products include those described in Fig. 3 and by Fuchs, et al., supra.

[0054] As mentioned above, the antigens of ILTV include glycoproteins such as gB, gC, gD, gE, gG, gH, gl, gj, gK, gL and gM, and are reported to play a crucial role in virus entry and replication; see also Poulsen, D.J., and C.L. 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, T.C., 2013. Identification and functional analysis of membrane proteins gl), gE, gl, and pUS9 of infectious laryngotracheitis virus. AVIAN DISEASES, 57:416-426; andBendezu, J., Ruiz S.M., Montesinos, R., Guevara, R.C., Rojas-Neyra, A., Pauyac- Antezana, K., Fernandez-Diaz, M., 2019. Glycoprotein G production profile during infectious laryngotrachetis (ILTV) infection, PEGS ONE 14(8):e0219475.

[0055] Glycoprotein B is involved in ILTV virus penetration into the host cell and immunogenic for both 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).

[0056] Glycoprotein D is responsible for ILTV binding to receptors for vims entry into the host cell. Further description of gD, gE, gl and other ILTV antigens is incorporated by reference to: Pavlova, S., Veits, J., Mettenleiter, T.C., 2013. Identification and functional analysis of 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 laryngotrachetisi (ILTV) infection, PLOS ONE 14(8):e0219475.

[0057] Glycoprotein E (gE) and glycoprotein I (gl) are essential for ILTV cell-to-cell spread that impacts virulence, therefore, polynucleotides encoding gE and gl specific antibodies and cellular responses may be incorporated into the saRNA as disclosed herein to reduce viral load.

[0058] 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 subfamily C, CC and CXC, and hence prevent the interaction between chemokines and their receptors. It also blocks binding of chemokine to glycosaminoglycans, which is necessary for in vivo chemokine activity'. The vCKBP of ILTV (gG), during early stages of infection, induces innate immune responses by recruiting particular subsets of immune cells. A saRNA construct as disclosed herein may be designed to express a non-functional gG protein that has reduced ability' to bind cytokines or inhibit innate immune responses.

[0059] Envelope glycoproteins mainly gC, rather than gB, gD, gH, and gL, are considered to mediate ILTV attachment with host cell receptors and help in the fusion of the viral envelope to the host cell membrane. A saRNA construct as described herein may be designed to induce immune responses against gC.

[0060] Immune responses to Glycoprotein C may help in “Differentiating Infected from Vaccinated Animals” or DIVA. RNA encoding gC may be incorporated into saRNA (containing other ILTV antigens such as gB, gD, gl, and gE. The saRNA as disclosed herein mav also omit RNA encoding gC. Quantitative PCR may be used to detect copies of ILTV genes on tracheal or ora / pharyngeal swabs, such genes encoding gC or gB, gD, gl, and gE. Such methods are incorporated by reference to Callison, et ah, Development and validation of a real-time Taqman PCR assay for the detection and quantitation of infectious laryngotracheitis virus in poultry, J. VIROL. METHODS, 2007, 139(1), 31-38. “Distinguishing infected from unvaccinated animals” or DIVA may be done by measuring immune responses to gC. If gC is omitted from a saRNA based vaccine, then the qPCR test may be used to differentiate between vaccinated (no gC positive samples) and ILTV infected chickens would have gC positive samples.

[0061] In some embodiments, a saRNA may express a full-length ILTV antigen such as would appear during a natural infection. In other embodiments a fragment of an ILTV antigen may be expressed. One example of such a truncated polypeptide antigen is gBdel which is a shorter fragment of gB. Cloning full-length gB has been difficult but the inventors found that a truncated version of gB had increased stability in a vectored system. In gB-del the transmembrane domain is deleted so that the protein is secreted rather than resident in the cell membrane.

[0062] Platform. This term encompasses a framework into which polynucleotides encoding exogenous antigens may be inserted. It also encompasses a construct or saRNA comprising the framework and sequences encoding exogenous antigens such as saRNA that expresses one or more than one ILTV antigens / immunogens.

[0063] A platform can include viral vector platforms such as a harmless viruses engineered to encode a polynucleotide encoding one or more ILTV antigens of interest and carry the polynucleotide into a cell, such as a porcine cell. Such viral vectors may be replicating or nonreplicating and cause a target cell to produce an ILTV antigen from the polynucleotide carried by the viral vector.

[0064] This term also covers nucleic acid platforms including messenger RNA that encodes 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 cause the target cell to produce an ILTV antigen from the polynucleotide encoding the ILTV antigen in the nucleic acid platform. Typically, such an RNA platfomi is non-infectious as it can lack the structural protein sequences required to produce an infectious virus. This term also encompasses DNA platforms that likewise carry DNA encoding a saRNA encoding one or more ILTV antigens and other nucleic acid elements to be delivered to a target cell where the express ILTV antigens. Such DNA platforms may comprise a plasmid or replicon carrying the polynucleotide(s) encoding saRNA capable of expressing the ILTV antigen(s) of interest.

[0065] Alphavirus. Advantageously, an alphavirus platform is used to express ILTV antigens. The Alphavirus genus belongs to the Togaviridae family and contains 28 virus species (Griffin, ENCYLOPEDIA OF VIROLOGY, 2008, 101-107). Alphavirus is a positive-sense RNA virus that is taxonomical distinct from the ILTV which is a double-stranded DNA virus. Other features of alphavirus are described by and incorporated by reference to 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 - 9; :https: / / doi.org / 10.1017 / S 1466252312000011; 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, each incorporated by reference.

[0066] Alphavirus platform. Alphavirus vectors have demonstrated high levels of transient heterologous gene expression both in vitro and in vivo and, therefore, possess attractive features for vaccine development. The most commonly used delivery vectors are based on three singlestranded encapsulated alphaviruses, namely Semliki Forest virus, Sindbis virus and Venezuelan equine encephalitis virus. An alphavirus platform that is advantageously used in many embodiments of technology disclosed herein encodes ILTV antigens in place of alphavirus structural proteins. However, it contains the alphavirus replication sequences such as nspl-nsp4 which can server to amplify RNA encoding the ILTV antigens. In some embodiments, the alphavirus vectors are based on Semliki Forest virus, Sindbis virus, or Venezuelan equine encephalitis virus platforms. Alphavirus platforms are described by Lundstrom, K., Alphavints- based antigen preparation. METHOEJS MOI.. BIOL 2021 , 2183-81; Lundstrum, K., Alphavirusbased 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.

[0067] In some embodiments, an RNA platform or saRNA as disclosed herein may have its nucleotides modified to stabilize the platform, increase its half-life, increase its cellular uptake or expression, or reduce unwanted immune responses, for example, uracil in the platform RNA sequence may be replaced 1 -methyl-3 ’-pseudouridylyl (canonical name is Nl- Methylpseudouridine) to reduce host immune responses to the platform once it is administered. The platform may comprise modified RNA in which some nucleosides are replaced by other naturally modified nucleosides or by synthetic nucleoside analogues.

[0068] The compositions and methods disclosed herein may comprise two or more of said platforms, wherein each platform encodes ILTV antigens from a different ILTV genotype or strain. Nine genotypes (I-IX) of ILTVs have been recognized using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method with three viral alleles (gB, gM and UL47 / gG). Further description of ILTV genotypes and genotyping is described by, and incorporated by reference to, Spatz, S. J., et al., MinlON sequencing to genotype US strains of infectious laryngotracheitis virus , AVIAN PATHOLOGY, 2019, 48(3), 255.

[0069] Non-alphavirus platforms. Non-limiting examples of modified non-alphavirus RNA replicons or platforms include modified RNA replicons of virus species belonging to Togaviridae family, Flaviviridae family, Orthomyxoviridae family, Rhabdoviridae family, or Paramyxoviridae family. Accordingly, in some embodiments, the modified non-alphavirus RNA replicon includes a modified RNA replicon of a negative- strand RNA virus. Suitable negative-strand RNA virus species include but are not limited to viral species of the families Orthomyxoviridae, Rhabdoviridae, and Paramyxoviridae. In some embodiments, the modified non-alphavirus RNA replicon includes a modified RNA replicon of a positive-strand virus species belonging to the Togaviridae family or Flaviviridae family. In some embodiments, the modified non-alphavirus RNA replicon includes a modified RNA replicon of a positive-strand virus species belonging to the Arterivirus genus of the Arteriviridae family. Suitable arterivirus species include, but are not limited to, species of Equine arteritis virus (EAV), Porcine respiratory and reproductive syndrome virus (PRRS virus), Lactate dehydrogenase elevating virus (LDV), Simian hemorrhagic fever virus (SHFV), and wobbly possum disease virus (WPDV). Other platforms are described by and incorporated by reference to Kamrud, et al.. U.S. 2018 / 0171340 Al which also describes capsid enhancers and stem loop structures.

[0070] Expression cassettes. In some embodiments, the sequence encoding saRNA further includes one or more expression cassettes, wherein each of the expression cassettes comprises a promoter operably linked to a sequence coding for a gene of interest (GOI). In some embodiments, the saRNA comprises at least one, two, three, four, five, or six expression cassettes. In some embodiments, at least one of the one or more expression cassettes is operably linked downstream of the second nucleic acid sequence encoding the at least one nonstructural viral protein or a portion thereof. In some embodiments, at least one of the one or more expression cassettes further comprises a third nucleic acid sequence encoding one or more structural elements of a viral capsid enhancer, wherein the third nucleic acid sequence is operably linked upstream to the coding sequence for the GOI. In some embodiments, the saRNA further includes a coding sequence for an autoprotease peptide operably linked downstream to the third nucleic acid sequence and upstream to the coding sequence for the GOI.

[0071] A platform may comprise or form part of a deliver)' systems containing naked DNA in buffer with or without adjuvant, DNA coupled to nanoparticles and / or formulated into adjuvant containing compounds or inserted into live bacterial or viral vectors such as Adenovirus, adeno- associated virus (AAV), alphavirus, poxvirus and herpesvirus.

[0072] Other types of platforms include the nucleic acid components of a whole virus, a live attenuated whole virus, inactivated virus (c.g. by chemical or radiation), viral vectors and in other nucleic acid (RNA or DNA) based platform 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 including by the saRNA disclosed herein. Platforms encoding more than one ILTV antigens are designed to permit more natural processing and association of ILTV antigens with each other, for example, as a complex between different viral antigens which forms virus-neutralizing epitopes not present on the individual proteins making up a complex.

[0073] 5’ Cap. The five-prime cap (5’ cap) is a specially altered nucleotide on the 5' end of some primary' transcripts such as precursor messenger RNA. This process, known as mRNA capping, is highly regulated and vital in the creation of stable and mature messenger RNA. Many preferred platforms as disclosed herein contain a 5’ cap. 5’ caps are well known. The saRNA platform as disclosed herein preferably has a 5’ cap.

[0074] 5 ’ UTR. The 5' untranslated region (also known as 5' UTR, leader sequence, transcript leader, or leader RNA) is the region of a messenger RNA (mRNA) that is directly upstream from the initiation codon. This region is important for the regulation of translation of a transcript by differing mechanisms. A platform as disclosed herein usually contains a 5 ’UTR often encoding a leader peptide. In some embodiments, the 5’ UTR, promoters, 3’ U TR or poly- A sequences are derived from alphavirus or from ILl'V. The saRNA platform as disclosed herein preferably comprises a 5’ UTR.

[0075] Alphavirus 26S promoter. The 26S promoter, which is located between the two ORFs on the alphavirus negative-sense RNA, is recognized by the alphavirus non-structural proteins for transcription of a sub-genomic mRNA from which structural proteins, such as exogenous ILTV antigens, are translated. Various alphavirus 26S promoter sequences or their equivalents may be used in the platforms disclosed herein. Each 26S promoter will generate subgenomic mRNAs. A number of subgenomic mRNAs produced from a 26S promoter may be about 10X more numerous than those produced from the full-length replicon RNA. 26S promoters are known, described by and incorporated by reference to Vander Veen, et ah. Alphavirus replicon vaccines, Cambridge University Press, 2012. A saRNA may comprise a single 26S promoter upstream of the at least one polynucleotide encoding ILTV antigen. A 26S alphavirus promoter can transcribe subgenomic mRNA encoding ILTV antigens and used to transcribe multiple polynucleotides encoding ILTV antigens. In other embodiments, 26S promoters may be paired upstream of polynucleotides encoding ILTV antigens. The saRNA disclosed herein preferably comprises a 26S promoter or a functionally equivalent promoter.

[0076] 6K or 6K~like protein. Alphavirus 6K proteins are small (58-61 amino acids), hydrophobic, and associate with membranes. Cleavage of 6K protein residues occurs via cellular proteases to generate structural proteins. A linker sequence encoding a 6K protein can be interposed between adjacent sequences coding for ILTV antigens. Cleavage of the translated 6K linker sequences produces discrete ILTV antigens. A 6K linker sequence imposed between a first and second gene of interest can direct a second gene of interest to the ER / Golgi compartments. In some embodiments, such as those comprising saRNA encoding two or more ILTV antigens, the saRNA will comprise a polynucleotide sequence encoding a 6K linker.

[0077] Ribosome stalling or skipping refers to an alternate mechanism of translation in which a specific peptide prevents the ribosome from covalently linking a new inserted amino acid, and let it continue translation. This results in apparent co-translational cleavage of die polyprotein. This process is induced by a "2A-like”, or CHYSEL (cis-acting hydrolase element) sequence which comprises a non-conserved sequence of amino-acids with a strong alpha-helical propensity followed by the consensus sequence -D(V / I)ExNPCi 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 2 A peptides that typically are 18-22 amino acid residues in length and which can induce ribosomal skipping during translation of a protein. Peptides in this family include P2A, E2.A, F2A, and T2A. Further description of this process as well as picornaviridae, iflaviradae, tetraviridae, dicistorviridae and reovriridat apparent cleavage sequences and their corresponding UniProt numbers is found at, 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 saRNA encoding two or more ILTV antigens, the saRNA will comprise a polynucleotide sequence that causes ribosome stalling or skipping.

[0078] Furin cleavage site. Furin is a protease that is ubiquitous in mammalian metabolism. Furin cleavage sites occur naturally in many viruses. Polynucleotides encoding other known furin cleavage sites may be inserted between ILTV antigen coding sequences in the platforms disclosed herein. Once a polyprotein is expressed, furin present in the host cell can cleave at these sites producing discrete ILTV antigens. In some embodiments, such as those comprising saRNA encoding two or more ILTV antigens, the saRNA will comprise a polynucleotide sequence encoding a 6K linker.

[0079] 3’ UTR. The three prime untranslated region (3'-UTR) is the section of messenger RNA (mRNA) that immediately follows the translation termination codon. The 3 '-UTR often contains regulatory regions that post-transcriptionally influence gene expression. A platform as disclosed herein usually contains a 3 'UTR. A 3’ UTR or 5’ UTR may be derived from ILTV, from alpha virus or from other sources. An alphavirus replicon, such as a VEE replicon, typically needs to have an alphavirus 5’ UTR and a 3’ UTR in order to replicate as these sequences are promoters for production of both + and - strand RNAs driven by the nsP 1 -4 complex. In many embodiments, a saRNA as disclosed herein will contain a 3’ UTR or poly- A sequence.

[0080] Pofy-A. Polyadenylation is the addition of a poly(A) tail to an RNA transcript, typically a 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 aids in transcription termination, export of the mRN A from the nucleus, and translation. The poly-A may be endogenous to the virus from which the platform is derived, such as endogenous to alphavirus or in other embodiments, may be exogenous. A poIy(A) tail is important for the nuclear export, translation and stability of mRNA. Polyadenylation sites for many alphaviruses are known.

[0081] The term "antigen” as used herein and as commonly used in the field of immunology refers to an "antibody generating” molecule or a molecule that induces an antigen-specific T cells, i.e. a substance, which can elicit an adaptive immune response. An antigen is thus a molecule binding to an antigen-specific receptor, either a T-cell or a B-cell receptor. An antigen is usually a polypeptide, but it can also be a polysaccharide or a lipid, possibly combined with a protein or polysaccharide carrier molecule. For the purpose of the various aspects and embodiments of the present invention, the antigen is a polypeptide, i.e. an amino acid sequence. In the case of binding to a T-cell receptor, the antigen is presented to the respective T-cell receptor via an antigen- presenting cell as an antigenic peptide bound to a histocompatibility molecule on the surface of the antigen presenting cell, wherein the antigenic peptide has been processed in advance by the antigen presenting cell. Antigen presentation by professional antigen-presenting cells (APC) is the first step towards the initiation of an adaptive immune response carried out by naive T lymphocytes. Thus, an "antigen" as used herein refers to a molecule, such as a protein or a polypeptide, comprising one or more epitopes that will stimulate a host's immune system to make a humoral and / or cellular antigen-specific response.

[0082] An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The epitopes of protein antigens are divided into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope. Conformational and linear epitopes interact with the paratope based on the 3-D conformation adopted by the epitope, which is determined by the surface features of the involved epitope residues and the shape or tertiary structure of other segments of the antigen. A conformational epitope is formed by the 3-D conformation adopted by the interaction of discontinuous amino acid residues. In contrast, a linear epitope is formed by the 3-D 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, whereas 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, whereas MHC class II molecules present longer peptides, 13-17 amino acids in length, and non-classical MHC molecules also present non-peptidic epitopes such as glycolipids.

[0083] An epitope that induces vints-neutralizlng response may induce antibodies that bind to ILTV in a manner that inhibits, reduces, or blocks infection, or progression of infection. A neutralizing antibody or antigen-binding molecule may block interactions with the receptor or may bind to a viral capsid in a manner that inhibits uncoating of the genome. The term "neutralizing antibodies” or "neutralizing antigen-binding molecules" also includes antibodies or antigenbinding molecules that are able to prevent infection of a pathogen, such as a virus, by facilitating a cytokine response or by facilitating uptake and removal by an immune cell. In particular, the term "neutralizing antibodies" includes antibodies (or fragments or derivatives thereof) capable of inhibiting or blocking infection (or progression of infection) of a pathogen by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Only a small subset of the many antibodies that bind a virus are capable of neutralization. Platforms incorporating 6K or 6K-like linkers are used to produce epitopes to which virus neutralizing antibodies or other response are directed.

[0084] Antigen processing is an immunological process that prepares antigens for presentation to special cells of the immune system called T lymphocytes. This process involves two distinct pathways for processing of antigens from an organisms own (self) proteins or intracellular pathogens (e.g, viruses), or from phagocytosed pathogens (e.g., bacteria); subsequent presentation of these antigens on class I or class II major histocompatibility complex (MHC) molecules is dependent on which pathway is used. Routes of antigen processing are disclosed by Fig. 4 and include presentation of peptide fragments via MHC class 1 or 2 molecules and exocytosis of ILTV polypeptides

[0085] A monovalent vaccine is meant to refer to a vaccine designed to immunize against a single ILTV antigen or single type of ILTV virus, whereas polyvalent or multivalent vaccines aim to immunize against several strains or genotypes of ILTV or against a mixture of different ILTV antigens. In some embodiments, a vaccine as disclosed herein may comprise a single platform or mixture of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more platforms each encoding antigens of a different ILTV strain. In other embodiments, a vaccine may comprise a platform encoding the same antigen from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more ILTV strains. Analogs or variants of the polynucleotides or polypeptides disclosed herein may have different degrees of sequence identity or similarity to said polynucleotides or polypeptides. BLASTN may 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 to a reference polynucleotide such as a polynucleotide comprising ILTV genomic, subgenomic, or antigen-coding sequences. A representative BLASTN setting modified to find highly similar sequences uses an Expect Threshold of 10 and a Wordsize of 28, max matches in query range of 0, match / mismatch scores of 1 / -2, and linear gap cost. Low complexity regions may be filtered or masked. Default settings of a Standard Nucleotide BLAST are described by and incorporated by reference to hypertext transfer protocol secure: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LIN K_LOC=blasthome (last accessed June 7, 2022).

[0086] 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 ammo acid, such as a 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 midrange sequences, and BLOSUM80 for more distantly related sequences. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score. BL ASTP “Identities” shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP “Positives” shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity or similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. A representative BLASTP setting that uses an Expect Threshold of 10, a Word Size of 3, BLOSUM 62 as a matrix, and Gap Penalty of 11 (Existence) and 1 (Extension) and a conditional compositional score matrix adjustment. Other default settings for BLASTP are described by and incorporated by reference to the disclosure available at: hypertext transfer protocol secure: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&PAGE_TYPE=

[0087] BlastSearch&LINK_LOC=blasthome (last accessed June 7, 2022). Analogs or variants of a polynucleotide may include those with 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or more deletions, substitutions, or insertions of nucleotides into a polynucleotide such as those disclosed herein. Such analogs may be based on a genomic virus sequence, on a sequence of a specific viral gene, or on sequences of other elements of a platform such as 3’ or 5’ UTR, 6k or 6k-like sequences, or any other sequence disclosed herein.

[0088] Analogs or variants of polypeptides include those with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or more deletions, substitutions, or insertions of amino acid residues into a polypeptide, such as those disclosed herein. Such 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 platform 6k or 6k- like sequences or any other encoding sequence disclosed herein.

[0089] Fragment or functional fragment as used herein refers to shorter or truncated segments of a longer nucleic acid or polypeptide sequence, preferably which retain at least one function of the whole nucleic acid or polypeptide, such as an epitope. Functional immunogenic fragments are thus suitable for vaccination purposes. Generally, a fr agment of a nucleic acid encoding an ILTV antigen will encode at least one epitope thereof and a fragment of a ILTV antigen will comprise at least one epitope of the antigen. Examples of polypeptide fragments include fragments of longer ILTV polypeptides 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 encoding the peptide fragments described above.

[0090] Pharmaceutically acceptable carriers. A ILTV vaccine usually comprises a platform encoding one or a multiple ILTV antigens and at least one pharmaceutically acceptable carrier, excipient or diluent. Stabilizers or agents that prevent degradation of RNA 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 can be sterile, and the formulation suits the mode of administration. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. Any of the common pharmaceutical carriers, such as sterile saline solution or sesame oil, can be used. The medium can also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like. Other media that can be used with the compositions and methods provided herein are normal saline and sesame oil. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In some embodiments, the carrier may be a particle, nanoparticle, liposome, lipoplex or lipid nanoparticle, for example, as described by and incorporated by reference to Kamrud, et al., US 2018 / 0171340 A.

[0091] LION. Lipid InOrganic Nanoparticle (LION). In some embodiments, the platform is administered as a nanoemulsion particle that has a hydrophobic core and comprises a mixture of a liquid oil and one or more inorganic solid nanoparticles. The nanoemulsion particle can also be referred to herein as Lipid InOrganic Nanoparticles (LIONs). The liquid oil is mixed with the one or more inorganic nanoparticles to form a hydrophobic core. The liquid oil is typically metabolizable. Suitable liquid oil can be a vegetable oil, animal oil, or synthetically prepared oil. In some embodiments, the liquid oil is a fish oil. In some embodiments, the liquid oil is a naturally occurring or synthetic terpenoid. In some embodiments, the liquid oil is squalene, triglyceride (such as capric / caprylic triglyceride or myristic acid triglyceride), vitamin E, lauroyl polyoxylglyceride, monoacylglycerol, soy lecithin, sunflower oil, soybean oil, olive oil, grapeseed oil, or a combination thereof. In one embodiment, the liquid oil is squalene, triglyceride (such as capric / caprylic triglyceride or myristic acid triglyceride), vitamin E, lauroyl polyoxylglyceride, monoacylglycerol, soy lecithin, or a combination thereof. In one embodiment, the liquid oil is squalene, triglyceride (such as capric / caprylic triglyceride or myristic acid triglyceride), sunflower oil, soybean oil, olive oil, grapeseed oil, or a combination thereof. In some embodiments, the liquid oil is squalene (either naturally occurring or synthetic, optionally in combination with any of the above listed liquid oils. The inorganic nanoparticles may be formed from one or more same or different metals (any metals including transition metal), such as from metal salts, metal oxides, metal hydroxides, and metal phosphates. Examples include silicon dioxide (SiO2), iron oxides (Fe3O4, Fe2O3, FeO, or combinations thereof), aluminum oxide (A]2O3), aluminum oxyhydroxide (A10(0H)), aluminum hydroxyphosphate (Al(OH)x(PO4)y), calcium phosphate (Ca3(PO4)2), calcium hydroxyapatite (Caio(P04).6(OH)2), iron gluconate, or iron sulfate. In some embodiments, the inorganic solid nanoparticle is a metal oxide, such as a transition metal oxide. In one embodiment, the inorganic solid nanoparticle is an iron oxide, for instance, magnetite (Fe3O4), maghemite (.gamma. -Fe2O3), wustite (FeO), hematite (a-Fe2O3), or combinations thereof. In some embodiments, the inorganic solid nanoparticle is a metal hydroxide, such as an aluminum hydroxide or aluminum oxyhydroxide. The inorganic solid nanoparticle may contain a reporter element detectable via imaging methods to allow for imaging and tracking the resulting nanoemulsion particles in the body. For instance, the inorganic solid nanoparticle may contain a reporter element detectable via magnetic resonance imaging (MRI), such as a paramagnetic, superparamagnetic, ferrimagnetic or ferromagnetic compound. Exemplary inorganic solid nanoparticle materials that are MRI-detectable are iron oxides, iron gluconates, and iron sulfates. The inorganic solid nanoparticle typically has an average diameter (number weighted average diameter) ranging from about 3 nm to about 50 nm. For instance, the inorganic solid nanoparticle can have an average diameter of about 5 nm, about 10 nm, about 15 mu, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, or about 50 nm. The inorganic solid nanoparticle may be surface modified before mixing with the liquid oil. For instance, if the surface of the inorganic solid nanoparticle is hydrophilic, the inorganic solid nanoparticle may be coated with hydrophobic molecules (or surfactants) to facilitate the miscibility of the inorganic solid nanoparticle with the liquid oil in the "oil" phase of the nanoemulsion particle. Phosphate- terminated lipids (such as phosphatidylated lipids), phosphorous-terminated surfactants, carboxylate-terminated surfactants, sulfate-terminated surfactants, or amine-terminated surfactants can be used for surface modification of the inorganic solid nanoparticle. Typical phosphate-terminated lipids or phosphorous-terminated surfactants are trioctylphosphine oxide (TOPO) or distearyl phosphatidic acid (DSPA). Typical sulfate-terminated surfactants include but not limited to sodium dodecyl sulfate (SDS). Typical carboxylate-terminated surfactants include oleic acid. Typical amine terminated surfactants include oleylamine. In one embodiment, 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 the liquid oil to form the hydrophobic core. In one embodiment, the inorganic solid nanoparticle is a metal hydroxide, such as an aluminum hydroxide or aluminum oxyhydroxide, and a phosphate-terminated lipid or a surfactant, such as oleic acid, oleylamine, SDS, TOPO or DSPA is used to coat the inorganic solid nanoparticle, before it is mixed with the liquid oil to form the hydrophobic core. The lipids used to form nanoemulsion particles can be cationic lipids, anionic lipids, neutral lipids, or mixtures thereof. In some embodiments, the lipids used are cationic lipids. For example, positively charged lipids that can have favorable interactions with negatively charged bioactive agent (such as DNAs or RNAs) may 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 (DD A) ; 1,2 -dimyristoyl-3 -trimethylammoniumpropane

[0092] (DMTAP), dipalmitoyl(C16:0)trimethyl ammonium propane (DPTAP); distearoyltrimethylanmionium propane (DSTAP); N-[l-(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); 1 , 1 '-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino (ethyl) (2- hydroxydodecyl)amino)ethyl)piperazin- 1 -yl)ethyl)azanediyl)bis(dodecan— 2-ol) (C 12-200); and combinations thereof. A typical cationic lipid is DOTAP. Other examples for suitable lipids include, but are not limited to, the phosphatidylcholines (PCs), such as di stearoylphosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), l-palmitoyl-2- oleoylphosphatidylcholine (POPC), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylcholine (DMPC), etc,; phosphatidylethanolamines (PEs), such as 1,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), etc.; phosphatidylglycerol (PGs); and PEGylated lipids including PEGylated version of any of the above lipids (e.g., DSPE-PEGs). The nanoemulsion particle can further contain one or more surfactants, which can be a hydrophobic surfactant or a hydrophilic surfactant. In some embodiments, the nanoemulsion particle further comprises a hydrophobic surfactant. In some embodiments, the nanoemulsion particle further comprises a hydrophilic surfactant. In one embodiment, the nanoemulsion particle further comprises a hydrophobic surfactant and a hydrophilic surfactant. Suitable hydrophobic surfactants include those having a hydrophilic- lipophilic balance (HLB) value of 10 or less, for instance, 5 or less, from 1 to 5, or from 4 to 5. An exemplary hydrophobic surfactant is a sorbitan ester (such as sorbitan monoester or sorbitan trimester). For instance, the hydrophobic surfactant can be a sorbitan ester having a HLB value from 1 to 5, or from 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. Exemplary sorbitan triesters include sorbitan tristearate and sorbitan trioleate. Suitable hydrophilic surfactants include those polyethylene oxide-based surfactants, for instance, a polyoxyethylene sorbitan ester (polysorbate). In some embodiments, the hydrophilic surfactant is a polysorbate. 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 embodiment, the hydrophilic surfactant is polysorbate 80. The nanoemulsion particle can have an oil-to-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-to-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-to-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 liquid oil, from about 0.001% to about 10% w / v inorganic solid nanoparticle, from about 0.2% to about 10% w / v lipid (e.g., cationic lipid), from about 0.25% to about 5% w / v hydrophobic surfactant (e.g., sorbitan ester), and from about 0.5% to about 10% w / v hydrophilic surfactant. In certain 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 phosphorous-terminated surfactant, a carboxylate-terminated surfactant, a sulfate-terminated surfactant, or an amine- terminated surfactant, and a liquid oil containing naturally occurring 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 naturally occurring 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 can comprise from about 0.2% to about 40% w / v squalene, from about 0.001% to about 10% w / v iron oxide nanoparticles, from about 0.2% to about 10% w / v DOTAP, from about 0.25% to about 5% w / v sorbitan monostearate, and from about 0.5% to about 10% w / v polysorbate 80. In one embodiment, the LION particle comprises from about 2% to about 6% w / v squalene, from about 0.01% to about 1% w / v iron oxide nanoparticles, from about 0.2% to about 1% w / v DOTAP, from about 0.25% to about 1% w / v sorbitan monostearate, and from about 0.5%) to about 5% w / v polysorbate 80. In certain 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 phosphorous-terminated surfactant, a carboxylate-terminated surfactant, a sulfate- terminated surfactant, or an amine-terminated surfactant, and a liquid oil containing naturally occurring 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 aluminum hydroxide or aluminum oxyhydroxide nanoparticles optionally coated with TOPO, and a liquid oil containing naturally occurring 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 can comprise from about 0.2% to about 40% w / v squalene, from about 0.001% to about 10% w / v aluminum hydroxide or aluminum oxyhydroxide nanoparticles, from about 0.2% to about 10% w / v DOTAP, from about 0.25% to about 5% w / v sorbitan monostearate, and from about 0.5% to about 10% w / v polysorbate 80. In one embodiment, the LION particle comprises from about 2% to about 6% w / v squalene, from about 0.01% to about 1% w / v aluminum hydroxide or aluminum oxyhydroxide nanoparticles, from about 0.2% to about 1% w / v DOTAP, from about 0.25% to about 1% w / v sorbitan monostearate, and from about 0.5%) to about 5% w / v polysorbate 80. Nanoparticles and nanoemulsions have been described in the literature and the terms are used herein to refer to those particles having a size less than 1000 nanometers. The nanoemulsion particle (LION) typically has an average diameter (z-average hydrodynamic diameter, measured by dynamic light scattering) ranging from about 20 nm to about 200 nm. In some embodiments, the z-average diameter of the LION particle ranges from about 20 nm to about 150 nm, from about 20 nm to about 100 nm, from about 20 nm to about 80 nm, from about 20 nm to about 60 nm. In some embodiments, the z-average diameter of the LION particle ranges from about 40 nm to about 200 nm, from about 40 nm to about 150 nm, from about 40 nm to about 100 nm, from about 40 nm to about 90 nm, from about 40 nm to about 80 nm, or from about 40 nm to about 60 nm. In one embodiment, the z-average diameter of the LION particle is from about 40 nm to about 80 nm. In one embodiment, the z-average diameter of the LION particle is from about 40 nm to about 60 nm. The average polydispersity index (PDI) of the nanoemulsion particles (LIONs) can range from about 0.1 to about 0.5. For instance, the average PDI of the 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.

[0093] Modes of administration. In some embodiments, the polynucleotides or compositions disclosed herein are formulated for in vivo delivery. For administration to an avian the composition according to the present application can be given by any enteral or parenteral route, which includes inter alia intramuscularly, by scarification of the skin or the wing web, intranasally, intradermally, subcutaneously, orally, by aerosol, topically, by spraying an avian, by gel drop or any combination thereof.

[0094] In some embodiments, the saRNA is formulated for intramuscular, subcutaneous, intraorgan, or intradermal delivery. In some embodiments, the polynucleotide or composition is administered subcutaneously, intravenously, intramuscularly, intra-articularly, intra-synovially, intrasternally, intrathecally, intrahepatically, intrathymically, into a sex organ, intralesionally, intracranially, intraventricularly, orally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Other delivery methods, such as those targeting specific tissues or avians at different stages of infection as described by Fig. 1 may be used. EM BODIMENTS

[0095] Generic description of saRNA. One embodiment of this technology is directed to selfamplifying RNA (saRNA) that encodes at least one infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvinis 1, GaHV-1) polypeptide, ILTV polypeptide variant, or an immunogenic fragment or epitope thereof which comprises in any order (a) a first nucleic acid sequence comprising nsPl , 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, ILTV polypeptide variant, or immunogenic fragment or epitope thereof. The saRNA 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 poly A tail. Typically, the saRNA when incorporated into a target cell expresses the at least one ILTV polypeptide, ILTV polypeptide variant, or an antigenic or immunogenic fragment or epitope of any thereof. saRNA platform. Preferably, the saRNA forms part of an alphavirus platform. The platform for saRNA expressing ILTV antigens or immunogens may be selected from the group consisting of Sindbis virus (SINV), Chickungunya vims (CHIKV), Semliki Forest Virus (SFV), Ross River Vims (RRV), Sagiyama vims (SAGV), Getah vims (GETV), Middleburg virus (MIDV), Bebam virus (BEBV), O'nyong nyong vims (ONNV), Ndumu (NDUV), and Barmah Forest vims (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 vims (EEEV). One example of the saRNA platform that is not commercially available for others to use is described by Maine, et al., 2020. saRNA element order. In some preferred embodiments, the saRNA comprises a 5 ’ cap, a 5’ UTR upstream of the first nucleic acid sequence, 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 optionally 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 ILTV antigen or immunogen variants or fragments, or alternative promoters or terminators. In some embodiments, the saRNA further comprises after the 5’ UTR a polynucleotide encoding nspI, followed by a downstream loop (DLP), followed by an autoprotease, such as P2A; see Fig. 5 which depicts a vector encoding the saRNA. Additional saRNA elements. In some embodiments additional elements may be incorporated into the saRNA. These include elements that produce two or more separate ILTV antigens or immunogens, such as sequences encoding 6K proteins or ribosomal skipping / stalling sequences, or protease sites. These may also include elements that enhance transcription of the saRNA or enhance ILTV antigen or immunogen expression, such as DLP loops. They may include polynucleotides encoding immunogenic carrier proteins, adjuvants, or cytokines.

[0096] Protein separation sites. In some embodiments, the saRNA as disclosed herein further comprises a nucleic acid sequence encoding at least one protein separation site, such as a 6K ribosome stalling or skipping site. Typically, these sites are positioned between nucleic acids encoding different proteins so that when the protein is expressed from the saRNA it may be expressed in, or easily converted to, an individual ILTV antigen or immunogen.

[0097] A protein separation site may comprise 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 enhancer. In some embodiments, when a DLP or capsid enhancer is present, the protein separation sites may be positioned between the at least one viral capsid enhancer or DLP and the second nucleic acid sequence. In some embodiments, the one viral capsid enhancer or downstream loop (“DLP”) is positioned immediately downstream of the 5’UTR or of 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. The saRNA as disclosed herein in some embodiments further comprises a nucleic acid sequence encoding a genetically modified cross-reacting material (CRM) of diphtheria toxin, tetanus toxoid (T), meningococcal outer membrane protein complex (OMPC), diphtheria toxoid (D), or H. influenzae protein D (HiD), wherein the subsequent nucleic acid sequence is optionally fused.

[0100] Immunomodulatory protein. The saRNA as disclosed herein may further comprise a nucleic acid sequence encoding, at least one immunomodulatory protein including but not limited to chemokines CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCLI2, 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 colonystimulating factor 3 (CSF 3); proteins that an intracellular pattern recognition receptor (PRR): Sendai virus-derived oligonucleotide that imitates Sendai virus defective interfering (DI) particles (SeVDI) or another PRR triggering protein; or combinations thereof.

[0101] Protein trafficking elements. The saRNA disclosed herein may further comprise elements that help traffic the expressed ILTV antigens or immunogens to particular cellular or extracellular compartments, for example, direct an expressed antigen to the endoplasmic reticulum and Golgi apparatus, to lysosomes, to cell membrane, or outside of the cell. An saRNA as described above may comprise a polynucleotide encoding a 6k or 6k-like cleavage site or a furin-cleavage site between polynucleotide sequences encoding ILTV antigens which can be trafficked to the endoplasmic reticulum and produce one or more glycosylated antigens.

[0102] In some embodiments, the saRNA platforms as disclosed herein comprise polynucleotides encoding leader or signal peptides, ILTV a 6k or 6k-like cleavage site, a linker encoding a furin- cleavage site, or that comprise P2A or T2A. External and cytoplasmic domains of membrane associated antigens are typically separated by a transmembrane domain.

[0103] Deduction of RNA, DNA or amino acid sequences from corresponding genetic information and use of genetic code. The nucleic acid sequences disclosed herein, such as those given by SEQ ID NOS: 1-5 encompass both RNA and DNA versions of these sequences. An RNA sequence is easily deduced from the corresponding DNA sequence. Similarly, amino acid sequences of ILTV immunogens or antigens may be deduced from the corresponding DNA or RNA sequences using the genetic code.

[0104] Accession Numbers of ILTV nucleic acids and polypeptides. The sequences of precursor, processed, and mature forms of the proteins named below as well as their variants; their modified or glycosylated residues of these proteins; and the subcellular locations of these proteins are described by and incorporated by reference to the database entries accessed by these accession numbers (last accessed September 1, 2022). Information disclosed by the GENBANK accession number entries that is not reproduced below is expressly incorporated by reference..

[0105] The section below describes accession numbers describing the nucleotide (e.g. RNA or DNA) or amino acid (protein) sequences of ILTV polypeptides. However, the nucleotides and amino acid sequences disclosed herein are not limited to these sequences. In some instances, these sequences can be further modified to produce variant sequences, such as sequences having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more deletions, substitutions, or insertions of nucleotides or amino acid residues or sequences having a certain degree of sequence identity to the sequences described by these accession, numbers. Other modifications include glycosylation or other chemical of amino acid residues, removal of glycosylation from a residue or its replacement by a non-glycosylated amino acid residue, or structural modifications including multimerization or alternative protein folding.

[0106] Specific ILTV antigens. In some embodiments, the second nucleic acid sequence of the saRNA disclosed herein encodes at least one ILTV polypeptide encoding gB, gB-del, gC, gD, gE, gG, gH, gl, gj, gK, gL or gM. Specific ILTV antigen sequences include those of gB (AGS36506.1), gC (AAA16957.1), gD (AAA98925.1), gE (AGS36509.1), gG (AGC50883.1), gH (AGF43577.1), gl (BAA33OO3.1; BAA33004.1; BAA33005.1, BAA33006.1, BAA33007.L 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.L CAA65891.1 , CAA65892.1, CAA65893.1, CAA65894.1, CAA65895.1, CAA65896.1, CAA65899.1 , CAA65900.1 ) and / or gM (ABX59489.1 ), or an isoform thereof, variant thereof, or an immunogenic fragment or epitope thereof. The sequences described by the accession numbers above are expressly incorporated by reference.

[0107] One example of the gB-del sequence, where gB is entirely or partially deleted, the deletion comprises all or part of gB as described by (AGS36506.1). SEQ ID NO: 2 also describes a polynucleotide comprising gB-del.

[0108] Specific sequences comprising ILTV antigens or modified antigens, such as gBdel, are described by SEQ ID NOS: 1, 2, 3, 4 and 5.

[0109] Variants of Specific ILTV antigens. In some embodiments, the second nucleic acid sequence of the saRNA disclosed herein encodes 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 to the unmodified ILTV antigen or immunogen sequence. A variant have at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99. 99.5, 99.9, or <100% sequence identity' or sequence similarity' to a 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 ), BAA33OO5.1, BAA33006.1, BAA33007.1, and / or BAA33008.1), gL (CAA65897.1 , CAA65898.1 , CAA65890.1 , CAA65891.1, CAA65892.1, CAA65893. L CAA65894.1, CAA65895.1, CAA65896.1, CAA65899.1, CAA65900.1) and / or gM (ABX59489.1). Similarly variants may be based on the sequences described by SEQ ID NOS: 1-5.

[0110] Fragments / Epitopes. An ILTV antigen or immunogen encoded by the saRNA disclosed herein may be a functional fragment of an ILTV antigen or immunogen or a variant thereof. In some embodiments, the saRNA will encode a polypeptide comprising a cellular epitope ranging in length from 13-17 residues or humoral epitope ranging in length from 8-11 residues. An epitope may be linear or conformational.

[0111] DNA construct encoding saRNA . One embodiment of the invention is a DNA construct that encodes that saRNA as disclosed herein. The DNA construct may be a plasmid, viral vector. This construct may be transformed into a cell where it can replicate or where it can transcribe saRNA.

[0112] Single ILTV antigen. In some embodiments, the saRNA will encode a single ILTV antigen or immunogen, such as one antigen or immunogen selected from encoding gB, gB-del, gC, gD, gE, gG, gH, gl, gJ, gK, gL or gM or other ILTV translation products such as those described by Fig. 3.

[0113] Multiple ILTV antigens. In some embodiments, the saRNA will encode two, three, four or more ILTV antigens or immunogens, such as two or more selected from encoding gB, gB-del, gC, gD, gE, gG, gH, gl, gJ, gK, gL or gM or other ILTV translation products such as those described by Fig. 3. The saRNA sequences encoding the 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 a saRNA encodes two or more antigens, the coding sequences may be separated by sequences encoding protein separation sites, such a sequence encoding a 6K protein, ribosome stalling site, or protease cleavage site, such as a site recognized by an endogenous protease.

[0114] Specific saRNA constructs include but are not limited to those described below.

[0115] In some embodiments, the second nucleic acid sequence encodes gB or a variant or fragment thereof.

[0116] In some embodiments, the second nucleic acid sequence encodes gBdel or a variant or fragment thereof. In some embodiments, the second nucleic acid sequence encodes gD or a variant or fragment thereof.

[0117] In some embodiments, the second nucleic acid sequence encodes gE or a variant or fragment thereof.

[0118] In some embodiments, the second nucleic acid sequence encodes gl or a variant or fragment thereof.

[0119] In some embodiments, the second nucleic acid sequence encodes gE-6K-I or a variant or fragment thereof.

[0120] In some embodiments, the second nucleic acid sequence encodes gB, gBdel, gD, or gE linked via 6K to gl; encodes gB, gBdel, or gD, linked via 6K to gE; or encodes gB or gBdel, linked via 6K to gD.

[0121] In some embodiments, the second nucleic acid sequence encodes at least two different polypeptides selected from gB, gBdel, gD, gE, and / or gl.

[0122] In some embodiments, the second nucleic acid sequence encodes gl and gE.

[0123] In some embodiments, the second nucleic acid sequence encodes gB or gBdel, gD, gl, and gE-

[0124] In some embodiments, the second nucleic acid sequence encodes gB or gBdel, gD, gl, gE and gC.

[0125] In some embodiments, a combination of gB or gB-del, gl and gE (in any order) includes the following specific ordering 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.

[0126] In other embodiments, a combination of gD, gl and gE (in any order) includes the following specific ordering 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.

[0127] In one embodiment, immunization of saRNA encoding gD alone only protected about 25% of chickens. However, gl was found to produce the strongest immune response. Thus, a preferred saRNA may include a gene encoding gl alone or in combination with other ITLV genes such as gD.

[0128] Compositions containing saRNA. Another aspect of this technology is directed to a composition comprising the saRNA (or saRNAs) as disclosed herein in combination with a pharmaceutically acceptable carrier or excipient. Such a carrier or excipient may comprise at least one of lipids, surfactants, polyamino acids, proteins, polymeric particles, self-assembled particles, composite nanoparticles of conjugated phospholipids, or inorganic particles (such as silica particles), or combinations thereof.

[0129] In some embodiments, advantageously, this composition may be formulated with one or more saRNAs and a pharmaceutically acceptable carrier or excipient comprising lipid inorganic nanoparticles (LION).

[0130] 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 a 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 that comprises a mixture of a liquid oil and one or more inorganic nanoparticles, one or more lipids and, optionally, one, two, or more surfactants.

[0131] Treatment with saRNA. Another aspect of this technology is directed to a method for reducing the severity of ILTV or comorbid condition(s) in an avian, such as a chicken, comprising administering to a subject in need thereof an saRNA that encodes at least one ILTV antigen or an immunogenic fragment, epitope, or variant of any thereof, as disclosed herein; a plasmid or DNA construct encoding the saRNA; a cell containing the DNA construct; or a saRNA composition as disclosed herein.

[0132] Reduction of severity of ILTV infection or prophylaxis. In some embodiments of this method the reducing the severity of ILTV comprises reducing dyspnea or hemorrhage associated with ILT at least one of a symptom of blood of tire beak or nares depression, and difficulty breathing compared to an unvaccinated control avian (Koski et al. 2015) as shown in reduction of clinical score in Fig. 8B and Fig. 13B.

[0133] In some embodiments of this method the reducing the severity comprises reducing weight loss of vaccinated avians compared to unvaccinated control avians.

[0134] In other embodiments of this method the reducing the severity' comprises reducing mortality of vaccinated avians compared to unvaccinated control avians.

[0135] In other embodiments of this method the reducing the severity comprises reducing transmission of ILTV compared to transmission of ILTV from an unvaccinated avian. In other embodiments of this method the reducing the severity comprises reducing the number of abnormal or thin-shelled eggs or increasing a number of eggs laid compared to those produced by an unvaccinated avian.

[0136] Avian subjects. In some embodiments, the saRNA as disclosed herein is administered to Gallus gallus domesticus (domestic chicken) or other avian which may or may not have been previously exposed to ILTV. The avian may be a breeder, broiler or layer chicken.

[0137] In some embodiments, this method comprises treating an avian which has not been exposed to ILTV, for example, to induce immunological protection against ILTV.

[0138] In other embodiments, this method comprises treating an avian who has been exposed or who is at risk of exposure to ILTV.

[0139] In some embodiments of this method the avian is a carrier of ILTV.

[0140] In some embodiments, the saRNA or saRNAs are administered intramuscularly or into a wing web.

[0141] In some embodiments of this method the saRNA administered orally, muscosally, or via the respiratory system.

[0142] In some embodiments of this method the saRNA administered subcutaneously, intravenously, or parenterally.

[0143] In some embodiments of this method it further comprises administering a drug or biologic that reduces the severity of ILTV.

[0144] In some embodiments of this method the saRNA administered the saRNA is administered in a nanoemulsion comprising a hydrophobic core that comprises a mixture of a liquid oil and one or more inorganic nanoparticles, one or more lipids and, optionally, one or more surfactants.

[0145] In some embodiments saRNA may be administered in combination with LION.

[0146] In one embodiment the saRNA when administered enhances acquired cellular or humoral immunity of a vaccinated animal compared to an unimmunized control. In another embodiment, the vaccine enhances levels of ILTV neutralizing antibodies compared to an unimmunized control.

[0147] In still yet another embodiment, the saRNA enhances cellular immunity to ILTV compared to an unimmunized control.

[0148] In another embodiment, the saRNA enhances innate immunity of a vaccinated animal or does not substantially inhibit innate immunity of the vaccinated animal compared to that of a nonimmunized control animal, hr some embodiments, the methods further comprises administering to the animal at least one agent that enhances innate or adaptive immunity against ILTV selected from the group consisting of a ILTV protein recognized by the animal’s pattern recognition proteins, interferon, or other agent that enhances an innate immune response, for a time and under conditions suitable for inducing an innate immune response to ILTV virus; and then administering the at least one platform for a time and under conditions suitable for inducing 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)-I-like receptors (RLRs), Toll-like receptors 3, 7, and 8 (TLR3, TLR7, and TLR8), interferon regulatory factor 3 (IRF-3), IRF7, NF-KB , Nodlike (NLRs), and C-type lectin (CLRs) receptors). In another embodiment, the at least one agent comprises type 1 interferon (e.g. IFN-a and P); a cytokine that induces Thl cell differentiation, including but not limited to IL-12 or IFN-y; 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 by not limited to an antibody to IL- 10 or an Spl inhibitor including, but not limited to, mithramycin or mithramycin analog MTMOX32E.

[0149] Method for making saRNA. Another aspect of this technology is directed to a DNA construct encoding the saRNA as disclosed herein or a cell comprising such a DNA construct; see Fig. 5 which describes one possible construct. Such a DNA construct may be employed to transcribe saRNA as disclosed herein. Another aspect of this technology is directed to a method for making the saRNA disclosed herein, comprising operably inserting into an alphavirus platform a nucleic acid sequence that expresses at least one ILTV polypeptide or an immunogenic fragment or epitope thereof, or a variant thereof.

[0150] This method may further comprise operably inserting 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 stalling site or protein separation site, a DLP loop, an immunogenic carrier, an immunomodulatory protein, trafficking moiety, or a cytokine.

[0151] A cell comprising a vector encoding the saRNA as disclosed herein may 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 non-mammalian cell types, including cultured cell lines, may also incorporate such a vector. Codon modification. This method may further comprise replacing at least one degenerate codon in the nucleic acid sequence which encodes 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 expression ILTV polypeptides, polypeptide fragments or variants, or increases the stability of the saRNA.

[0152] In one embodiment, the one or more polynucleotides or other nucleic acid sequences of the saRNA platform or its polynucleotide components are codon modified based on the codon usage in the host cell or based on decreasing or increasing their GC content. Various functions and methods for codon modification may be used including those described by, and incorporated by reference to Hanson, G., Colter, J. Codon optimality, bias and usage in translation and mRNA decay. NAT REV MOL CELL BIOL 19, 20-30 (2018). Hypertext transfer protocol secure: / / doi.org / 10.1038 / nrm.2017.91_; or hypertext transfer protocol secure: / / en.wikipedia.org / wiki / Codon_usage_bias#Effect_on_transcription_or_gene_expression (last accessed June 7, 2022) and by the references cited therein.

[0153] In another embodiment, the one or more polynucleotides or other nucleic acid sequences of the platform or its polynucleotide components are selected to attenuate formation of secondary RNA structures which reduce the expression of the at least one polynucleotide encoding a ILTV antigen; provide or enhance secondary structures that stabilize the stability of mRNAs encoding ILTV antigens; or to otherwise control the translation and relative abundance of multiple encoded ILTV antigens.

[0154] In another embodiment, the one or more polynucleotides or other nucleic acid sequences of the saRNA platform or its polynucleotide components are selected to increase the expression of the at least one polynucleotide encoding a ILTV antigen. For example, the codons encoding one ILTV antigen may be selected to increase the relative abundance (or stoichiometric amount), of that antigen with respect to another ILTV antigen.

[0155] In some embodiments, this method produces a platform comprising a self-amplifying RNA engineered for resistance to innate cellular immune responses.

[0156] EXAMPLE 1

[0157] Comparison of saRNA vaccine expressing different ILTV immunogens Six groups of twenty chickens (Gallus domesticus} were immunized with various combinations of saRNA. Chickens were immunized and boosted Intramuscular (IM) / Intramuscular, or WingWeb (WW) / Intramuscular. . A placebo vaccinated group is not administered vaccine. After immunization, a virulent challenge dose (as described in Fig. 7) is administered by intratracheal and ILTV (LT) clinical signs were observed.

[0158] Fig. 7 describes saRNA vaccine design and testing. Vaccine combinations described in Fig. 7 includes between 0.1 and 5 pg of each recited gene of interest, for example for the first group “gB, gD, gE, gl”.

[0159] Fig. 7 also describes the route of administration for each combination of saRNAs encoding ILTV antigens. For example, the first, second and third groups were vaccinated and boosted IM / IM. In some embodiments, subcutaneous priming and or boosting may be used.

[0160] Fig. 8A discloses percent protection against ILTV clinical signs and clinical scores observed in vaccinated chickens and mortality as described in Example 1.

[0161] Fig. 8B presents results of use of different modes or administration of the SRV-LT constructs. Route: vaccination / boost. IM: intamuscular; WW: wingweb; SC: subcutaneous.

[0162] ILTV shedding by vaccinated chickens onto oropharyngeal swabs was measured using qPCR equivalent to 50% egg infectious dose of the ILTV challenge virus; see Fig. 9. DPC: days post-challenge.

[0163] Intramuscular vaccination and boost with 0.1-5 pg each of saRNAs encoding gB, gD, gE and gl eliminated ILTV shedding 6 days post-challenge and intramuscular vaccination and boost with 0.1-5 pg each of saRNAs encoding gD, gE and gl eliminated ILTV shedding in 95% of the chickens 6 days post challenge. Wingweb vaccination with these same saRNAs stopped 50% of chickens from shedding.

[0164] These results demonstrate the efficacy of saRNA vaccines disclosed herein for ameliorating clinical symptoms of ILT, reducing mortality from ILTV, and attenuating or eliminating shedding of ILTV.

[0165] EXAMPLE 2

[0166] Comparison of saRNA vaccine expressing different ILTV immunogens

[0167] Seven groups of twenty chickens (Gallus domesticus} were immunized with saRNA platforms encoding the ILTV antigens described by Fig. 11. Animals were vaccinated at six weeks of age and boosted three weeks later at nine weeks of age. All animals were challenged by intratracheal administration of a virulent ILTV on week

[0168] 12.

[0169] A Western blot showed that saRNA encoded ILTV proteins recognized by anti-ILTV serum, was stably expressed, see Fig. 12.

[0170] Protection against ILT clinical signs, ILT clinical sign severity, and mortality for each group were observed and shown by Fig. 13.

[0171] ILTV shedding three and six days post-challenge was measured and shown in Fig. 14. All placebo vaccinated birds shed ILTV on day 3 and day 6 post-challenge. However, 75-95% of chickens vaccinated and swabbed at 6 dpc were ILTV negative excepted those chickens that received the (gl-VEE linker-gE), gD-Low dose vaccination and placebo-vaccinated, ILTV challenged chickens.

[0172] On day six post-challenge, birds immunized and boosted IM / IM with high dose gl-VEE 6K linker-gE + gD and gB-del were 95% negative for shedding.

[0173] Birds immunized and boosted IM / IM with high dose gD + gE + gl or with high dose gl- VEE 6K linker-gE + gD were 90% negative for ILTV shedding on day six post-challenge and those immunized with low dose gl-VEE 6K linker-gE + gD were 80% negative.

[0174] Birds immunized and boosted IM / IM with high dose gl-VEE 6K linker-gE + gD were 90% negative for ILTV shedding on day six post-challenge.

[0175] Birds immunized and boosted IM / IM with gB-del alone were 75% negative for ILTV shedding.

[0176] Antibodies to ILTV antigens were measured using BioChek ILT ELISA which measures the amount of antibodies to the ILTV in the serum of chickens. Results are shown in Fig. 15. After boost, four of the seven groups had anti-ILTV antibody levels greater than the threshold value with high dose IM / IM saRNAs encoding gD, gE and gl exhibiting tire highest titers.

[0177] The mortality in each group was determined. A 25% mortality was observed in the placebo vaccinated group and 5% mortality in the group receiving low dose gl- VEE-6K-linker-gE + gD. None of the birds in the other saRNA-immunized groups died and all of these groups exhibited at least 90% clinical sign protection.

[0178] These results demonstrate the efficacy of the saRNA vaccines disclosed herein for ameliorating clinical symptoms of ILT, reducing mortality from ILTV, and reducing shedding of ILTV. Terminology’. Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0179] Unless expressly stated, the terms limit the meaning of such terms unless specifically indicated.

[0180] While aspects of the present disclosure have been described in conjunction with the specific embodiments thereof that are proposed as examples, alternatives, modifications, and variations to the examples may be made. The description and specific examples, while indicating embodiments of the technology, are intended for purposes of illustration only and are not intended to limit the scope of the technology. Moreover, recitation of multiple embodiments having stated features is not intended to exclude other embodiments having additional features, or other embodiments incorporating different combinations of the stated features. Specific examples are provided for illustrative purposes of how to make and use the compositions and methods of this technology and, unless explicitly stated otherwise, are not intended to be a representation that given embodiments of this technology have, or have not, been made or tested.

[0181] As used herein, the words "preferred" and "preferably" refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of 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.

[0182] It should be noted that, as used in the specification and the appended 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 Graphic Sys., Inc. 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).

[0183] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “ / ”. A and / or B includes A, B, and (A + B).

[0184] As used herein in the specification including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “substantially”, “about” or “approximately,” even if the term does not expressly appear. Claim language may refer to exact values (e.g., “10%”) unless further specified (c.g., “about 10%”) The phrase “about” or “approximately” may be used when describing magnitude and / or position to indicate that the value and / or position described is within a reasonable expected range of values and / or positions. For example, a numeric value may have a value that is + / - 0.1% of the stated value (or range of values), + / - 0.2% of the stated value (or range of values), + / - 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 numerical range recited herein is intended to include all sub-ranges and values subsumed therein.

[0185] Any numerical range recited herein is intended to include all sub-ranges and values subsumed therein. Where a range of values is provided, it is to be understood that each intervening value between an upper and lower limit of the range and any other stated or intervening value in that stated range is encompassed within the disclosure. Where the stated range includes upper and lower limits, ranges excluding either of those limits are also included.

[0186] Disclosure of values and ranges of values for specific parameters (such as temperatures, molecular weights, weight percentages, etc.) are not exclusive of other values and ranges of values useful herein. It is envisioned that two or more specific exemplified values for a given parameter may define endpoints for a range of values that may be claimed for the parameter. For example, if Parameter X is exemplified herein to have value A and also exemplified to have value Z, it is envisioned that parameter X may have a range of values from about A to about Z. Similar ly, it is envisioned that disclosure of two or more ranges of values for a parameter (whether such ranges are nested, overlapping or distinct) subsume all possible combination of ranges for the value that might be claimed using endpoints of the disclosed ranges. For example, if parameter X is exemplified herein to have values in the range of 1-10 it also describes subranges for 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 as some examples. A range encompasses its endpoints as well as values inside of an endpoint, for example, the range 0-5 includes 0, >0, 1, 2, 3, 4, <5 and 5.

[0187] As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word "include," and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology. Similarly, the terms "can" and "may" and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present invention that do not contain those elements or features.

[0188] Although the terms “first” and “second” may be used herein 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 discussed below could be termed a second feature / element, and similarly, a second feature / element discussed below could be termed a first feature / element without departing from the teachings of the present invention.

[0189] It will also be understood that, when a feature or element is referred to as being “connected”, “attached” or “coupled” to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being “directly connected”, “directly attached” or “directly coupled” to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.

[0190] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference, especially referenced is disclosure appearing in the same sentence, paragraph, page or section of the specification in which the incorporation by reference appears.

[0191] The citation of references herein does not constitute an admission that those references are prior art or have any relevance to the patentability of the technology disclosed herein. Any discussion of the content of references cited is intended merely to provide a general summary of assertions made by the authors of the references and does not constitute an admission as to the accuracy of the content of such references.

Claims

CLAIMS1. Self-amplifying RNA (saRNA) that encodes at least one infectious laryngotracheitis virus (1LTV; Gallid alphaherpesvirus 1, GaHV-1) polypeptide, ILTV polypeptide variant, or an immunogenic fragment or epitope thereof; which comprises in any order:(a) a first nucleic acid sequence comprising nsPl, 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, ILTV polypeptide variant, or immunogenic fragment or epitope thereof, such as gl; and(c) wherein the saRNA comprises 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; which saRNA when incorporated into a cell expresses the at least one ILTV polypeptide, ILTV polypeptide variant, or an immunogenic fragment or epitope of any thereof.

2. The saRNA of claim 1 , wherein the alphavirus is: an Old World alphavirus 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 vims (EEEV).

3. The saRNA of claim 1 or 2 that comprises in the following order the 5’ cap, the 5’ UTR, nsPl , nsP2, nsP3, nsP4, the 26S promoter, the second nucleic acid sequence, the 3’LTTR and the poly A tail, and optionally, a terminator.

4. The saRNA of claims 1, 2 or 3 that further comprises after the 5’ UTR a polynucleotides encoding nspl, followed by a downstream loop (DLP), followed by a P2A autoprotease.

5. The saRNA of any one of claims 1-4, wherein the second nucleic acid sequence encodes an ILTV polypeptide comprising gB, gC, gD, gE, gG, gH, gl, gj, gK, gL and / or gM, or a variant thereof, or an immunogenic fragment or epitope thereof.

6. The saRNA of any one of claims 1-3, wherein the second nucleic acid sequence encodes at least one ILTV polypeptide comprising gB (AGS36506.1), gC (AAA16957.1), gD (AAA98925.1), gE (AGS36509.1), gG (AGC50883.1), gH (AGF43577.1), gl (BAA33003.1 ; BAA33004.1; BAA33OO5.1, BAA33006.1, BAA33007.1, and / or BAA33008.1), gJ (AGS36512.1), gK (BAA33OO3.1 ; 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.L CAA65895.1, CAA65896.1, CAA65899.1, CAA65900.1) and / or gM (ABX59489.1), or a variant thereof, or an immunogenic fragment or epitope thereof.

7. The saRNA of claim 6, wherein the second nucleic acid sequence encodes a variant of an ILTV polypeptide containing 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 insertions, substitutions, or deletions to the unmodified sequence of gB, gC, gD, gE, gG, gH, gl, gJ, gK, gL and / or gM; or wherein said variant has at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99. 99.5, 99.9, or <100% sequence identity a ILTV polypeptide comprising gB (AGS36506.I), gC (AAAI 6957.1), 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), gE (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).

8. The saRNA any one of claims 1-7, wherein the second nucleic acid sequence encodes an ILTV polypeptide comprising gB, gD, gE, and / or gl, or a variant or fragment thereof.

9. The saRNA of any one of claims 1-7, wherein the second nucleic acid sequence encodes gB or a variant or fragment thereof.

10. The saRNA of any one of claims 1-7, wherein the second nucleic acid sequence encodes gBdel or a variant or fragment thereof.

11. The saRNA of any one of claims 1 -7, wherein the second nucleic acid sequence gD or a variant or fragment thereof.

12. The saRNA of any one of claims 1-7. wherein the second nucleic acid sequence encodes gE or a variant or fragment thereof.

13. The saRNA of any one of claims 1 -7, wherein the second nucleic acid sequence encodes gl or a variant or fragment thereof.

14. The saRNA of any one of claims 1-7, wherein the second nucleic acid sequence encodes gE- 6k-I or a variant or fragment thereof.

15. The saRNA of any one of claims 1-7, wherein the second nucleic acid sequence encodes gB, gBdel, gD, or gE linked via 6k to gl; encodes gB, gBdel, or gD, linked via 6k to gE; or encodes gB or gBdel, linked via 6k to gD.

16. The saRNA of any one of claims 1-7, wherein the second nucleic acid sequence encodes gl and gE.

17. The saRNA of any one of claims 1-7, wherein the second nucleic acid sequence encodes gB or gBdel, gD, gl and gE.

18. The saRNA of any one of claims 1-7, wherein the second nucleic acid sequence encodes gB or gBdel, gD, gl, and / or gE; and gC.

19. The saRNA of any one of claims 1-7 that further comprises a nucleic acid sequence encoding at least one protein separation site or at least one cleavage site for an endogenous or exogenous protease which is positioned between segments of the second nucleic acid sequence that encode different IL TV antigens.

20. The saRNA of any one of claims 1-7, wherein the second nucleic acid sequence encodes at least two different polypeptides selected from gB, gC, gD, gE, gG, gH, gl, gj, gK, gL, and / or gM; wherein each sequence encoding an ILTV antigen is separated by a 6k sequence or another protein separation site.

21. The saRNA of claims 1-7, wherein the second nucleic acid sequence encodes at least two different polypeptides selected from gB, gBdel, gD, gE, and / or gl.

22. The saRNA of any one of claims 1-21, which further comprises at least one viral capsid enhancer or downstream loop (“DLP”) which, optionally, is immediately downstream of the 5’UTR or of the 26S promoter.

23. The saRNA of claims 1-21 that further comprises a protein separation site, ribosome stalling site, or protease cleavage site between the at least one viral capsid enhancer or DLP and the second nucleic acid sequence.

24. The saRNA of any one of claims 1 -23 which comprises at least one internal ribosome entry site (IRES) that is upstream of the second nucleic acid sequence encoding gB, gC, gD, gE, gG, gH, gl, gj, gK, gL and / or gM polypeptides, variants thereof, or fragments or epitopes thereof.

25. The saRNA of claims 1-23, further comprising a nucleic acid sequence encoding an immunogenic carrier, wherein the nucleic acid sequence encoding the immunogenic carrier is optionally bound to the second nucleic acid sequence in the same open reading frame and expresses a fusion protein comprising gB, gC, gD, gE, gG, gH, gl, gj, gK, gL and / or gM polypeptides; or immunogenic fragments, epitopes, or variants thereof, and the immunogenic carrier.

26. The saRNA of claims 1-25 that further comprises a nucleic acid sequence encoding a genetically modified cross-reacting material (CRM) of diphtheria toxin, tetanus toxoid (T), meningococcal outer membrane protein complex (OMPC), diphtheria toxoid (D), or H influenzae protein D (HiD), wherein the subsequent nucleic acid sequence is optionally fused.

27. The saRNA of claims 1-26 further comprising a nucleic acid sequence encoding, at least one immunomodulatory protein including but not limited to chemokines CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, CCL14, CCL19, CCL20, CCL2I, 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-1 1, IL- 12, IL- 12 A, 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 (CSF 3); proteins that an intracellular pattern recognition receptor (PRR): Sendai virus-derived oligonucleotide that imitates Sendai virus defective interfering (DI) particles (SeVDI) or another PRR triggering protein; or combinations thereof.

28. A DNA construct encoding the saRNA of any one of claims 1-27.

29. A cell comprising the DNA construct of claim 28.

30. A composition comprising the saRNA of any one of claims 1-27 in combination with a pharmaceutically acceptable carrier or excipient.

31. The composition of claim 30, wherein the pharmaceutically acceptable carrier or excipient comprises at least one of lipids, surfactants, polyamino acids, proteins, polymeric particles, self-assembled particles, composite nanoparticles of conjugated phospholipids, or inorganic particles (such as silica particles), or combinations thereof.

32. The composition of claim 30 or 31 , wherein the pharmaceutically acceptable carrier or excipient comprises lipid inorganic nanoparticles (LION).

33. The composition of any one of claims 30-32 in a form suitable for administration orally, to a mucous membrane, or to a respiratory system.

34. The composition of any one of claims 30-32 in a form suitable for intramuscular, wingweb, intravenous, or other parenteral administration.

35. The composition of any one of claims 30-32 in a particulate or nanoparticulate form.

36. The composition of any one of claims 30-32 in the form of a liposome, lipoplex, or lipid nanoparticle (LNP).

37. The composition of any one of claims 390-32 in the form of a nanoemulsion comprising a hydrophobic core that comprises a mixture of a liquid oil and one or more inorganic nanoparticles, one or more lipids and, optionally, one or more surfactants.

38. A method for reducing the severity of ILTV or comorbid condition(s) in an avian comprising: administering to a subject in need thereof an saRNA that encodes at least one ILTV antigen or an immunogenic fragment, epitope, or variant of any thereof, according to any one of claims 1-24; a plasmid or DNA construct according to claim 28; a cell according to claim 29; or a composition to any one of claims 30-37.

39. The method of claim 38, wherein reducing the severity of ILTV comprises reducing at least one of: a symptom of sneezing, head shaking, lethargy, discharge from the eyes and nostrils, and difficulty breathing compared to an unvaccinated control avian.

40. The method of claim 38, wherein reducing the severity of ILTV comprises reducing weight loss of vaccinated avians compared to unvaccinated control avians.

41. The method of claim 38, wherein reducing the severity of ILTV comprises reducing mortality of vaccinated avians compared to unvaccinated control avians.

42. The method of claim 38, wherein reducing the severity of ILTV comprises reducing transmission of ILTV to vaccinated avians compared to unvaccinated control avians.

43. The method of claim 38, wherein reducing the severity of ILTV comprises reducing transmission of ILTV to unvaccinated avians compared to transmission of ILTV from unvaccinated avian.s44. The method of claim 38, wherein the avian is Gallus gall us domesticus (domestic chicken).

45. The method of any one of claims 38-44, wherein the avian has not been previously exposed to ILTV.

46. The method of any one of claims 38-44, wherein the avian has been previously exposed to ILTV.

47. The method of any one of claims 38-44, wherein the avian is a carrier of ILTV.

48. The method of any one of claims 38-47, wherein the construct is administered orally, muscosally, or via the respiratory system.

49. The method of any one of claims 38-44, to a wing web, or otherwise parenterally.

50. The method of any one of claims 38-49, wherein the saRNA is administered in a nanoemulsion comprising a hydrophobic core that comprises a mixture of a liquid oil and one or more inorganic nanoparticles, one or more lipids and, optionally, one or more surfactants.

51. A method for making the construct of any one of claims 1-24, comprising operably inserting into a saRNA platform a nucleic acid sequence that expresses at least one ILTV polypeptide or an immunogenic fragment or epitope thereof, or a variant thereof.

52. The method of claim 51, further comprising operably inserting 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 stalling site or protein separation site, a DLP loop, an IRES, an immunogenic carrier, n immunomodulatory protein, or a cytokine.

53. The method of claim 51 or 52, wherein the at least one ILTV polypeptide expressed by the saRNA is folded differently or is post-translationally or epigenetically modified compared to said ILTV polypeptide expressed in the cytoplasm or expressed via the endoplasmic reticulum and Golgi apparatus.

54. The method of any one of claims 51-53, further comprising replacing at least one degenerate codon in the nucleic acid sequence which encodes 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 expression ILTV polypeptides, polypeptide fragments or variants, or increases the stability of the saRNA.

55. The method of any one of claims 51-54, wherein the platform comprises a self-amplifying RNA engineered for resistance to innate cellular immune responses.

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