Methods and compositions for astrovirus replicons

Abstract

The present invention provides recombinant replicons and methods of using the recombinant replicons to express a secreted protein of interest in order to induce an enhanced immune response.

Description

[0001] This application is a divisional application. The application date of the original application is June 10, 2020, the application number is (202080041231.0), and the name is “Methods and compositions of astrovirus replicons”. Technical Field

[0002] The present invention relates to recombinant replicons for eliciting improved immune responses and methods of using the same. Background Art

[0003] A fundamental aspect of using vaccines to generate immunity is to deliver an immunogenic agent to a subject in order to elicit a response from the subject's immune system. Specifically, effective stimulation of the subject's adaptive immune system is important in developing long-term immunity to specific pathogens. One approach to immunization against viral pathogens involves the introduction of independently replicable viral genetic material, i.e., replicons, into host cells, thereby causing the expression and secretion of antigenic proteins that stimulate the immune system.

[0004] Current replicon technology was originally conceptualized and developed based on the ability to package RNA replicons in virus-derived nucleocapsids with or without envelope glycoproteins, known as viral replicon particles (VRPs). This approach is necessarily limited to larger viruses with sufficient packaging capacity that would allow the incorporation of large amounts of exogenous genomic information. To meet this requirement, two commonly used VRPs are derived from alphaviruses or flaviviruses, both of which have relatively large genomes (>11 kilobases) and particle sizes (>60 nm).

[0005] The emergence of non-viral delivery of RNA using various formulations that can prevent RNA degradation makes it possible to develop naked RNA replicons using positive-strand RNA viruses containing infectious RNA genomes. There is no need to package the replicons in viral particles, and there is no need to use replicons derived from larger viruses. However, this technology is still focused on the use of alpha virus and flavivirus replicons, which is likely due to the extensive use of the two viral replicons in history. In addition, naked RNA replicons based on alpha viruses have been proven to be effective as vaccine platforms because they have robust antigen expression kinetics, which are attributed to the use of subgenomes encoding heterologous genes of interest and robust induction of the host's IFN-mediated antiviral state and resistance to the host's IFN-mediated antiviral state. However, since it is desirable to reduce the effective dose of replicon material in order to best avoid potential toxicity problems, genome size remains a consideration. In addition, when smaller constructs are used, nucleic acid manipulation using recombinant DNA technology is greatly simplified, resulting in greater gene tractability and faster candidate vaccine development. Therefore, the development of replicons with smaller genomes that exhibit the same or similar beneficial properties as existing platforms remains a desirable goal.

[0006] The present disclosure overcomes shortcomings in the art by providing recombinant Astrovirus replicons that meet this desirable profile and are effective in inducing an increased immune response. Summary of the invention

[0007] According to the description, each embodiment includes a recombinant replicon nucleic acid, the recombinant replicon nucleic acid comprising: a first open reading frame, the first open reading frame comprising a.) a subgenomic nucleic acid sequence encoding a protein of interest that can be secreted by a cell; b.) a second open reading frame, the second open reading frame comprising a nucleic acid sequence encoding a first astrovirus nonstructural protein (nsP1a) and comprising a hypervariable region; and c.) a third open reading frame, the third open reading frame comprising a nucleic acid sequence encoding a second astrovirus nonstructural protein (nsP1b) and comprising a subgenomic promoter, the subgenomic promoter being positioned to facilitate initiating transcription of the subgenomic nucleic acid sequence. In some embodiments, the recombinant replicon nucleic acid has the following structure: c.→b.→a. In some embodiments, the recombinant replicon nucleic acid has a 7-methylguanylate cap at its 5' end.

[0008] In some embodiments, the first open reading frame further comprises a subset of nucleic acid sequences encoding an Astrovirus structural protein (VP90). In specific embodiments, the subset consists of 5 to 50 nucleotides. In other specific embodiments, the subset consists of 30 nucleotides.

[0009] In some embodiments, the recombinant replicon nucleic acid further comprises an Astrovirus conserved sequence element that begins within the first open reading frame and extends beyond the 3' end of the first open reading frame.

[0010] In some embodiments, the Astrovirus genotype of the hypervariable region is different from the Astrovirus genotype of the recombinant replicon nucleic acid. In some of these embodiments, the Astrovirus genotype of the recombinant replicon nucleic acid is HAstV VII, and the Astrovirus genotype of the hypervariable region is HAstV IV.

[0011] In some embodiments, the third open reading frame comprises a translation upstream ribosome binding site.

[0012] In some embodiments, the first open reading frame further comprises a nucleic acid sequence encoding a peptide having ribosome skipping properties. In a specific embodiment, the peptide is a 2A peptide (T2A) from the capsid protein of Thosea asigna virus.

[0013] Other embodiments include a nanoparticle comprising any of the above-mentioned recombinant replicon nucleic acids. In some of these embodiments, the nanoparticle consists essentially of a nanostructured lipid carrier containing the recombinant replicon nucleic acid. Other embodiments include a formulation comprising a plurality of the nanoparticles in a pharmaceutically acceptable carrier.

[0014] In some embodiments, a method of treating a subject to induce immunity in the subject comprises administering the above formulation to the subject and thereby eliciting an immune response in the subject. In some of these embodiments, the immune response comprises CD4+ T cell activation.

[0015] In some embodiments, a composition includes any of the above-described recombinant replicon nucleic acids in a pharmaceutically acceptable carrier.

[0016] In some embodiments, an isolated cell comprises any of the above-described recombinant replicon nucleic acids. In other embodiments, a method of delivering a therapeutic amount of a protein of interest to a subject comprises administering to a subject an effective amount of the isolated cell, wherein the protein of interest is a therapeutic protein, and the cell secretes the protein of interest and thereby delivers a therapeutic amount of the protein of interest to the subject.

[0017] In some embodiments, a method of secreting a protein of interest from a cell comprises introducing any of the above-described recombinant replicon nucleic acids into the cell under conditions whereby the protein of interest is secreted, and wherein the cell is in cell culture, whereby the protein is secreted from the cell. Some embodiments further comprise the step of harvesting the protein of interest from the cell culture. Another embodiment comprises a composition comprising the protein of interest produced by this method. In still other embodiments, a method of delivering a therapeutic protein of interest to a subject comprises administering the composition to the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A -C shows the organization of the human Astrovirus genome and the importance of this organization for the expression of proteins of interest. Figure 1A A schematic diagram of the genome organization of human Astrovirus (HAstV) comprising three open reading frames (ORFs), ORF 1a, ORF 1b and ORF 2, is shown, and the positions of highly conserved sequence elements flanking ORF 2 that are proposed to play a role in subgenomic transcription and translation are also shown. Figure 1B A table depicting four HAstV replicons is shown. Luciferase (nLUC) is encoded and each has one of four combinations of modifications to the flanking sequences derived from HAstV ORF2 and the 3' untranslated region (3'UTR) adjacent to ORF2. Figure 1C Shown by Figure 1B Expression of nLUC by the replicons described in 293T cells transfected with 100 ng of the replicon and alphavirus replicons encoding nLUC or Zika virus (ZIKV) antigens as positive and negative controls, respectively, and harvested 24 hours later.

[0019] Figure 2A -G shows a comparison between nanostructured lipid formulated replicons in their ability to induce an immune response in C57B1 / 6 mice injected intramuscularly. Figure 2A Shown is a schematic diagram of an alphavirus-derived replicon featuring a subgenome encoding a gene of interest (GOI). Figure 2B Shown are the results of plaque reduction neutralization assays performed on alphavirus replicons carrying the ZIKV gene (four doses of NLC formulated replicons) as well as naked replicon controls and mock injected controls. Figure 2C Shown are the percentages of two different antigen-specific CD8 T cells induced by alphavirus replicons. Figure 2DShown are the levels of two different antigen-specific CD4 T cells induced by alphavirus replicons. Figure 2E Shown from Figure 1B Schematic representation of one of the astroviral replicons (5'-HAstV-3') having a subgenome encoding a GOI. Figure 2F Shown are the combined percentages of antigen-specific CD4 T cells induced by alphavirus replicons and astrovirus replicons encoding two GOIs: ZIKV NS3 antigen and Mycobacterium tuberculosis antigen ID- 93. Also shown are the results from ID-93 protein alone and in the presence of GLA-SE adjuvant. Figure 2G Shown are anti-hemagglutinin immunoglobulin G ELISA titers after a single dose of an unadjuvanted linear epitope (PR8 hemagglutinin (HA) subunit) given to naive mice and mice previously primed with an astrovirus replicon encoding a partial sequence of the hemagglutinin gene.

[0020] Figure 3A -D shows the effect of ORF 2 sequence length on the predicted replicon secondary RNA structure and gene expression. Figure 3A The predicted secondary structure of a 400 nucleotide (nt) region encompassing the 3' end of ORF 1b and the 5' end of ORF 2 of wild-type (WT) HAstV is shown. The triple hairpin structure is depicted with light and medium lines, with the ORF 2 start codon boxed. Figure 3B Shown is the predicted secondary structure of a 167-nt region in the 5′-3′ HAstV replicon encompassing the first 9 nt of ORF 2, depicting a clear loss of the triple hairpin structure. Figure 3C The predicted secondary structure of the 188-nt region encompassing the first 30 nt of ORF 2 (depicted as a light line at the lower right of the structure) is shown, in which the triple hairpin structure is apparently stabilized. Figure 3D The results for the replicon with the first 9 nt of ORF 2 are shown. Figure 1B Results of luciferase assays in 293T cells with three replicons of and three replicons containing the first 30 nt of ORF 2 with the following: unmodified flanking sequence (5'-30nt-3'); modified 5' flanking sequence (Δ5'-30nt-3'); and unmodified flanking sequence with the addition of the ribosomal skipping sequence of the 2A (T2A) ribosomal skipping sequence of the cleaved thrush virus (5'-30nt-T2A-3').

[0021] Figure 4A -C shows the results of a coupled transcription and translation assay for detecting subgenomic transcription and translation in alphavirus and astrovirus replicons. Figure 4ASubgenomic transcription in CaCo-2 cells infected with wild-type HAstV is shown. Figure 4B Shown are the results of quantitative reverse transcription polymerase chain reaction (qRT-PCR) and luciferase assays following transfection of 293T cells with the 5'-30nt-T2A-3' replicon encoding nLUC and an uncapped control. Figure 4C Shown are the results of qRT-PCR and luciferase assays following transfection of 293T cells with alphavirus replicons encoding nLUC and uncapped controls.

[0022] Figure 5 Shown is a comparison of gene expression in two cell types between 5'-30nt-T2A-3' replicons in which the hypervariable region (HVR) of ORF 1a belongs to the native genotype (HVR-VII) or the divergent genotype (HVR-IV).

[0023] Fig. 6A -B shows the results of a bicistronic reporter assay used to study the ORF 2 translation control mechanism, in particular translation termination-reinitiation. Fig. 6A A summary of selected mutations of ORF 1b is shown. Figure 6B The results show that the Fig. 6A Results of dual luciferase assays performed on BHK cell lysates 24 h after transfection with each plasmid as described in .

[0024] Fig. 7A -B shows the dependence of downstream ORF expression on the upstream ORF sequence. Fig. 7A A depiction of a plasmid construct with a deletion in ORF 1b is shown. Figure 7B Shown are the results of dual luciferase assays performed in BHK cells 24 hours after transfection with 100 ng of each plasmid. DETAILED DESCRIPTION

[0025] As used herein, "replicon nucleic acid" or "replicon" refers to a region of a ribonucleic acid (RNA) molecule or RNA that replicates from a single replication origin. The term "replicon nucleic acid" refers to a replicon nucleic acid that has been altered by human intervention. As a non-limiting example, a recombinant nucleic acid molecule: 1) has been synthesized or modified in vitro, such as using chemical or enzymatic techniques (e.g., by using chemical nucleic acid synthesis or by using enzymes for replication, polymerization, exonucleoside, endonucleoside, connection, reverse transcription, transcription, base modification (including, for example, methylation) or recombination (including homology and site-specific recombination) of nucleic acid molecules); 2) contains essentially unconnected connecting nucleotide sequences; 3) has been engineered using molecular cloning techniques so that it lacks one or more nucleotides relative to a naturally occurring nucleic acid molecule sequence; and / or 4) has been manipulated using molecular cloning techniques so that it has one or more sequence changes or rearrangements relative to a naturally occurring nucleic acid sequence.

[0026] The term "nucleic acid sequence" refers to the sequence of a nucleic acid molecule. The nucleotide base nomenclature as set forth in 37C.FR § 1.822 is used herein. Nucleic acid molecules can be any length, including but not limited to between 3Kb and 50Kb, such as between 3Kb and 40Kb, between 3Kb and 40Kb, between 3Kb and 30Kb, between 3Kb and 20Kb, between 5Kb and 40Kb, between 5Kb and 40Kb, between 5Kb and 30Kb, between 5Kb and 20Kb, or between 10Kb and 50Kb, such as between 15Kb and 30Kb, between 20Kb and 50Kb, between 20Kb and 40Kb, between 5Kb and 25Kb, or between 30Kb and 50Kb. Nucleic acid molecules can also be, for example, more than 50kb.

[0027] The term "open reading frame" (ORF) means a nucleic acid sequence consisting of a continuous stretch of codons starting with a start codon (usually AUG) and ending with a stop codon (usually UAA, UAG or UGA). More than one open reading frame can exist in a single nucleic acid molecule, and an open reading frame can overlap with another open reading frame on the same molecule. For example, the nucleic acid sequence of one open reading frame can contain the start codon of another open reading frame.

[0028] The term "Astroviral 5' untranslated region (5'UTR)" means a fragment of the Astroviral genome comprising a nucleic acid sequence located upstream of the start AUG of the open reading frame ORF 1a.

[0029] By "Astroviral 3' untranslated region (3'UTR)" is meant a fragment of the Astroviral genome comprising the nucleic acid sequence located downstream of the stop codon of the open reading frame ORF2.

[0030] A "subgenomic promoter" is a promoter that directs transcription of a subgenomic messenger RNA as part of the replication process. Such a promoter may have a wild-type sequence or a sequence that has been modified from the wild-type sequence but retains promoter activity.

[0031] The term "conserved sequence element (CSE)" describes an RNA element with similar position, sequence and secondary structure in the genomes of all known human astroviruses.

[0032] As used herein, an "isolated cell" is a cell or cell population that has been removed from the environment in which the cell naturally exists and / or has been altered or modified from the state in which the cell exists in its natural environment. An isolated cell can be, for example, a cell in cell culture. An isolated cell can also be a cell that can be in an animal and / or introduced into an animal, and wherein the cell has been altered or modified, for example, by introducing an alpha virus particle into the cell.

[0033] "Subject" includes, but is not limited to, warm-blooded animals such as humans, non-human primates, horses, cows, cats, dogs, pigs, rats, and mice.

[0034] Some embodiments provide compositions (e.g., pharmaceutical compositions) comprising a replicon encapsulated in a supramolecular structure to form a nanoparticle, wherein a plurality of such nanoparticles are dispersed in a pharmaceutically acceptable carrier. In particular embodiments, the nanoparticles include a replicon encapsulated in a lipid-based nanoparticle. In a specific aspect, the nanoparticles include a replicon encapsulated in a nanostructured lipid carrier (NLC). An example of a suitable NLC is described in Erasmus et al., Molecular Therapeutics 26(10):2507-2522 (2018).

[0035] "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject with the selected nanoparticles without causing a substantial deleterious biological effect or interacting in a deleterious manner with any of the other components of a composition comprising the other components. Pharmaceutically acceptable carriers are suitable for administration or delivery to humans and other subjects. As is well known to those skilled in the art, the carrier will naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject (see, e.g., Remington's Pharmaceutical Science; latest edition). Pharmaceutical formulations such as vaccines or other immunogenic compositions may include an immunogenic amount of the disclosed Astrovirus replicons and a pharmaceutically acceptable carrier. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sterile pyrogen-free water and sterile pyrogen-free saline solutions.

[0036] The administration of various compositions (e.g., nucleic acids, nanoparticles, pharmaceutical compositions) can be accomplished by any of several different approaches. The compositions can be administered intramuscularly, subcutaneously, intraperitoneally, intradermally, intranasally, intracranially, sublingually, intravaginally, intrarectally, orally or topically. The compositions can also be administered transdermally by skin scarification or by a patch or liquid. The compositions can also be delivered subcutaneously in the form of a biodegradable material that releases the compositions over a period of time. The compositions can also be delivered intramuscularly by injection.

[0037] Nucleic acids, nanoparticles and pharmaceutical compositions can be used in methods for delivering secreted proteins of interest to cells, which can be cells in a subject. Therefore, some embodiments provide a method for introducing an effective amount of the nucleic acid, nanoparticle and / or composition of the embodiments into a cell. A method for delivering an effective amount of the nucleic acid, nanoparticle and / or composition of the embodiments to a subject is also provided. According to well-known gene therapy protocols, such methods can be used to confer a therapeutic effect on cells and / or subjects.

[0038] Astrovirus replicons offer an attractive alternative by combining their smaller genome size with those features offered by alphavirus replicons: a subgenomic RNA replication strategy and a delayed but robust IFN induction. The astrovirus replicon machinery is encoded by approximately 4 kb RNA, whereas those of alphavirus or flavivirus origin are encoded by approximately 8 kb RNA. This reduces the effective dose in terms of copy number by approximately 2-fold.

[0039] As used herein, "effective amount" refers to the amount of a composition or formulation sufficient to produce a desired effect, which may be a therapeutic effect. The effective amount will vary with the age of the subject, the general condition; the severity of the condition being treated; the specific agent being administered; the duration of treatment; the nature of any concurrent treatment; the pharmaceutically acceptable carrier used; and similar factors within the knowledge and expertise of those skilled in the art. Appropriately, the effective amount in any individual case can be determined by those skilled in the art by reference to relevant texts and literature and / or by using routine experiments. (See, for example, Remington, The Science and Practice of Pharmacy (20th ed. 2000)).

[0040] The replicon RNA compositions described herein are administered in a manner compatible with dosage formulations and in an amount that is prophylactically and / or therapeutically effective. 4 to 10 10 In the range of unit doses (e.g., 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10 The amount of the active ingredient to be administered depends on: the subject to be treated, the route of administration or delivery of the particles, the immunogenicity of the expressed product, the type of effector immune response desired, and the degree of protection desired. The effective amount of the active ingredient required to be administered or delivered may depend on the judgment of a physician, veterinarian or other health practitioner and may be specific to a given subject, but such determination is within the skill of such practitioners.

[0041] The disclosed compositions and formulations can be administered in a single dose regimen or a multiple dose regimen. A multiple dose regimen is a regimen in which the main course of administration may comprise 1 to 10 or more individual doses, followed by other doses administered at subsequent time intervals as needed to maintain and / or enhance the desired effect (e.g., therapeutic response).

[0042] The term "therapeutic amount" refers to an amount sufficient to confer a modulating effect (e.g., a therapeutic response) on a subject suffering from a condition, disease, or disorder, which modulating effect, for example, can be a beneficial effect, including an improvement in the subject's condition (e.g., an improvement in one or more symptoms), a delay or slowing of the progression of a condition, a delay in the onset of a condition, disease, or disorder, and / or a change in any of the clinical parameters of the condition, disease, or disorder, etc., as are well known in the art. Another example of a therapeutic response contemplated in the present disclosure is increasing resistance to pathogenic disease by stimulating the subject's immune system.

[0043] As used herein, "a", "an", and "the" may mean one or more than one, depending on the context in which they are used. For example, "a" cell may mean one cell or a plurality of cells. Furthermore, as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted in an alternative form ("or").

[0044] It should be understood that the foregoing detailed description is given by way of illustration only and that modifications and variations may be made therein without departing from the spirit and scope of the present invention.

[0045] Examples

[0046] The following examples are provided to illustrate certain disclosed embodiments and should not be construed as limiting the scope of the disclosure in any way.

[0047] Example 1 - Materials and Methods

[0048] Preparation of replicon RNA

[0049] Various astrovirus replicon sequences were synthesized and cloned into plasmids downstream of the T7 promoter and upstream of the hepatitis delta virus ribozyme sequence, T7 terminator, and NotI restriction site. To prepare RNA for downstream studies, the purified plasmids were linearized by restriction digestion with NotI enzyme and then purified by phenol-chloroform and ethanol precipitation. The linearized template was then used for RNA transcription using T7 polymerase and purified by LiCl precipitation and ethanol wash. The RNA transcripts were then capped using vaccinia virus capping enzyme and purified by LiCl precipitation and ethanol wash.

[0050] Example 2 - Protein expression from replicons with selected modifications

[0051] The wild-type (WT) human astrovirus (HAstV) replication machinery consists of 5' and 3' untranslated regions (UTRs) and the nonstructural proteins nsP1a and nsP1b, encoded by two overlapping open reading frames, ORF 1a and ORF 1b, respectively, which are processed by the ribosomal frameshift machinery. The 3' end of ORF 1b contains a proposed subgenomic promoter that is assumed to initiate transcription of subgenomic RNAs mediated by proteins translated from ORFs 1a and 1b. The structural proteins encoded by ORF 2 are thought to be translated from this subgenomic RNA, whose start codon overlaps with the 3' end of ORF 1b. In addition, there is a highly conserved stem-loop sequence that begins at the 3' end of ORF 2 and ends in the 3'UTR. See Figure 1A .

[0052] Four replicons containing combinations of these conserved sequence elements were developed to test whether the four replicons were important for ORF2 expression. The 5'-3' replicon contained intact 5' and 3' conserved sequence elements. The other replicons contained synonymous mutations in ORF 1b that silenced the start methionine of ORF 2, (Δ5'), deletions of the conserved 3' ORF 2 sequence (Δ3'), or both (Δ5'-Δ3'). ORF 2 contained a luciferase (nLUC). Figure 1B .

[0053] To test the effect of sequence elements on ORF 2 expression, 293T cells were transfected with 100 ng of each replicon and alphavirus replicons encoding nLUC or Zika virus (ZIKV) antigens as positive and negative controls, respectively, and nLUC expression was measured 24 hours later. Figure 1C The results of this test are shown in . Although the intact 5' and 3' sequences enhanced nLUC expression 5-fold compared to the Δ5'-Δ3' counterpart and about 25-fold compared to background, the expression level was about 17,000-fold lower than the alphavirus positive control, suggesting that additional nucleic acid sequences may be important for enhancing ORF 2 expression.

[0054] Example 3 - CD4+ T cell induction by recombinant astrovirus replicons

[0055] Next, the ability of the 5'-3' HAstV replicons to induce T cell responses to the Mycobacterium tuberculosis antigen ID-93 or the Zika virus NS3 antigen was evaluated. To prepare these constructs, sequences encoding the ID-93 or Zika virus NS3 proteins were synthesized and cloned into the 5'-3' HAstV replicon ( between the AvrII restriction site and the PvuI restriction site) using Gibson assembly. Figure 2E ) and an alphavirus replicon derived from the Venezuelan equine encephalitis virus TC-83 strain between the PflMI restriction site and the SacII restriction site ( Figure 2A ). Capped RNA was then prepared as described in Example 1 above and transfected into BHK cells to confirm antigen expression by Western blot (data not shown). After confirming antigen expression, the replicons were formulated in nanostructured lipid carriers and 1 μg of each replicon was administered to C57BL / 6 mice by a single intramuscular injection. For the ID-93 replicon, additional controls for the protein subunits alone and with adjuvant (GLA-SE) were included to compare T cell responses between the replicons and a more traditional vaccine preparation, which has previously been shown to induce potent antigen-specific CD4 + T cell responses. Fourteen days after a single injection, spleens were harvested and stimulated with MHC-II restricted peptides derived from Zika virus NS3 or mycobacterium ID-93 antigens and stained for analysis by flow cytometry ( Figure 2F ).

[0056] The results shown in FIG. 2 demonstrate that although non-viral delivery of alphavirus-derived replicons encoding bacterial or viral antigens drives potent CD8+ and antibody responses to the encoded antigens after a single intramuscular injection ( Figure 2B -D), but astrovirus-derived replicons encoding the same antigen drove significantly higher antigen-specific CD4+ T cell responses ( Figure 2F ).

[0057] To test whether these CD4+ T cell responses to linear epitopes can enhance antibody responses to the entire protein subunit, C57Bl / 6 mice were primed with a single dose of an astrovirus replicon encoding a 15 amino acid (aa) sequence of the hemagglutinin (HA) gene, which is conserved among seasonal influenza virus subtypes and previously shown to be reactive in C57Bl / 6 mice. Twenty-one days later, a single dose of unadjuvanted recombinant PR8 HA subunit protein was administered to astrovirus RNA primed mice as well as naive mice, and anti-HA IgG ELISA titers were compared. The anti-HA ELISA titers (mean = 1:2200) of mice primed with astrovirus RNA encoding the conserved 15aa sequence were significantly higher (5.5-fold) compared to the anti-HA ELISA titers (mean = 1:400) of mice receiving protein alone. Figure 2G ).

[0058] Example 4 - Effect of ORF 2 sequence composition on expression and translation

[0059] A. ORF 2 sequence length

[0060] The predicted secondary RNA structure of the 400-bp region of WT HAstV containing the proposed subgenomic RNA promoter was evaluated and compared with the predicted structure of the 5'-3' nLUC replicon containing the first 9 nucleotides (nt) of ORF 2 of Examples 2 and 3. Figure 3A In the figure, a triple hairpin structure can be clearly observed (shown as a structure presented with light and medium lines), where the start codon of ORF 2 is boxed. The predicted secondary structure of the 5'-3' replicon with the first 9 nt of ORF 2 ( Figure 3B In the figure (shown in Figure 2), there are no two hairpin structures, only hairpins that are conserved between the WT sequence and the replicon sequence (depicted in light lines). By including an additional 21 nt from ORF 2 (depicted in light lines at the bottom right of the figure), the three-hairpin structure appears to be stabilized in the prediction ( Figure 3C ).

[0061] To evaluate the role of these 30 nt in HAstV replication, additional HAstV replicons containing the 30-nt sequence with or without synonymous mutations in ORF 1b were constructed (5'-30nt-3' replicons or Δ5'-30nt-3' replicons, respectively). For the 5'-30nt-3' replicon encoding the N-terminal 10 amino acids of ORF 2, the ribosomal skipping sequence of the 2A (T2A) ribosomal skipping sequence of the lycopodiella virus was inserted before the nLUC gene to prepare the 5'-30nt-T2A-3' replicon.

[0062] After in vitro transcription and blocking of RNA, 293T cells were transfected with the same dose of each construct, each of which contained two Venezuelan equine encephalitis virus replicons (one Venezuelan equine encephalitis virus replicon (VEE-nLUC) containing the nLUC gene and another Venezuelan equine encephalitis virus replicon (VEE-ZIKV) containing the ZIKV antigen gene as positive and negative controls, respectively. 24 hours after transfection, luciferase assays were performed to quantify heterologous gene expression. Figure 3D The results shown in support the conclusion that the 30 nt enhances heterologous gene expression and that the ORF 2 start codon is involved in efficient translation initiation, with the detected luciferase activity being more than 16,000-fold above background levels, approaching within 30-fold of alphavirus replicating viral RNA.

[0063] B. Effects of the identified ORF 2 sequences on translation

[0064] Having demonstrated the importance of the first 30 nt in ORF 2 expression, we next sought to determine the role of this sequence element in subgenomic transcription.

[0065] The quantitative reverse transcription (qRT) PCR assay was designed to quantify the genomic and subgenomic copies accumulated during astrovirus replication and to validate the assay in the context of WT astrovirus replication. CaCo-2 cells were infected with WT astrovirus at a multiplicity of infection of 0.1 and cell lysates were collected at 0, 4, 8, 12 and 24 hours post infection. RNA was then extracted and run in a qRT-PCR assay together with T7-transcribed RNA from an infectious clone of the same virus to be used as a standard curve. A 5-fold excess of subgenomic transcripts could be detected compared to genomic transcripts starting at 8 hours post infection ( Figure 4A ), recapitulating previously published northern blot data and confirming that this assay can indeed detect subgenomic transcripts.

[0066] Next, this assay was applied in the context of replicons that do not encode structural genes and cannot spread between cells, and the assay also allows for simple quantification of ORF 2 expression coupled to transcription. As a positive control, a similar qRT-PCR assay was designed to detect both genomic and subgenomic levels of alphavirus replicons. Although alphavirus replicons exhibited excessive subgenomic transcription starting 8 hours after transfection, consistent with nLUC expression ( Figure 4B), but the astrovirus replicon (5'-30nt-T2A-3') did not clearly show subgenomic transcription exceeding genomic transcription, and interestingly, nLUC expression was detectable as early as 30 min after transfection ( Figure 4B ).

[0067] These results suggest that: 1) the sequence elements tested are insufficient to mediate subgenomic RNA transcription, and 2) HAstV utilizes an alternative ORF 2 expression mechanism that allows ORF 2 to be translated early independently of subgenomic RNA transcription. Similar observations have been made for caliciviruses that also utilize subgenomic messages to translate their structural genes. Early expression of structural genes independent of subgenomic transcription has been described in bovine norovirus. This may also indicate an important role for structural gene expression in RNA replication.

[0068] Example 5 - Effect of ORF 1a chimera on ORF 2 gene expression

[0069] Regions of ORF 1a, termed hypervariable regions (HVRs), were examined for differences in genomic and subgenomic transcription. HVRs derived from genotype IV HAstV were associated with higher viral titers in clinical samples and differences in the subgenomic to genomic ratio.

[0070] Using the 5'-30nt-T2A-3' replicon described above as the backbone (genotype VII HVR), the HVR was replaced with the HVR of genotype IV HAstV and transfected into CaCo2 cells and BHK cells. Figure 5 As shown in , while no differences in ORF 2 expression were detected in BHK cells, small but significant differences were detected on CaCo-2 cells, supporting previously published data observed for wt virus on CaCo-2 cells. The difference in results between cell lines suggests a role for HVR in host range. Interestingly, HVR chimeras demonstrated lower toxicity in E. coli, resulting in higher plasmid yields, which may prove useful in downstream applications of astrovirus replicons.

[0071] Example 6 - Mechanism of subgenomic transcription-independent ORF 2 translation

[0072] Given the evolutionary relationship between caliciviruses and astroviruses, we next tested whether astroviruses utilize translation termination reinitiation (TTR) between ORF 1b and ORF 2 that allows subgenomic transcription-independent translation of ORF 2 early in the replication cycle in a similar manner to caliciviruses. To test this hypothesis, bicistronic reporter genes encoding Renilla luciferase and firefly luciferase separated by an 808 bp region of ORF 1b and ORF 2 of HAstV-1 were generated in the first and second ORFs, respectively. Fig. 6A ). A series of mutations were then performed to test the importance of ORF 1b stop codons and ORF 2 start codons, because TTR in Calicivirus has been shown to depend on the position of the upstream ORF stop codon, rather than the downstream ORF start codon. As a negative control, a stop codon (3' stop) was inserted at the end of ORF 1b immediately before the ORF 2 start codon. After transfection of BHK cells with 100ng of each plasmid, a dual luciferase assay was performed to first measure firefly activity, then quench and detect Renilla activity ( Figure 6B ). The downstream ORF (firefly) expression was then normalized to the upstream ORF (Renilla) expression relative to the negative control (3' termination).

[0073] The WT ORF 1b / ORF 2 sequence resulted in a 12-fold increase in downstream ORF expression relative to a 3' termination negative control. Although the type of ORF 1b stop codon did not appear to be critical for downstream ORF expression, changing the position by replacing the stop codon with TGG, encoding tryptophan, thereby causing the ORF 1b reading frame to extend an additional 34 codons before termination appeared to eliminate downstream ORF expression. Finally, replacing the start codon of ORF 2 with ACG did not appear to affect downstream ORF 2 expression. These findings are consistent with TTR in Calicivirus.

[0074] The mechanism of TTR in caliciviruses has been shown to depend on the binding of complementary sequences in the host 18s ribosomal RNA to upstream sequences in the calicivirus genome, known as translation upstream ribosome binding sites (TURBS), allowing disengaged ribosomes to reinitiate translation of downstream ORFs. To identify the potential location of such a sequence in astrovirus ORF 1b, we next used a bicistronic reporter system ( Fig. 7A ). In addition, mutations were generated within a TURBS-like sequence located in the del3 mutant to see if the sequence was important for downstream ORF expression. The results showed that while the TURBS-like sequence did not appear to significantly affect downstream ORF expression, a 200 bp sequence located within the del3 mutant ( Figure 7B). Interestingly, this 200 bp sequence is expected to form Figure 3C The triple hairpin structure depicted in .

[0075] Equivalent

[0076] The foregoing written description is considered sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and examples detail certain embodiments and describe the best mode contemplated by the inventor. However, it should be understood that no matter how detailed the foregoing may appear in text, the embodiments may be practiced in many ways and should be interpreted in accordance with the appended claims and any equivalents thereof.

[0077] As used herein, the terms refer to values, including, for example, integers, fractions, and percentages, whether or not explicitly stated. The terms generally refer to a range of values ​​(e.g., + / - 5-10% of the range listed) that one of ordinary skill in the art would consider equivalent to the listed value (e.g., having the same function or result). When terms such as at least and etc. precede a list of values ​​or ranges, the terms modify all values ​​or ranges provided in the list. In some cases, the terms may include values ​​rounded to the nearest valid figure.

[0078] Sequence Listing

[0079] The following sequence listing provides a list of sequences disclosed herein. It should be understood that if a DNA sequence (including Ts) is referenced relative to an RNA, then the Ts should be replaced by Us (with or without modification depending on the context), and vice versa.

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

Claims

1. A recombinant replicon nucleic acid comprising: a. a first open reading frame comprising a subgenomic nucleic acid sequence encoding a protein of interest that can be secreted by a cell; b. a second open reading frame comprising a nucleic acid sequence encoding a first astrovirus nonstructural protein (nsP1a) and comprising a hypervariable region; and c. A third open reading frame comprising a nucleic acid sequence encoding a second Astrovirus nonstructural protein (nsP1b) and comprising a subgenomic promoter positioned so as to initiate transcription of the subgenomic nucleic acid sequence.

2. The recombinant replicon nucleic acid of claim 1, wherein the first open reading frame further comprises a subset of nucleic acid sequences encoding an Astrovirus structural protein (VP90).

3. The recombinant replicon nucleic acid of claim 2, wherein the subset consists of 5 to 50 nucleotides.

4. The recombinant replicon nucleic acid of claim 3, wherein the subset consists of 30 nucleotides. The recombinant replicon nucleic acid according to claim 1 , which has the following structure: c.→b.→a.

6. The recombinant replicon nucleic acid of claim 1, further comprising an Astrovirus conserved sequence element starting within the first open reading frame and extending beyond the 3' end of the first open reading frame.

7. The recombinant replicon nucleic acid of claim 1, having a first Astrovirus genotype, wherein the hypervariable region has a second Astrovirus genotype different from the first Astrovirus genotype.

8. The recombinant replicon nucleic acid according to claim 7, wherein the first Astrovirus genotype is HAstVVII and the second Astrovirus genotype is HAstV IV.

9. The recombinant replicon nucleic acid of claim 1, wherein the third open reading frame comprises a translation upstream ribosome binding site.

10. The recombinant replicon nucleic acid according to any one of the preceding claims, having a 7-methylguanylate cap at its 5' end.

11. The recombinant replicon nucleic acid of any one of the preceding claims, wherein the first open reading frame further comprises a nucleic acid sequence encoding a peptide having ribosome skipping properties.

12. The recombinant replicon nucleic acid according to claim 11, wherein the peptide having ribosome skipping properties is a 2A peptide (T2A) from the capsid protein of Thosea asigna virus.

13. A nanoparticle comprising the recombinant replicon nucleic acid according to any one of the preceding claims.

14. The nanoparticle of claim 13, consisting essentially of a nanostructured lipid carrier containing the recombinant replicon nucleic acid.

15. A formulation comprising a plurality of nanoparticles according to any one of claims 13 or 14 in a pharmaceutically acceptable carrier.

16. A composition comprising the recombinant replicon nucleic acid according to any one of claims 1 to 11 in a pharmaceutically acceptable carrier.

17. An isolated cell comprising the recombinant replicon nucleic acid of any one of claims 1 to 11.

18. A method of treating a subject to render said subject immune, said method comprising administering to said subject a formulation according to claim 16 and thereby eliciting an immune response in said subject.

19. The method of claim 18, wherein the immune response comprises CD4+ T cell activation.

20. A method for secreting a protein of interest from a cell, the method comprising introducing a recombinant replicon nucleic acid according to any one of claims 1 to 11 into the cell, wherein the cell is in cell culture, under conditions whereby the protein of interest is secreted, whereby the protein is secreted from the cell.

21. The method of claim 20, further comprising the step of harvesting the protein of interest from the cell culture.

22. A composition comprising the protein of interest produced by the method of claim 20.

23. A method of delivering a therapeutic protein of interest to a subject, the method comprising administering to the subject the composition of claim 22.

24. A method of delivering a therapeutic amount of a protein of interest to a subject, the method comprising administering to the subject an effective amount of the isolated cells of claim 17, wherein the protein of interest is a therapeutic protein, and the cells secrete the protein of interest and thereby deliver a therapeutic amount of the protein of interest to the subject.

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