Method for enhancing self-amplifying RNA mediated protein expression

Abstract

The present invention relates to isolated self-amplifying RNA (saRNA) constructs and the uses thereof. The invention relates to self-amplifying RNA, which encode an RNA-dependent-RNA- polymerase (RdRP), a heterologous protein, and a viral suppressor of RNAi protein, as well as methods and uses thereof.

Description

[0001] METHOD FOR ENHANCING SELF-AMPLIFYING RNA MEDIATED PROTEIN EXPRESSION

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to isolated self-amplifying RNAs (saRNAs) and methods for enhancing self-amplifying RNA mediated protein expression. Expression of a B2 protein from a viral-genome based saRNA increases the virus’ ability to express a gene of interest in stem cells and somatic cells.

[0004] BACKGROUND

[0005] Following the success of messenger RNA (mRNA)-based vaccines to tackle the global COVID-19 pandemic, there is a tremendous interest in RNA technologies and in their optimisation. Selfamplifying RNA (saRNA) holds great promise as a novel strategy for efficient vaccine design since its ability to replicative within the cells allows for high and long-term expression of an antigen in vivo and thereby necessitates a lower dose to confer protective immunity compared to non-replicative mRNAs.

[0006] saRNA technology is often derived from alphaviruses, the sole genus of the Togaviridae, which comprises human pathogens such as Chikungunya virus (CHIKV) and Venezuela equine encephalitis (VEEV). The alphavirus genome is a ~12 kilobases (kb) long, single-stranded RNA of positive (+) polarity and comprises two open reading frames (ORFs). The 5’ ORF encodes for a polyprotein, which after processing, results in four non-structural proteins (NSP 1-4) that form the RNA-dependent-RNA-polymerase (RdRP, replicase) and transcription complex. The 3’ ORF is expressed from a subgenomic mRNA (sgmRNA) produced from an internal promoter located on the negative (-)-sense RNA replication intermediate. The sgmRNA encodes for all the structural proteins, which together with newly synthetised genome form new viral particles. In alphavirus genome-based saRNA, all the structural genes are replaced by a gene of interest (GOI) thereby removing its ability to produce viral particles while maintaining its ability to self-amplify via its encoded RdRp in a target cell. It therefore requires a much lower dose of saRNA to express an antigen and to confer protection compared to mRNA.

[0007] However, saRNAs induce a strong innate immune response that is a double-edged sword for its efficiency since it plays a crucial role for its adjuvanticity yet it also induces cell-intrinsic innate antiviral mechanisms detrimental for saRNA stability, replication, transcription and / or translation. In the process of self-amplification within the cells, RdRp generates double-stranded RNA (dsRNA) replication intermediates that trigger innate immune responses such as the interferon (IFN) system. Vertebrates cells are equipped with cytosolic nucleic acid sensors that continuously survey signs of viral infections. The presence of viral RNAs within the cytosol is sensed by the RIG-I-like receptors (RLRs) that comprise RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation factor 5) and LGP2 (laboratory of genetics and physiology 2). RIG-I recognizes 5’ tri or di-phosphorylatedmoieties at base-paired extremities found in certain viral genomes, while MDA5 recognizes long dsRNA molecules which is often a product of viral replication. Both RIG-I and MDA5-mediated innate immune responses are modulated by LGP2. Upon recognition of viral RNAs, RLRs activate the mitochondrial adaptor MAVS and thereby induce a signalling cascade leading to the activation of transcription factors IRF3 and IRF7 (interferon regulatory factor 3 and 7) and NF-KB (nuclear factor kappa-light chain enhance of activated B cells) that eventually induce the expression of proinflammatory cytokines and IFN-I, respectively. While proinflammatory cytokines are key for triggering an inflammatory response in the activated tissue, IFN-I binds to its cognate receptor IFNAR (IFN-a / b receptor) and signals through the JAK / STAT (Janus Kinase / signal transducer and activator of transcription) pathway to induce the transcription of hundreds of interferon-stimulated genes (ISGs). These include proteins such as cytokines and chemokines and co-stimulatory molecules that will favour the initiation of adaptive immunity. The ability of saRNA to produce dsRNA hence confers strong intrinsic adjuvanticity which contribute to an increased immunogenicity compared to non- replicative mRNAs. However, ISGs also comprise proteins with antiviral activities that target various stages of the viral life cycle and that can affect saRNA efficiency. For example, one such ISG encodes for the serine / threonine kinase PKR (protein kinase RNA-dependent) which plays a central role in the antiviral effects mediated by IFN. Albeit being constitutively expressed at steady-state, PKR is upregulated by IFN and get activated upon binding to dsRNA of viral or endogenous origin. Upon recognition of dsRNA, PKR undergoes dimerization and autophosphorylation to form an active kinase. PKR then phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (eiF2a), which causes a general block of translation thereby preventing cellular and viral proteins production. Consistent with a deleterious effect of the IFN response, protein expression derived from saRNA was increased in vivo upon co-administration with an inhibitor of type I IFN signalling (Blakney, A. K, et al. Innate Inhibiting Proteins Enhance Expression and Immunogenicity of Self-Amplifying RNA. Mol Ther 29, 1174-1185 (2021)). Likewise, the immunogenicity of saRNA was increased in MAVS-deficient mice unable to signal through the RLRs compared to wild-type animals (Tregoning, J. S. et al. Formulation, inflammation, and RNA sensing impact the immunogenicity of self-amplifying RNA vaccines. Mol. Ther. - Nucleic Acids 31, 29-42 (2023)). Furthermore, c / s-encoded expression of some viral proteins that inhibit the IFN pathway was shown to enhance immunogenicity (Blakney, A. K. et al, 2021).

[0008] The presence of dsRNA in the cytosol elicits another innate antiviral pathway based on RNA interference (RNAi). This pathway acts as the main antiviral defence mechanisms in plants and invertebrates and recent work shows that this ancestral mechanism is also active in mammals. In antiviral RNAi, the endoribonuclease enzyme Dicer cleaves viral dsRNA replicate intermediates into viral small interfering RNA (siRNAs) of 21 to 23 nucleotides (nt) in length. These viral siRNAs are then loaded into the RNA-induced silencing complex (RISC) composed of Argonaute (Ago) proteins and mediate the sequence-specific cleavage of complementary target viral RNAs. Mammalian antiviral RNAi has been shown in embryonic and tissue stem cells, which naturally have an attenuated IFN response and therefore rely on RNAi as well as on the constitutive expression of asubset of ISGs to counteract viruses. Notably, stem cells express a specific isoform of Dicer that exhibits an increased ability to cleave dsRNA in vitro and a heightened antiviral activity. In differentiated cells, which display a potent IFN response, this isoform of Dicer is expressed at low levels and antiviral RNAi activity is further diminished by IFN-dependent mechanisms.

[0009] saRNAs have the potential to be incredibly useful for transgene expression, yet face problems such as the need to avoid RNAi activity and the need to balance stimulating the innate immune system (by stimulating cytokines, chemokines and co-stimulatory molecules that will favour the initiation of adaptive immunity) without also causing the innate IFN pathway to decrease the stability of the saRNA.

[0010] There is therefore a need to develop saRNAs which do not induce RNAi activity, and maintain their self-adjuvanticity while remaining stable.

[0011] SUMMARY OF THE INVENTION

[0012] In a first aspect of the invention, there is provided an isolated self-amplifying RNA (saRNA) comprising:

[0013] (a) a nucleic acid sequence encoding an RNA-dependent-RNA-polymerase (RdRP); (b) a nucleic acid sequence encoding a heterologous protein; and

[0014] (c) a nucleic acid sequence encoding a viral suppressor of RNAi (VSR) protein.

[0015] In a second aspect of the invention, there is provided a composition comprising a saRNA according to the first aspect of the invention.

[0016] In a third aspect of the invention, there is provided a pharmaceutical composition comprising a saRNA according to the first aspect of the invention.

[0017] In a fourth aspect of the invention, there is provided a vector encoding the saRNA according to the first aspect of the invention.

[0018] In a fifth aspect of the invention, there is provided a cell comprising a saRNA according to the first aspect of the invention or a vector according to the fourth aspect of the invention.

[0019] In a sixth aspect of the invention, there is provided a vaccine comprising a saRNA according to the first aspect of the invention, or a vector according to the fourth aspect of the invention.

[0020] In a seventh aspect of the invention, there is provided a method of modulating expression of a heterologous protein in a cell, comprising introducing to the cell a saRNA according to the first aspect of the invention or a vector according to the fourth aspect of the invention.In an eighth aspect of the invention, there is provided a method of producing induced pluripotent stem (iPS) cells, comprising the steps of:

[0021] (a) introducing to a somatic cell a saRNA according to the first aspect of the invention or a vector according to the fourth aspect of the invention, encoding one or more reprogramming factors; and

[0022] (b) expressing the one or more reprogramming factors in the cell to initiate dedifferentiation of the somatic cell into an iPS cell.

[0023] In a ninth aspect of the invention, there is provided a method of inducing an immune response in a subject, comprising administering a saRNA according to the first aspect of the invention, a composition according to the second aspect of the invention, a pharmaceutical composition according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a vaccine according to the sixth aspect of the invention to a subject.

[0024] In a tenth aspect of the invention, there is provided a saRNA according to the first aspect of the invention, a composition according to the second aspect of the invention, a pharmaceutical composition according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a vaccine according to the sixth aspect of the invention for use in a method of inducing an immune response in a subject.

[0025] In an eleventh aspect of the invention, there is provided a saRNA according to the first aspect of the invention, a composition according to the second aspect of the invention, a pharmaceutical composition according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the fifth aspect of the invention for use in medicine.

[0026] In a twelfth aspect of the invention, there is provided a method of treating a disease or a condition in a subject, comprising administering a saRNA according to the first aspect of the invention, a composition according to the second aspect of the invention, a pharmaceutical composition according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the fifth aspect of the invention. This aspect of the invention also extends to the use of a saRNA according to the first aspect of the invention, a composition according to the second aspect of the invention, a pharmaceutical composition according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the fifth aspect of the invention for the manufacture of a medicament for the treatment of a disease or a condition in a subject.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1. (A, B) Mean Fluorescence Intensity (MFI) analysis of GFP-positive cells. Mouse embryonic stem cells (mESCs) E14 were transfected with CHIKV-GFP-STOP or -B2 saRNAs. Flow cytometrywas used to monitor the GFP expression. MFI of GFPhighpopulation is represented at (A) 16, 24 and 48 hours or (B) 3, 7, 10 and 16 hours after transfection of saRNAs.

[0029] Figure 2. (A) Percentage of GFP-positive cells and (B) MFI of GFPhighpopulations after transfection of mESCs with CHIKV-GFP-B2 (red / left) or CHIK-GFP-B2 R59Q (orange / right) saRNAs. (C) Small RNA deep sequencing analysis was performed in mESCs from A, B and the abundance of 20 to 24-nucleotide long reads mapping the genome of Nodamuravirus (NoV) was determined.

[0030] Figure 3. Percentage of GFP-positive (GFP+) mouse embryonic stem cells (mESCs) transfected with either CHIKV-GFP-B2 (red) or -STOP (blue) or with transfection reagent alone (black).

[0031] Figure 4. (A) MFI of GFPhighpopulation of HeLa cells transfected with CHIKV-GFP-STOP or -B2 monitored at 16, 24 and 48 hours after transfection. (B) Area under the curve for GFPhighMFI values obtained in (A). (C, D) Immunofluorescence of HeLa cells 24 hours after transfection of CHIKV-GFP-B2 or-R59Q in the presence of DMSO (C) or ruxotilinib (D) showing GFP and DAPI.

[0032] Figure 5. (A, B) MFI of GFPhigh(A) or percentage of GFP-positive cells (B) HeLa cells transfected with STOP, B2, or B2-R59Q expressing saRNA in the presence of DMSO, ruxotilinib or IFN-a recombinant proteins 24 hours after transfection. (C) The expression of vector-derived GFP (left) or NSP1 (right) mRNA were monitored by qRT-PCR analysis 24 hours after transfection using oligo-dT for RT reaction.

[0033] Figure 6. (A) Hela cells were either non-transfected (mock) or transfected with poly (l: C), STOP, B2, or B2-R59Q expressing CHlKV-derived saRNA. IFN-stimulated-genes IFIT1 (left), IFIT2 (middle) or Rig-I (right) gene expression was analysed by qRT-PCR 24 hours post-transfection. (B) HEK 293 cells WT or knockout for both RIG-I and MDA-5 (RIG-I- / -MDA-5- / -) were transfected with B2 or B2-R59Q-expressing CHIKV saRNA and the MFI within GFPhighcells were monitored by flow cytometry analysis. The data are from 3 independent experiments where each point represents the average of technical replicates. Connected dots correspond to values obtained from the same biological replicate. ****P < 0.0001 [two-way ANOVA], (C) The accumulation of negative genome (negative-strand RNA genome) was monitored by strand-specific qRT-PCR analysis on GFP (left) or NSP1 (right) sequence 24 hours post transfection of the corresponding saRNA. (D) Same as in A, but here the level of pro-inflammatory cytokine CCL5 was assessed by qRT-PCR analysis.

[0034] Figure 7. (A) Western blot analysis using the indicated antibodies of RIG-I- / -MDA-5- / -293 cells, stimulated with a RIG-I agonist (a 5’ triphosphate hairpin RNA, 3p-hpRNA) and a MDA-5 agonist (poly (l: C), a dsRNA analogue) - PCNA served as a loading control. (B) Percentage of GFP-positive RIG-I- / -MDA5- / -cells compared to wild-type 24 hours post-transfection of CHIKV-GFP-B2 saRNA.Figure 8. (A) MFI of GFPhighpopulation 24 hours post-transfection of THP1-2R-wt or IFNAR- / -cells transfected with CHIKV-GFP-STOP, -B2 or-R59Q. (B) Immunostimulatory activity of different saRNA vectors in THP1-2R-wt or-IFNAR- / -cells by measuring Lucia Luciferase in the supernatant. For each experiment in (A), Lucia luciferase activity was measured. Data in all panels are from 4 independent experiments where each point represents the average of technical replicates. Connected dots correspond to values obtained from the same biological replicate. ***P < 0.001 [two-way ANOVA],

[0035] Figure 9. THP1 cells containing a secreted embryonic alkaline phosphatase (SEAP) reporter gene under a NF-kB-dependent promoter were transfected with CHIKV-GFP-STOP and CHIKV-GFP-B2. The NF-kB response was then monitored 24 hours post-transfection by quantifying the level of SEAP in the supernatant. Data is from 4 independent experiments where each point represents the average of technical replicates.

[0036] Figure 10. (A) CHIKV-GFP-STOP (blue / left), CHIKV-GFP-B2 (red / middle) or CHIKV-GFP-B2 R59Q (orange / right) were transfected in either HEK 293 or HEK 293T that are either wild-type (WT) or knockout for Dicer (Dicer- / -) or double knockout for both Dicer and PKR (Dicer- / -; PKR- / -). Flow cytometry was used to monitor MFI of GFPhighpopulation 24 hours post-transfection. (B) HeLa cells knockout for PKR (PKR- / -) cells were transfected with CHIKV-GFP-B2 (red / left) or -R59Q (orange / right). (C) Western Blot analysis of PKR and B2 expression in Hela wild-type (HeLa-wt) and PKR KO (PKR- / -) cells 24 hours after transfection with the corresponding CHIKV-GFP-saRNA. Vinculin served as a loading control. GFP-MFIhighwas measured in four (A) or three (B) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001 [two-way ANOVA].

[0037] Figure 11. (A) HEK293T cells knockout for both Dicer and PKR gene (Dicer- / -- PKR- / -) were transfected with plasmids encoding different FLAG-tagged PKR encoding plasmids and 1 day later with either CHIKV-GFP-B2 (red / left) or -R59Q (orange / right) saRNA. The MFI of GFP was measured in the FLAG-positive cells 24 hours after transfection and are represented as percentage (%) of GFP MFI relative to B2 expressing vector for each condition. Statistical analyses were calculated based on the MFI-GFP values of three independent experiments. (B) THP1-wt cells were transfected with CHIKV-GFP-B2 (red / top) or-R59Q (orange / bottom) in the presence of increasing concentration of the PKR inhibitor C16. Data represent for independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 [two-way ANOVA]

[0038] Figure 12. (A) Western blot analysis with the indicated antibodies of THP1 cells that are either nontransfected (NT) or transfected with CHIKV-GFP-B2 or CHIKV-GFP-B2 R59Q saRNA. PCNA served as a loading control. (B) HeLa cells were transfected with CHIKV-GFP-B2 or -R59Q saRNA. Levels of phosphorylated eiF2a (P-eiF2a) within the GFP-positive cells were assessed by flow cytometry 24 hours post-transfection. Data are representative of four independent experiments (C) As in B, but the level of phosphorylated eiF2a (P-eiF2a) was analysed by confocal microscopy.Figure 13. HeLa wild-type (wt) or knockout for (PKR- / -) cells were transfected with CHIKV-GFP-saRNA encoding NoV-B2, R59Q mutant, human enterovirus 71 (HEV71) 3A or vaccinia virus E3L proteins. The MFI within the GFP-MFIh'9hpopulation was determined by flow cytometry 24 hours posttransfection. Data represents four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001 [two-way ANOVA],

[0039] Figure 14. (A) Western Blot analysis with the indicated antibodies of HEK 293 cells or HEK 293T that are wild-type (wt), single knockout for Dicer (Dicer- / -) or double knockout for both Dicer and PKR (Dicer- / -PKR- / -). Cells were either non-transfected (NT) ortransfected with CHIKV-GFP-B2 orCHIKV-GFP-B2 R59Q saRNA and analysed by western blot 24 hours later. PCNA served as a loading control. (B) Western Blot analysis of HEK293 Dicer- / -PKR- / -transfected with plasmids encoding different FLAG-tagged PKR-encoding plasmids and either CHIKV-GFP-B2 or -R59Q saRNA. (C) THP1-IFNAR- / -cells were transfected with CHIKV-GFP-B2 (red / top) or-R59Q (orange / bottom) in the presence of increasing concentration of the PKR inhibitor C16. GFP-MFIh'9hvalues of four independent experiments were plotted. *P < 0.05 [two-way ANOVA], (D) Fold increase of GFP-positive THP1 cells in the presence of PKR inhibitor C16. (E) HeLa cells were transfected with CHIKV-GFP-B2 or-R59Q saRNA. Levels of phosphorylated eiF2a (P-eiF2a) within the GFP-positive cells were assessed by flow cytometry 24 hours post-transfection. Data represents four independent experiments.

[0040] Figure 15. (A) Confocal microscopy images of HeLa cells transfected with saRNA encoding GFP-STrEP-B2 and stained to visualise the nucleus (blue), STrEP-tag (white) and NSP1 (magenta). Red square in panel (A-d) corresponds to a magnification of the green square in the same image. (B and C) Confocal microscopy images of HeLa PKR- / -cells transfected with saRNA encoding GFP-STrEP-B2 (B) or GFP-STrEP-R59Q (C). (D) Quantification of dsRNA particles in 13 cells transfected with saRNA encoding GFP-B2 orGFP-R59Q. (E) Distribution of dsRNA particles in different layers of the cells in the Z-axis. Histograms represent the mean with SEM of dsRNA particles present in each segment of 10 individual cells. *P < 0.05, **P < 0.01 [two-way ANOVA],

[0041] Figure 16. THP1 cells transfected with saRNA encoding GFP-STrEP-B2 (A) or GFP-STrEP-R59Q (B) were analysed 24 hours later by confocal microscopy to visualise the nucleus (blue / DAPI), STrEP-tag (redZa-STrEP(a)) and GFP (greenZGFP(b)). GFP and StrEP stainings are merged for both figures to reveal colocalization in yellow (Merge(a+b)).

[0042] Figure 17. (A) HeLa, HEK 293 and HEK 293T cells were transfected with VEEV-GFP-B2 (red / left) or -R59Q (orange / right). Flow cytometry was used to monitor MFI of GFPhighpopulation 24 hours after transfection. (B) HeLa PKR- / -cells were transfected with VEEV-GFP-B2 (red / left) or -R59Q (orange / right). (C) THP1-2R-wt or (D) IFNAR- / -cells were transfected with CHIKV-GFP- or VEEV-GFP-B2 (red / left) or -R59Q (orange / right). (E) Immunostimulatory activity of different saRNA vectors in wild-type (wt) or IFNAR knockout (IFNAR- / -) THP1 reporter cells (THP1-2R) by measuring LuciaLuciferase in the supernatants of (C) and (D). (F) Flow cytometry was used to assess nascent protein synthesis by monitoring the incorporation of a methionine analogue, L-homopropargylglycine (HPG) in HeLa cells transfected with VEEV-derived saRNA expressing GFP-B2 or GFP-R59Q. (G and H) Western blot analysis of SARS-CoV2 Spike protein expression in HEK293T.17 (G) or HeLa (H) cells 24 hours after transfection of CHIKV-SARS-CoV2- or VEEV-SARS-CoV2-B2 or -R59Q expressing saRNA. (I) Western blot analysis of HEK293T cells transfected with CHIKV and VEEV saRNA encoding the wild-type or the R59Q B2 mutant and the influenza A virus hemagglutinin (HA) protein. Vinculin served as a loading control. Data from all flow cytometry experiments are from three independent experiments where each point represents the average of technical replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 [two-way ANOVA],

[0043] Figure 18. Comparison of the percentage of GFP-positive cells 24 hours post-transfection of HEK 293T, HEK 293, THP1 and HeLa cells with either CHIKV-GFP-B2 (black) and VEEV-GFP-B2 (grey) saRNAs. Data represent the means and standard deviations (s.d) of x independent experiments.

[0044] Figure 19. (A) Schematic representation of different constructs used to express B2 upstream or downstream of SARS-CoV2 Spike protein. (B) Western blot analysis of Spike protein expression for the different constructs in (A). Vinculin served as a loading control. Expression of Spike protein was quantified using ImageJ by calculating the area under the curve of the bands relative to Spike expression with the CHIKV-Spike-STOP.

[0045] Figure 20. Schematic representation of viral replicon particle (VRP) production. BHK-21 cells were co-electroporated with saRNA encoding either GFP-STOP, GFP-B2, or the GFP-R59Q mutant, together with in vitro-transcribed mRNAs encoding the chikungunya virus Capsid and Envelope proteins. VRPs were collected from cell supernatants 24 h post-electroporation and titrated.

[0046] Figure 21. (A) Representative fluorescence microscopy images of BHK-21 cells transduced with VRPs at a multiplicity of infection of 10, showing GFP expression. (B) MFI of GFP-positive BHK-21 cells as quantified by flow cytometry. Data are shown as mean ± SEM / technical replicates.

[0047] DETAILED DESCRIPTION

[0048] The inventors have surprisingly shown that through the cis-expression of a viral suppressors of RNA interference (VSR), e.g. NoV B2 or FHV B2, transgene expression from saRNA was increased without impacting on ISGs and pro-inflammatory cytokines. Hence, they provide a unique tool to potentially increase saRNA-vaccine efficiency by both boosting expression of its transgene (i.e. antigen) while preserving its self-adjuvanticity.

[0049] In addition, the saRNA technology is not only a promising approach for vaccine design but also for gene delivery and regenerative medicine. saRNA was recently used to express the fourreprogramming factors (Oct4, Klf4, SOX2 together with c-myc or GLIS1), which was shown to constitute an efficient approach to generate human induced-pluripotent stem cell from fibroblasts (Yoshioka, N. et al. Efficient Generation of Human iPSCs by a Synthetic Self-Replicative RNA. Cell Stem Cell 13, 246-254 (2013)). However, the activation of the IFN response is deleterious for reprogramming. The inventors have found that the VSR proteins described herein increase protein expression from saRNA without impacting on the IFN response, and may therefore boost the efficiency of iPSCs generation, as well as have implications for the development of efficient regenerative medicine therapies. Further, proteins exhibiting structural similarities to the VSR proteins described herein may be able to increase protein expression from saRNA without impacting on the IFN response.

[0050] The inventors surprisingly found that the cis-expression of a VSR protein (e.g. NoV B2 or FHV B2) enhances saRNA-mediated protein expression not only in differentiated cells, but also in stem cells, offering a versatile approach to boost saRNA efficiency in both pluripotent and differentiated cells. Stem cells have an attenuated IFN response and instead rely on IFN-independent mechanisms such as antiviral RNAi to protect themselves against viruses. The cis-expression of viral proteins impacting on IFN induction and / or IFN signalling would therefore not provide a beneficial effect on saRNA efficiency in stem cells. In contrast, the present invention can enhance protein expression from saRNA in stem cells, which have implications to boost heterologous expression of genes for stem cell therapy.

[0051] Though not wishing to be bound by theory, a mechanism by which the present invention confers an enhancement of saRNA is by decreasing the translational shutdown that is otherwise observed in cells receiving current saRNA design. The present invention not only alleviates the translation inhibition occurring on the saRNA’s transgene but also reduces the general block in protein synthesis occurring on host cellular messenger RNAs thereby reducing its impact on the recipient cells.

[0052] Antiviral RNAi activity has been observed in differentiated cells notably with mutant viruses that are deficient in viral suppressors of RNAi (VSR). These virus-encoded proteins or RNAs inhibit the RNAi pathway and are necessary for efficient virus multiplication and spread. An example of VSR are the B2 proteins encoded by the Nodaviridae family, which includes Flock-House virus (FHV) and Nodamura virus (NoV). While FHV infects only insects, NoV causes lethality in suckling mice, pigs and insects. The B2 proteins homodimerize to bind both synthetic or viral-derived dsRNAs and siRNAs and exert their VSR activity by sequestering them from the RNAi machinery. Accordingly, NoV mutants expressing either no B2 or a dsRNA-binding mutant of Nov B2 (NoV B2 R59Q) accumulate to lower levels than WT NoV during infections of mouse embryonic stem cells (mESCs) but also somatic cells (i.e. mouse embryonic fibroblasts) and in newborn mice. These infections are accompanied by the accumulation of canonical vsiRNAs and, in cells, NoV mutants’ replication is rescued upon inactivation of the RNAi pathway.Since the antiviral function of RNAi is active in mammalian stem cells and, to some extent, in differentiated cells, the inventors thought that this dsRNA-dependant pathway might impede saRNA. The inventors show herein that the VSR B2 protein from NoV encoded in cis within CHIKV and VEEV-derived saRNA increases protein expression in both mouse embryonic stem cells as well as various cell lines. While NoV B2 shielded saRNA from RNAi activity in mESCs, it conferred protection of saRNA in somatic cells by preventing PKR-mediated block of translation without impacting on the induction of the IFN response. Interestingly, the dsRNA produced by sa-RNA was preferentially localised at the periphery of the cell when NoV B2 was expressed suggesting that it sequesters dsRNA away from the cytosolic PKR pathway.

[0053] The inventors have demonstrated in stem cells and various cell lines that NoV B2 expressed from either a CHIKV genome or a VEEV genome-based saRNAs increases the expression of a transgene. In differentiated cells, the molecular mechanism by which B2 mediates this increase in protein expression were characterised.

[0054] Accordingly, described herein is an isolated self-amplifying RNA (saRNA) comprising, generally: a nucleic acid sequence encoding an RNA-dependent-RNA-polymerase (RdRP); a nucleic acid sequence encoding a heterologous protein; and a nucleic acid sequence encoding a viral suppressor of RNAi (VSR) protein. The isolated saRNA is capable of increasing the expression of a heterologous protein while preserving its self-adjuvanticity.

[0055] As used herein, the term “self-amplifying RNA” (saRNA) refers to a self-amplifying RNA, often derived from the genome of a positive-strand RNA virus such as Alphavirus. saRNAs are capable of replication within cells, which in turn allows expression of an encoded antigen to be modulated. saRNAs also possess self-adjuvant activity. Self-adjuvanticity refers to the ability of the saRNA to boost adaptive immunity, often by covalent linkage of antigen and adjuvant that experience simultaneous uptake by immune cells. Preservation of self-adjuvanticity refers to the ability of the saRNA to maintain its native level of self-adjuvanticity, even when functioning in an environment that suppresses viral adjuvanticity. The saRNA may be synthetic. The term “synthetic” refers to RNA, mRNA or saRNA that has been artificially synthesised as opposed to naturally produced within living cells. The term “nucleic acid sequence” refers to a succession of the nucleotide bases adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U). The nucleic acid sequence may comprise modified bases. Modified bases may comprise 2-Amino-deoxyadenosine (2-Amino-dA), 2'-dlnosine (2'-dl), 2'-dUridine (2'-dU), 2-Thio-dT, 4-Thio-dT, 4-Thio-Uridine, 5-Br-dC, 5-Br-dU, 5-Br-U, 5-Ethynyl-dU (5-EdU), 5-F-dU, 5-Formyl-dC, 5-Hydroxymethyl-dC(5-hm-dC), 5-Hydroxymethyl-dU(5-hm-dU), 5-l-dC, 5-l-dU, 5-lod-C, 5-lod-U, 5-Me-C, 5-Methyl-deoxycytidine(5-Me-dC), 5-Trifluormethyl-dU, 8-Oxo-dG, Aminoethyl-phenoxazine-deoxycytidine(AP-dC, G-Clamp), C-5Propynyl-deoxycytidine(pdC), C-5Propynyl-deoxyuridine(pdU), C8-Alkyne-dC, 8-Alkyne-dU, Dimethyl-2'-Deoxyadenosine, Formylindole, Inosine, invertedEnd(3'-3'), iso-dC(5-Me-iso-dC), N1-Methyl-Pseudouridine, N4-Ethyl-dC, N6, N6-Dimethyl-rA, N6-Me-A(m6A), N6-Me-dA, N-POMCaged-dT, Perylene-dU, or Pseudouridine. The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0056] The isolated saRNA may comprise a nucleic acid sequence encoding an RdRP. RdRP is an enzyme that catalyses the replication of RNA from an RNA template, which specifically catalyses synthesis of the RNA strand complementary to a given RNA template. The RdRP may be a polyprotein which is typically cleaved into four subunits, namely the four non-structural proteins (NSP 1-4). The isolated saRNA may comprise a nucleic acid sequence encoding a heterologous protein. As used herein, the term “heterologous protein” refers to a protein that is not native to the cell in which it is being expressed. Examples include a mammalian protein, an insect protein, an avian protein, a fish protein, a viral protein, a bacterial protein, a fungal protein, a protozoal protein or a parasitic worm protein, when not native to the cell in which the heterologous protein is being expressed. The heterologous protein may be considered as an antigen, capable of inducing an immune response in the organism in which it is expressed. The heterologous protein may, for example, comprise an antigen, a transcription factor (reprogramming factor), a therapeutic protein, an immunomodulator (e.g. cytokine) or a therapeutic protein. The antigen may be a viral protein such as the Spike (S) protein from SARS-CoV2 or a modified version having, for instance, a stabilised prefusion form of the S protein. The antigen may also be a F protein from Respiratory syncytial virus, or Hemagglutinin (HA) from Influenza virus. Examples of transcription factors include reprogramming factors for the generation of iPSCs (i.e. OCT4, KLF4, and SOX2 together with c-MYC or GLIS1). Examples of immunomodulators include cytokines used in potentiating the immune response (i.e. pro-inflammatory cytokine for cancer immunotherapy) such as interferon-alpha, interleukin-2 (IL-2). IL-15, IL-21, and IL- 12. Examples oftherapeutic proteins include coagulation factors such as factor VIII or factor IX for therapeutic use towards haemophilia and vascular endothelial growth factor A (VEGF-A) to induce regenerative angiogenesis in patients with cardiovascular disease.

[0057] The isolated saRNA may comprise a nucleic acid sequence encoding a VSR protein. As used herein, the term “viral suppressor of RNAi (VSR) protein” refers to a virus-encoded protein exhibiting the activity to block RNA interference (RNAi) inducing by long dsRNA, siRNA or virus replication. The VSR protein may be a viral B2 protein. VSRs efficiently inhibit host antiviral responses by interacting with the key components of cellular silencing machinery, often mimicking their normal cellular functions. The viral B2 protein is a potent suppressor of RNAi in animals and plants, and is a homodimer in solution that contains three a-helices per monomer.

[0058] The viral B2 protein may be from the family Nodaviridae. Nodaviridae is a family of nonenveloped positive-strand RNA viruses, for which vertebrates and invertebrates serve as natural hosts. Diseases associated with this family include viral encephalopathy and retinopathy in fish. TheNodaviridae family contains nine species, assigned to two genera, namely Alphanodavirus and Betanodavirus.

[0059] The viral B2 protein may be derived from a Nodamuravirus B2 (NoV B2) protein or a Flock House virus B2 (FHV B2) protein. Nodamuravirus is a +ssRNA virus of the family Nodaviridae and the genus Alphanodavirus, originally isolated from Culex tritaeniorhynchus. Nodamuravirus contains a nonenveloped virion that has a diameter of approximately 30 nm. The virion is made up of 180 copies of a single viral capsid protein. The virion is organised in T=3 icosahedral symmetry, wherein 60 triangular subunits are each made up of 3 viral capsid proteins. Nodamuravirus replicates by penetrating a host cell, uncoating and releasing viral genomic RNA into the cytoplasm. The viral RNA-1 is then translated to produce the RdRp protein, after which replication then begins in cytoplasmic viral factories. A dsRNA genome is synthesised from the genomic ssRNA(+) and further transcribed providing viral mRNAs / new ssRNA(+) genomes. The subgenomic RNA-3 is then transcribed along with the RNA-2, which encodes capsid protein alpha.

[0060] Flock house virus is a +ssRNA virus of the family Nodaviridae and the genus Alphanodavirus, originally isolated from Costelytra zealandica. Flock House virus is small, non-enveloped, icosahedral T=3 insect virus containing a bipartite positive sense ssRNA genome comprising two genes: RNA1 (3.1 kb) an RNA2 (1.4kb). RNA1 encodes the RNA-dependent RNA polymerase and also contains a frame-shifted subgenomic RNA 3 (369 nts) that encodes protein B2, responsible for inhibition of RNAi pathways. Viral entry into host cells occurs via receptor-mediated endocytosis. Receptor binding initiates a sequence of events during which the virus exploits the host environment in order to deliver the viral cargo in to the host cytosol. Receptor binding prompts the meta-stability of the capsid-proteins, the coordinated rearrangements of which are crucial for subsequent steps in the infection pathway.

[0061] The NoV B2 protein may comprise an amino acid sequence that is substantially homologous to the amino acid sequence of protein B2 [Nodamura virus], wherein the protein has an arginine residue at position 59 (R59) relative to the amino acid sequence of protein B2 [Nodamura virus]. Amino acid sequences with greaterthan 20% identity (for example 25%, 30%, 40%, 50%, 60%, 70%, 80%, 80%, 95% or 99%) are considered to be homologous sequences. As used herein, substantially homologous refers to sequences exhibiting at least 60% or 70%, preferably means at least 80%, more preferably at least 90%, and most preferably at least 95%, 96%, 97%, 98%, 99% or greater identity. In an embodiment of the invention the amino acid sequence has at least 80% or more (e.g.

[0062] 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% etc.) homology with the amino acid sequence of protein B2 [Nodamura virus]. The amino acid sequence of protein B2 [Nodamura virus] (Accession No. NP_077731) (SEQ ID NO: 1) is reproduced as follows:

[0063] MTNMSCAYELIKSLPAKLEQLAQETQATIQTLMIADPNVNKDLRAFCEFLTVQHQRAYRATNSLLIKPRVAAA LRGEELDLGEADVAARVRQLKQQLAELEMEIKPGHQQVAQVSGRRKAAAAAPVAQLGRVGWNE[ SEQ ID NO: 1]

[0064] As used herein, the terms homology and identity are interchangeable.

[0065] Sequence comparisons to determine homology can be carried out using readily available sequence comparison software. Examples include but are not limited to BLAST (see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4thEd - Chapter 18) and FASTA (Altschul et al., 1990 J. Mol. Biol.

[0066] 403-410). Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60). Having aligned the sequences, different ways of calculating percentage identity between the two sequences may include dividing the number of identities by: the length of the shortest sequence, the length of alignment, the mean length of sequence, the number of non-gap positions, or the number of equivalenced positions excluding overhangs.

[0067] The NoV B2 protein may comprise an amino acid sequence consisting of the amino acid sequence of protein B2 [Nodamura virus].

[0068] The FHV B2 protein may comprise an amino acid sequence that is substantially homologous to the amino acid sequence of protein B2 [Flock House virus], wherein the protein has an arginine residue at position 54 (R54) relative to the amino acid sequence of protein B2 [Flock House virus]. Amino acid sequences with greater than 20% identity (for example 25%, 30%, 40%, 50%, 60%, 70%, 80%, 80%, 95% or 99%) are considered to be homologous sequences. As used herein, substantially homologous refers to sequences exhibiting at least 60% or 70%, preferably means at least 80%, more preferably at least 90%, and most preferably at least 95%, 96%, 97%, 98%, 99% or greater identity. In an embodiment of the invention the amino acid sequence has at least 80% or more (e.g.

[0069] 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% etc.) homology with the amino acid sequence of protein B2 [Flock House virus]. The amino acid sequence of protein B2 [Flock House virus] (Accession No. NP_689446) (SEQ ID NO: 2) is reproduced as follows:

[0070] MPSKLALIQELPDRIQTAVEAAMGMSYQDAPNNVRRDLDNLHACLNKAKLTVSRMVTSLLEKPSWAYLEGKA PEEAKPTLEERLRKLELSHSLPTTGSDPPPAKL

[0071] [ SEQ ID NO: 2]

[0072] The FHV B2 protein may comprise an amino acid sequence consisting of the amino acid sequence of protein B2 [Flock House virus].

[0073] The saRNA may be an Alphavirus saRNA. Alphavirus is the sole genus in the Togaviridae family and has a positive-sense, single-stranded RNA genome. The alphaviruses are small, spherical, enveloped viruses with a genome of a single strand of positive-sense RNA. The total genome length ranges between 11,000 and 12,000 nucleotides, and has a 5’ cap and a 3’ poly-A tail. The four non-structural protein genes are encoded in the 5' two-thirds of the genome, while the three structuralproteins are translated from a subgenomic mRNA colinear with the 3' one-third of the genome. Species in the Alphavirus genus include: Aura virus, Barmah Forest virus, Bebaru virus, Caaingua virus, Cabassou virus, Chikungunya virus (CHIKV), Eastern equine encephalitis virus, Eilat virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Madariaga virus, Mayaro virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus, O'nyong'nyong virus, Pixuna virus, Rio Negro virus, Ross River virus, Salmon pancreas disease virus, Semliki Forest virus, Sindbis virus, Southern elephant seal virus, Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus (VEEV), Western equine encephalitis virus and Whataroa virus.

[0074] The saRNA may be a Chikungunya virus (CHIKV) saRNA or a Venezuelan encephalitis virus (VEEV) saRNA. CHIKV is a positive-sense single-stranded RNA virus with a genome of about 11.6kb, generally transmitted by arthropods. The virus consists of four nonstructural proteins and three structural proteins. The structural proteins are the capsid and two envelope glycoproteins: E1 and E2, which form heterodimeric spikes on the virion surface. E2 binds to cellular receptors in order to enter the host cell through receptor-mediated endocytosis. E1 contains a fusion peptide which, when exposed to the acidity of the endosome in eukaryotic cells, dissociates from E2 and initiates membrane fusion that allows the release of nucleocapsids into the host cytoplasm, promoting infection. The mature virion contains 240 heterodimeric spikes of E2 / E1, which after release, bud on the surface of the infected cell, where they are released by exocytosis to infect other cells.

[0075] VEEV is an enveloped virus that has a single-stranded, positive-sense RNA genome of about 11,5kb, generally transmitted by arthropods. The virion is spherical and approximately 70 nm in diameter, and has a lipid membrane with glycoprotein surface proteins spread around the outside. Surrounding the nuclear material is a nucleocapsid that has an icosahedral symmetry of T = 4, and is approximately 40 nm in diameter. Six serotypes of VEEV have been identified. The 5' end of the genome encodes four non-structural proteins, nsP1, nsP2, nsP3 and nsP4, and the 3' end is responsible for three structural proteins, the capsid and the envelope proteins E1 and E2. Nonstructural proteins participate in the replication of the viral genome involved in functions of the cytoplasm of the host.

[0076] The nucleic acid encoding the RdRP of the saRNA may be upstream of the nucleic acids encoding the VSR protein and heterologous protein. By “upstream” it is meant that the part of the RNA molecule in question is toward the 5’ end of the RNA molecule. Alternatively, the nucleic acid encoding the RdRP of the saRNA may be downstream of the nucleic acids encoding the VSR protein and heterologous protein. By “downstream” it is meant that the part of the RNA molecule in question is toward the 3’ end of the RNA molecule. The nucleic acid encoding the VSR protein may be upstream of the nucleic acid encoding the heterologous protein. Advantageously, positioning of the nucleic acid encoding the VSR protein upstream of the nucleic acid encoding the heterologous protein improves expression of the heterologous protein. The nucleic acid encoding the VSR protein may be downstream of the nucleic acid encoding the heterologous protein.The NoV B2 or FHV B2 protein may be expressed in cis. Alternatively, the NoV B2 or FHV B2 protein may be expressed in trans. Expression in cis refers to expression of a gene that is regulated by contiguous element that is on the same vector or section of nucleic acid as the gene. Expression in trans refers to expression of a gene that is regulated by a non-contiguous element that does not reside on the same vector as the gene. In trans expression may be controlled by an external vector or nucleic acid, for example by a second vector.

[0077] The NoVB2 and FHVB2 proteins share 47.81% identity in their DNA sequence and 24.64% identity in their amino acid sequence (amino acid sequence identity in the RNA-binding region of 38.1%). However, NoVB2 and FHVB2 share a very similar structure. This was discovered by superimposing the structures of NoVB2 and FHVB2 onto each other and then quantifying the similarity using the Root Mean Square Deviation (RMSD) value. RMSD is used as a quantitative measure of the similarity between two superimposed atomic coordinates. An RMSD value of less than 2 Angstrom (A) shows that two proteins are very similar in their structure. When superimposed, NoVB2 and FHVB2 have an RMSD value of 1.78 A. Accordingly, the conformational structures of the two proteins are highly similar. Similarities in protein structures can indicate similarities in protein functions and such structural similarities have been used to infer functional properties of proteins and to reveal distant evolutionary relationships between proteins exhibiting little or no similarity in their amino acid sequences. The high structural similarity between NoVB2 and FHVB2 suggests that they may function similarly, and may both be utilised interchangeably to modulate the expression of a heterologous protein. The VSR protein may be any VSR protein whose 3D structure only deviates by an RMSD of < 3 or < 2 Angstrom to the structure of NovB2 or FHV B2. The 3D structure of a protein may be generated by structural biological methods or computational modelling. Structural biological methods may be X-ray crystallography, electron microscopy, or nuclear magnetic resonance. Computational modelling may involve the use of protein structure prediction software such as AlphaFold, RoseTTAFold or Umol.

[0078] The saRNA may be provided in a plasmid construct. A plasmid construct refers to an artificially engineered DNA molecule that has been assembled into a plasmid vector. Plasmid constructs may contain specific DNA sequences, such as promoter regions, coding sequences and regulatory elements, which allow the expression of desired genes. Plasmid constructs generated by the inventors are detailed in Table 1. Plasmid constructs may encode saRNAs having the order of genetic elements as depicted in Figure 19A. The plasmid construct may comprise a constitutive promoter, or an inducible promoter. The constitutive promoter may be human p-actin, human elongation factor-la, chicken p-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, herpes simplex virus thymidine kinase, SV40, UBC, EF1 A, PGK and CAGG, or COPIA and ACT5C. The inducible promoter may be a heat shock promoter, IPTG, positive inducible alcohol and steroid regulated promoters, or Tetracycline (Tet). The plasmid construct may comprise abacteriophage promoter. The bacteriophage promoter may be SP6, T7 or T3. The bacteriophage promoter may allow for in vitro transcription of the plasmid construct.

[0079] In Table 1 there is shown: plasmids generated by including B2 proteins in CHIKV and VEEV. The “Order insert” refers to whether the gene of interest is located upstream or downstream of NoV B2 or FHV B2. The genes of interest correspond to: GFP, Firefly Luciferase, Spike-SARS-CoV-2, RBD-SARS2: Receptor binding domain of SARS-CoV-2, Hemagglutinin-Influenza: Antigen from influenza A virus.

[0080] Table 1:

[0081] Order insert

[0082] SEQ ID NO #plasmid Backbone First Second

[0083] 3 1 CHIKV GFP NoV-B2

[0084] 4 2 VEEV GFP NoV-B2

[0085] 5 3 CHIKV GFP FHV-B2

[0086] 6 11 VEEV GFP FHV-B2

[0087] 7 22 CHIKV Spike-SARS2 NoV-B2

[0088] 8 24 VEEV Spike-SARS2 NoV-B2

[0089] 9 26 CHIKV RBD-SARS2 NoV-B2

[0090] 10 30 CHIKV NoV-B2 RBD-SARS2

[0091] 11 33 CHIKV NoV-B2 Spike-SARS2

[0092] 12 37 VEEV NoV-B2 Spike-SARS2

[0093] 13 39 VEEV RBD-SARS2 NoV-B2

[0094] 14 41 VEEV NoV-B2 RBD-SARS2

[0095] 15 43 CHIKV NoV-B2 GFP

[0096] 16 45 VEEV NoV-B2 GFP

[0097] 17 49 CHIKV NoV-B2 HA-Influenza

[0098] 18 51 CHIKV NoV-B2 FireflyLuciferase 19 53 VEEV NoV-B2 HA-Influenza

[0099] 20 55 VEEV NoV-B2 FireflyLuciferase

[0100]

[0101] Preferred features of the second and subsequent aspects of the invention as for the first aspect mutatis mutandis.

[0102] In a second aspect of the invention, there is provided a composition comprising a saRNA according to the first aspect of the invention.

[0103] The composition may be adapted for administration by any appropriate route, for example the oral (including buccal or sublingual), topical (including buccal, sublingual or transdermal), or parenteral (including subcutaneous, intramuscular, intravenous, intra-arterial, intra-thecal, intra-pleural, intra-ophthalmological, intra-cardiac, intraperitoneal or intradermal) route. Suitable compositions mayinclude solid forms, such as pills, granules, capsules, powders and tablets, and liquid forms including solutions, elixirs, suspensions and syrups. Compositions suitable for parenteral administration include emulsions, suspensions and sterile solutions.

[0104] In a third aspect of the invention, there is provided a pharmaceutical composition comprising a saRNA according to the first aspect of the invention. The composition may optionally comprise a pharmaceutically acceptable carrier, excipient or diluent.

[0105] Pharmaceutical compositions adapted for parenteral administration include aqueous and nonaqueous sterile injection solution which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0106] Excipients which may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example. The compositions may be presented in unit-dose or multidose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

[0107] The pharmaceutical compositions may contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts (substances of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents, antioxidants or adjuvants. They may also contain therapeutically active agents in addition to the substance of the present invention.

[0108] The term “pharmaceutically acceptable carrier” or “diluent” refers to any known compound of combination of known compounds that are known to the skilled person to be useful in formulating pharmaceutical compositions. The pharmaceutically acceptable carrier may be a solid, in the form of a tablet or powder. The pharmaceutically acceptable carrier may be a liquid, used in the preparation of a solution, emulsion, suspension, syrup or elixir. The pharmaceutically acceptable carrier may be a lipid nanoparticle (LNP). The LNP may be modified to improve its stability, its efficiency of delivery, or its tissue targeting. The LNP may comprise ionizable lipids that facilitate targeting of specific cell types, tissue types or organs. The lipid may comprise a cationic helper lipid, a charged amphipathic phospholipid, a zwitterionic helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), a anionic helper lipid l-a-phosphatidylserine (PS), a cationic helper lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or the lipidoids 306Oiw, 2000iw, or 5140e,io. The LNP may comprise an antibody either within the LNP or protruding from the surface of the LNP. The LNP may bind a receptor specific to a cell to deliver its payload into the cell. The LNP may comprise an antibodythat binds an immune cell. The LNP may comprise a mRNA or saRNA as described herein. The saRNA as described herein may be packaged inside a LNP as described herein.

[0109] In a fourth aspect of the invention, there is provided a vector encoding the saRNA according to the first aspect of the invention. The vector may be a DNA molecule (e.g. a plasmid or a virus) that is used as a vehicle to carry a particular DNA or RNA segment into a host cell as part of a cloning or recombinant DNA or RNA technique. The vector may assist in replicating and / or expressing the inserted DNA or RNA sequence inside the host cell. The vector may also be a cosmid, a Lambda phage, a viral vector or an artificial chromosome (including a yeast artificial chromosome, a bacterial artificial chromosome or a human artificial chromosome), for example.

[0110] In a fifth aspect of the invention, there is provided a cell comprising a saRNA according to the first aspect of the invention or a vector according to the fourth aspect of the invention. Suitable examples of cells may include bacteria, fungi, archaea, plant, animal, slime moulds, protozoa and algae. The cell may be a somatic cell. A somatic cell can be any biological cell forming the body of a multicellular organism, other than a gamete, germ cell, gametocyte or undifferentiated stem cell. For example, the somatic cell may be a fibroblast cell. The cell may be a stem cell. A stem cell is an undifferentiated or partially differentiated cell that can change into a number of types of cell and proliferate indefinitely to produce more of the same stem cell. Examples of stem cells include embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, mesenchymal stem cells, haematopoietic stem cells, adult stem cells, totipotent stem cells and multipotent stem cells.

[0111] In a sixth aspect of the invention, there is provided a vaccine comprising a saRNA according the first aspect of the invention, or a vector according to the fourth aspect of the invention.

[0112] The vaccine may also be prepared using conventional vector carrier technologies. For example, presenting the one or more epitopes, hotspot regions or immunogenic portions on one or more replication-deficient adenovirus vectors, vesicular stomatitis virus vectors, influenza virus vectors or measles virus vectors.

[0113] The vaccine composition may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and / or adjuvants which enhance the effectiveness of the vaccine.

[0114] Examples of adjuvants which may be effective include but are not limited to: granulocytemacrophage colony-stimulating factor (GM-CSF), aluminium hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3 -hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI,which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene / Tween 80 emulsion. Further examples of adjuvants and other agents include aluminium hydroxide, aluminium phosphate, aluminium potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAB-dextran, blocked copolymers or other synthetic adjuvants. Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N. J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.). The saRNA of the invention may also be selfadjuvant. The adjuvant may comprise compounds that are derived from an LNP. The adjuvant may also comprise a LNP as described herein. The LNP as described herein or the saRNA as described herein may have intrinsic adjuvant activity that promotes induction of strong immune cell responses. The LNP as described herein may comprise an ionizable lipid.

[0115] A general advantage of a vaccine composition comprising a saRNA of the invention over extant vaccines such as mRNA vaccines is that a much lower dose of saRNA can be administered than mRNA.

[0116] In a seventh aspect of the invention, there is provided a method of modulating expression of a heterologous protein in a cell, comprising introducing to the cell a saRNA according to the first aspect of the invention or a vector according to the fourth aspect of the invention.

[0117] It is intended that the isolated saRNA is capable of modulating the expression of a heterologous protein. An modulation in expression may take the form of an increase in protein expression, whereby the amount of heterologous protein expressed is expressed as a percentage increase compared to a reference baseline value. An exemplified increase in heterologous protein expression may be at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500% at least 600%, at least 700%, at least 800% or at least 900% or more, when compared to a reference baseline value.

[0118] It is possible to measure modulation of expression of a heterologous protein by calculating the Area Under the Curve (AUC) ratio for protein expression when comparing an engineered saRNA to a control. For example, GFP expression can be monitored over a set time period (i.e. 48 hours) in cells transfected with either GFP and active B2 or GFP alone. It is also possible to measure modulation of expression of a heterologous protein by analytical techniques such as western blot. Techniques such as western blot allow quantitative analysis of protein levels and expression, such that it is possible to measure modulation of expression of a heterologous protein when compared with a control.The method of modulating expression of a heterologous protein may be an in vitro method. Such a method may therefore be performed outside of a living organism.

[0119] In an eighth aspect of the invention, there is provided a method of producing induced pluripotent stem (iPS) cells, comprising the steps of:

[0120] (a) introducing to a somatic cell a saRNA according to the first aspect of the invention or a vector according to the fourth aspect of the invention, encoding one or more reprogramming factors; and

[0121] (b) expressing the one or more reprogramming factors in the cell to initiate dedifferentiation of the somatic cell into an iPS cell.

[0122] iPS cells are a type of pluripotent stem cell that can be generated directly from a somatic cell. For example, the somatic cell may be a fibroblast cell. It is known that introduction of the genes Myc, Oct3 / 4, Sox2 and Klf4 (Yamanaka factors) encoding transcription factors can convert somatic cells into pluripotent stem cells. These genes are also known as reprogramming factors. Each of the factors can also be functionally replaced by related transcription factors, miRNAs, small molecules or saRNAs. The one or more reprogramming factors may comprise any of Oct4, KLf4 and SOX2, expressed together with c-myc or GLIS1. The method of produced iPS cells from somatic cells may be an in vitro method, whereby the method is performed outside of a living organism. Extant methods of reprogramming cells to pluripotency face challenges such as low efficiency of cell conversion, genomic insertion of the transcription factors, tumorigenicity and the expression of oncogenes and incomplete reprogramming. The term “initiate” refers to the ability of the reprogramming factors to induce de-differentiation of a somatic, or differentiated cell, into an iPS cell. Thus, the term “initiate” may be interchanged with the term “control”, as the reprogramming factors may be seen to control the de-differentiation of a somatic cell into an iPS cell. The terms “de-differentiate” or “dedifferentiation” refer to the process of a cell becoming less specialised. The terms also refer to the process of reversing normal differentiation. For example, de-differentiation may refer to the return of a somatic or differentiated cell, such as a fibroblast cell, into an iPS cell.

[0123] In a ninth aspect of the invention, there is provided a method of inducing an immune response in a subject, comprising administering a saRNA according to the first aspect of the invention, a composition according to the second aspect of the invention, a pharmaceutical composition according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a vaccine according to the sixth aspect of the invention to a subject.

[0124] As used herein, the term “subject” generally refers to an animal, preferably a mammal, in particular a human.Suitable routes of administration the oral (including buccal or sublingual), topical (including buccal, sublingual or transdermal), or parenteral (including subcutaneous, intramuscular, intravenous, intraarterial, intra-thecal, intra-pleural, intra-ophthalmological, intra-cardiac, intraperitoneal or intradermal) route.

[0125] In a tenth aspect of the invention, there is provided a saRNA according to the first aspect of the invention, a composition according to the second aspect of the invention, a pharmaceutical composition according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a vaccine according to the sixth aspect of the invention for use in a method of inducing an immune response in a subject.

[0126] The immune response induced by the saRNA, composition, pharmaceutical composition, vector or vaccine of the invention may be cellular or humoral in nature. The immune response induced may be adaptive or innate or both. An adaptive immune response may include the reaction of cells such as dendritic cells, T cells, B cells and antibodies which interact with the perceived antigen to neutralise said antigen. An adaptive immune response may cause the production of memory T and memory B cells specific to the antigen introduced to the immune system. An innate immune response may include the reaction of cells such as neutrophils, macrophages and monocytes, and soluble factors including cytokines and complement, to neutralise a perceived antigen. The immune response induced in a subject may be an anticancer immune response. The immune response induced in a subject may be against a pathogen. The pathogen may be a virus, a bacterium, a fungus, a protozoa or a parasitic worm. The immune response induced in a subject may be a form of vaccination. The immune response may be intended to be prophylactic or therapeutic.

[0127] In an eleventh aspect of the invention, there is provided a saRNA according to the first aspect of the invention, a composition according to the second aspect of the invention, a pharmaceutical composition according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the fifth aspect of the invention for use in medicine.

[0128] The term “for use in medicine” refers to all uses wherein there may be a prophylactic or therapeutic effect upon using the saRNA, composition, pharmaceutical composition, vector or cell of the invention. This may include preventing, slowing, interrupting, arresting or stopping of the progression of a disorder, or amelioration of one or more symptoms thereof, or may indicate a total elimination of all symptoms.

[0129] In a twelfth aspect of the invention, there is provided a saRNA according to the first aspect of the invention, a composition according to the second aspect of the invention, a pharmaceutical composition according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the fifth aspect of the invention for use in a method of treating a disease or condition in a subject.The terms “disease” and “condition” are interchangeable and refer to a disorder of structure or function in a subject, especially one that has a known cause and a distinctive group of symptoms, signs or anatomical changes. A disease may be caused by external factors such as pathogens, or by internal dysfunctions. The disease may be acquired or congenital. The disease may be acute or chronic. The disease may be idiopathic.

[0130] Definitions

[0131] Below are provided certain other definitions of terms, technical means, and embodiments used herein.

[0132] Aspects and embodiments described herein with the term “comprising” may include other features or steps within the scope. It is also understood that aspects and embodiments described as “comprising” also describes aspect and embodiments wherein the term “comprising” is replaced by the term “consisting essentially of’ or “consisting of’.

[0133] The phrase "selected from the group comprising" may be substituted with the phrase "selected from the group consisting of and vice versa, wherever they occur herein.

[0134] It is also understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and / or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

[0135] Similarly, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0136] Unless defined otherwise above, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, genetics and protein and nucleic acid chemistry described herein are those well-known and commonly used in the art, or according to manufacturer’s specifications.

[0137] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.The invention will now be further described by way of the following Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention, with reference to the Figures.

[0138] Those skilled in the art will appreciate that the present invention is defined by the appended claims and not by the Examples or other description of certain embodiments included herein.

[0139] EXAMPLES

[0140] Example 1

[0141] As a model of saRNA, a chikungunya virus (CHIKV) genome-based saRNA containing nsP1-4 genes was first engineered and, as a reporter gene, a green fluorescent protein (GFP) under the control of CHIKV subgenomic promoter was also engineered. Alphaviruses induce a general transcriptional shutoff and a rapid cytopathic effect as a strategy to inhibit the host antiviral response. To minimise these adverse effects, the nsP2 gene within the constructs harboured three-point mutations (ATL to ERR substitutions at position 674-676) that were previously shown to abolish the transcription shutdown and render CHIKV non-cytopathic, while preserving its replication. To assess the impact of antiviral RNAi on saRNA, the stop codon of the GFP was replaced by a P2A cleavage site and the coding sequence for Nodamura virus B2 gene (CHIKV-GFP-B2), which was shown to exhibit VSR activity in mammalian cells. As a control, a saRNA in which the B2 gene was replaced by a stop codon (CHIKV-GFP-STOP) was used.

[0142] Mouse embryonic stem cells (mESCs) were shown to exhibit a potent RNAi response when treated with synthetic dsRNA or upon viral infections. mESCs were therefore transfected with the engineered saRNA expressing or not NoV B2, and the level of GFP was then measured by flow cytometry at various timepoints post-transfection. The percentage of GFP-positive (GFP+) cells were similar at all time-points between cells transfected with either CHIKV-GFP-B2 or -STOP with a maximum of 15% GFP+cells 16 hrs post-transfection (Figure 3). Within the GFP+populations, self-replicating properties of the vectors were defined by gating on cells with mean fluorescent intensity (MFI) of 103or higher. When comparing both vectors, CHIKV-GFP-STOP reached approximately 10’000 AU, while B2 encoding vectors elicited significantly higher values 16 and 24 hours after transfection (Figure 1 A). After 48 hours, differences were no longer visible and, after 96 hours, no GFP positive cells were observed. Further examination at earlier timepoints showed that the level of GFP in mESCs transfected with CHIKV-GFP-B2 or CHIKV-GFP-STOP was similar 3 and 7 hours posttransfection, yet the B2-encoding saRNA expressed a higher level of GFP 10 hours post-transfection (Figure 1B). Hence, both vectors are similarly competent at early stages of RdRp activity, but between 7 and 10 hours, B2 expression enhances saRNA activity in mESCs.

[0143] Example 2

[0144] It was next addressed whether the dsRNA-binding activity of B2 was required for the observed increase in saRNA-derived GFP expression in mESCs. For this, an amino acid substitution ofArginine with Glutamine at position 59 (R59Q) of NoV B2 within the B2-expressing vector (CHIKV-GFP-R59Q) was introduced. This mutation was previously shown to decrease NoV ability to bind dsRNA and thereby abolish its VSR activity. It was found that NoV B2 dsRNA-binding activity was required for its ability to increase saRNA efficiency in mESCs since cells transfected with NoV B2 R59Q-encoding vector resulted in a both a lower percentage of GFP+cells (Figure 2A) and a lower MFI within the GFPhighcells compared to NoV B2-expressing vector (Figure 2A-2B). To test whether the c / s-encoded NoV B2 protein exhibited VSR activity and prevented the processing of saRNAs by Dicer into vsiRNAs, a small RNA deep sequencing analysis in saRNA-transfected mESCs was carried out. For cells transfected with either CHIKV-GFP-B2-wt, CHIKV-GFP-R59Q or with vehicle alone (mock), reads of 15-35 nucleotides (nt) in length represented 43.37%, 42.9% and 47.6% respectively of total reads. In mESCs transfected with CHIKV-GFP-B2, 3.6% of those 15-35-nt long reads aligned to the vector genome of which 18.2% were of 21-23 nt in length, the approximate size for Dicer cleavage products. For mESCs transfected with the NoV R59Q-expressing saRNAs, 4.05% of 15-35-nt long reads mapped the vector genome of which 20.2% corresponded to 21-23-nt long RNAs.

[0145] The saRNA reads were further analysed and their size distribution determined. In cells transfected with NoV B2 R59Q-expressing saRNA, an accumulation of 22-nt-long small RNAs derived from both the viral positive (+) and negative strands (-) was found, a feature of Dicer-derived small RNAs (Figure 2C). These viral (+) and (-) reads of 22-nt in length were reduced when saRNA were encoding NoV B2 WT, consistent with its role as an inhibitor of Dicer processing of viral dsRNA replication intermediates (Figure 2C). The viral 21-23-nt long reads along the viral genome were then mapped. It was found that most of the reads were derived from the (+)-strand genome for cells transfected with saRNAs expressing NoV B2 WT or mutant in line with the strong bias of (+)-strand RNA genome during alphavirus replication. However, cells with the NoV B2 R59Q saRNA exhibited much higher level of 21-23-nt long RNAs in the (-) strand of the vector. Most of these viral (-)-reads were located in the 5’ end of the vector genome with 34.7% of them located in the first 500 nucleotides This site corresponds to regions of viral dsRNA replication intermediates production through synthesis of (+)-genomic RNA previously shown to accumulate Dicer-mediated vsiRNAs during authentic alphavirus infections. The data therefore suggest that saRNAs are targeted by Dicer in mESCs leading to a reduction in gene expression. The c / s-expression of NoV B2 increased saRNA-mediated gene expression and concomitantly reduced the accumulation of viral-derived siRNAs.

[0146] Example 3

[0147] Antiviral RNAi activity has also been detected in differentiated mammalian cells upon infection with WT or VSR-deficient viruses. It was therefore tested whether NoV B2 impacted saRNA efficiency in cell lines. The efficiency of CHIKV-GFP-STOP and CHIKV-GFP-B2 in Hela cells was initially assessed. It was found that while the MFI of GFPhighof Hela cells transfected with CHIKV-GFP-STOP had a MFI within GFPhighcells of -3,000 AU, the GFP expression was robustly enhanced using CHIKV-GFP-B2 with cells reaching higher MFI than 105AU. The enhanced GFP expression of B2-expressing saRNA in Hela cells was higher at various timepoints post-transfection (Figure 4A-4B). It was then tested whether gene expression from CHIK-GFP-STOP saRNA could be increased by the co-expression of NoV B2 in trans. Hela cells were co-transfected with CHIKV-GFP-STOP saRNA and either a N-terminal Strep-tag NoV-B2 (1xSTreP-NoV B2) expressing plasmid or an empty plasmid. Intracellular staining of STrEP-tagged positive cells followed by flow cytometry analysis revealed that CHIKV-GFP-STOP saRNA expressed higher levels of GFP when NoV B2 was provided in trans. Thus, expression of NoV B2 in HeLa cells in cis or trans enhances saRNA-mediated gene expression. Similar to the observations in mESCs, this effect on saRNA depended on NoV B2’s ability to bind dsRNA as shown by fluorescent microscopy with the absence of GFP-expressing cells upon transfection with B2 R59Q-expressing saRNA (Figure 4C). Furthermore, while c / s-encoded expression of NoV B2 increased the MFI of GFP within the GFPhighpopulation, this enhancement was lost in cells transfected with saRNA encoding NoV B2-R59Q vector (Figure 5A). Strand-specific quantitative (q) RT-PCR on the (+)-strand genome of saRNAs supported these observations with cells transfected with CHIKV-GFP-B2 showing a higher level of (+)-sense RNA containing GFP or NSP1 coding sequences (Figure 5C). Interestingly, NoV B2 did not cause an increase in the complementary (-)-sense RNA genome (Figure 6C-6D). Thus, NoV B2 through its dsRNA binding ability enhances the (+)-sense RNA synthesis and mRNA production without enhancement of the (-)-sense template RNA.

[0148] Example 4

[0149] In contrast to mESCs, somatic cells are equipped with a potent type I IFN system. It was therefore addressed whether the enhancing effect of B2 on saRNA encompassed an inhibition of the IFN response. The efficiency of saRNAs was first tested in presence of ruxolitinib, a JAK1 / 2 inhibitor that blocks the type I IFN receptor IFNAR signalling pathway. It was found that the level of GFP expressed from CHIKV-GFP-R59Q did not increase in the presence of ruxolitinib (Figure 4D). Furthermore, the enhanced GFP level per cell caused by the cis- expression of NoV B2 was similar in both the presence or absence of ruxolitinib (Figure 5A). Consistent with the findings that NoV B2 increases saRNA-mediated gene expression independently of the type I IFN signalling, no significant difference was observed in the levels of prototypical ISGs or proinflammatory cytokine (IFIT1, IFIT2, RIG-I and CCL5) upon transfection with saRNA encoding either NoV B2 WT, B2 R59Q or no NoV B2 (Figure 6A and 6E). Furthermore, NoV B2’s ability to increase GFP expression from saRNA was intact in human embryonic kidney (HEK) 293 cells knockout (KO) for RIG-I and MDA-5 and therefore deficient in the RLR-mediated induction of the IFN response (Figure 6B). The RIG-I- / -MDA-5- / -293 cells were validated by western blot and functionally by stimulation with a RIG-I agonist (a 5’ triphosphate hairpin RNA, 3p-hpRNA) and a MDA-5 agonist (poly (l: C), a dsRNA analogue) (Figure 7A). Nevertheless, Rig-I- / -MDA5- / -cells had significantly higher percentage of GFP+cells (Figure 7B), which supports previous observations where ruxolitinib also enhanced the percentage of GFP+cells upon transfection of HeLa cells (Figure 5B) and pre-treatment of cells with recombinant IFN-I almost abolished the vectors’ gene expression regardless of the presence of B2 (Figure 5A).Example 5

[0150] To further characterise B2 enhancement ofsaRNA activity, CHIKV-GFP-B2 activity was assessed in THP1-Dual cells (THP1-2R), a human monocytic cell line, engineered to contain two reporter genes: i) a secreted luciferase under the control of an IRF-dependent promoter reporting the activation of the IFN response, and ii) a secreted embryonic alkaline phosphatase (SEAP) driven by a promoter activated by the NF-kB pathway. Upon transfection of THP1 cells with a saRNA encoding for NoV B2 (CHIKV-GFP-B2), there was increased GFP expression compared to saRNAs that either does not encode for NoV B2 (CHIKV-GFP-STOP) or express NoV B2 R59Q mutant (CHIKV-GFP-B2-R59Q) 24 hours post-transfection (Figure 8A). Concomitantly, no significant difference was observed in the activation of the IFN-I and NF-kB responses between cells transfected with the 3 different saRNAs with similar level of secreted luciferase reporter activity and SEAP, respectively (Figure 8B, Figure 9). Confirming that NoV B2 increases GFP expression independently of the IFN response, it was found that type I IFN receptor-deficient THP1-Dual cells (Jfnar2-7-THP1-2R) transfected with CHIKV-GFP-B2 exhibited a higher MFI in GFPhighcells compared to cells transfected with saRNA devoid of B2-encoding sequence or expressing B2 R59Q (Figure 8A). Interestingly, all three saRNAs were more efficient in Ifnar2- / -THP1 cells compared to WT, yet an additive effect was observed with the c / s-expression of NoV B2 on the level of GFP (Figure 8A). These data suggest that, while the activation of the IFN response impacts the efficiency of saRNAs, NoV B2 increases gene expression from saRNA without impacting on ISGs expression and by a mechanism independent of the immediate IFN activation.

[0151] Example 6

[0152] A further aim was to identify the molecular mechanism underlying B2 enhancement ofsaRNA activity. NoV B2 was shown to exhibit VSR activity not only in stem cells but also in somatic cells in the context of an authentic infection with NoV or with a NoV genome based replicon system. It was therefore tested whether the enhancement of GFP expression that was observed upon the cis expression of NoV B2 within a CHIKV-genome based saRNA similarly entails a protection from Dicer activity. To test this possibility, saRNAs expressing either NoV B2, NoV B2 R59Q or no B2 in previously described KO HEK293T lines lacking Dicer (Dicer- / -) or both Dicer and PKR (Dicer7-PKR- / -) were transfected. This latter was included in the analysis as it was shown that Dicer antiviral activity against alphaviruses such as SINV and SFV depends on the presence of PKR.

[0153] Western Blot analysis validated the lack of expression of either Dicer or both Dicer and PKR in HEK 293T KO cells (Figure 14A). Similar to the observations in HeLa and THP1 cells, transfection with CHIKV-GFP-B2 resulted in a higher MFI with the GFPhighpopulation in control lines HEK293 and HEK293T cells (Figure 10A). Unexpectedly, the restriction on saRNA lacking NoV B2 or expressing B2-R59Q was maintained in Dicer- / -cells and the enhanced expression of GFP by NoV B2 was not impacted (Figure 10A). Strikingly, this restriction on CHIKV-GFP-STOP and CHIKV-GFP-R59Q was abrogated in Dicer- / -PKR- / -cells with no difference in GFP level between saRNA expressing or not a functional NoV B2 (Figure 10A). These results suggest a crucial role of PKR on the observedrestriction of saRNA in somatic cells. To validate these observations, tests were next performed in PKR-deficient Hela cells (HeLa PKR- / -cells). It was confirmed that lack of PKR alone was sufficient to abrogate the restricted GFP expression of saRNAs in HeLa cells and no additive effect was observed in the presence of c / s-encoded NoV B2 (Figure 10B). Additionally, while the saRNA-mediated expression of NoV B2 R59Q was undetectable compared to NoV B2 WT in Hela cells, the production of both versions of NoV B2 were increased in Hela PKR- / -cells demonstrating the loss of restriction in these cells (Figure 10C). Thus, PKR restricts saRNAs and the c / s-encoded NoV B2 allows to alleviate this inhibition.

[0154] Example 7

[0155] To further characterise the mechanism by which PKR restricts saRNA, Dicer- / -PKR- / -HEK 2993T cells were complemented by transient transfection with FLAG-tagged constructs encoding either WT or mutant versions of PKR. An ATP-binding mutant PKR K296R unable to bind ATP, homodimerize and hence phosphorylate its targets was used. Also included was a catalytically dead PKR D414A mutant that harbours a mutation in its kinase catalytic site. One day post-transfection with the constructs, cells were transfected with CHIKV-GFP-B2 or -R59Q mutant followed by intracellular staining for FLAG expression to monitor the level of GFP in PKR-expressing cells by flow cytometry. Comparative analysis of the GFP MFI between FLAG+cells revealed that cells expressing PKR-WT caused a reduction of GFP expression from the NoV B2 R59Q-expressing saRNA relative to the B2 WT-expressing vector (Figure 11 A). In contrast, PKR mutants K296R or D414A overexpression did not reconstitute the restriction, suggesting that an active kinase activity is required for PKR restriction (Figure 11A). PKR-WT expression was low compared to PKR K296R and D414A mutants as shown by western blot analysis (Figure 14C). This was also observed by reduced FLAG staining in PKR-wt transfected cells as compared to PKR D414A. Yet, as expected, the phosphorylated form of PKR-wt (P-PKR) was detected upon transfection with saRNAs in contrast to both PKR mutants that were completely (PKR K296R) or partially (PKR D414) defective in turning into an active P-PKR form (Figure 14).

[0156] Example 8

[0157] To validate that saRNA are restricted by PKR enzymatic activity and that NoV B2 allows the saRNA to alleviate this inhibition, THP1 cells were transfected with CHIKV-GFP-B2 or -R59Q vectors in the presence of increasing concentrations of the PKR inhibitor C16, which acts by binding the ATP-binding site and thereby blocks PKR autophosphorylation. It was found that in cells transfected with CHIKV-GFP-R59Q the level of GFP within the GFPhighpopulations was enhanced in presence of increasing amounts of C16 (Figure 11 B). Conversely, cells transfected with CHIKV-GFP-B2 vector did not present any enhancement on GFPhighMFI and consequently non-significant difference in GFP level between both vectors was observed at 1 pM concentration (Figure 11 A). These results suggests that NoV B2 subverts PKR enzymatic activity, thus rendering C16 inhibitor’s activity redundant. Similar results were found using Ifnar2- / -THP1 cells, yet only 500nM of C16 was sufficient to reach non-significant difference in GFP level between CHIKV-GFP-R59Q and CHIKV-GFP-B2 vectors(Figure 14C). This higher sensitivity to C16 likely reflected the lower amount of PKR expression in Ifnar2- / -THP1 cells compared to THP1 WT with a steady-state level of PKR not amplified by the IFN signalling pathway. Interestingly, C16 treatment of THP-1 cells also enhanced up to 8-fold the percentage of GFP+ cells for both vectors in a dose-dependent manner suggesting that blocking the activity of the steady-state level of PKR impact on the permissivity of the cells to saRNAs (Figure 14D). These results show that catalytically active PKR restricts saRNA activity and the c / s-expression of NoV B2 alleviate this restriction.

[0158] Example 9

[0159] Next, the level at which the PKR pathway is inhibited by NoV B2 was examined. The activation of the PKR pathway is characterised by the auto-phosphorylation of PKR, which then phosphorylates eiF2a. THP1 cells were therefore transfected with CHIKV-GFP-B2 or CHIKV-GFP-R59Q vectors and the phosphorylation status of both PKR and eiF2a was assessed by western blot analysis. As previously observed upon reconstitution of PKR-wt in Dicer7-PKR- / -cells (Figure 14B), similar level of phospho-PKR were detected in THP1 cells transfected with either CHIKV-GFP-B2 or CHIKV-GFP-R59Q vectors (Figure 12A). In contrast, while eiF2a was phosphorylated upon transfection with CHIKV-GFP-R59Q, no phosho-eIF2α was detected with CHIKV-GFP-B2 and concomitantly B2 protein was readily detected (Figure 12A). To confirm that NoV B2 impacted on eiF2a phosphorylation, HeLa cells were transfected with CHIKV-GFP-B2 or -R59Q vectors and 24 hours later the level of P-elF2a was measured by flow cytometry in GFP+cells. It was found that cells containing CHIKV-GFP-R59Q exhibited high levels of P-elF2a, while cells transfected with CHIKV-GFP-B2 had no P-elF2a (Figure 12B). These levels of P-elF2a inversely correlated with the expression of GFP expressed from the saRNAs (Figure 14E). Confocal microscopy validated these observations and showed the presence of P-elF2a in GFP+cells transfected with CHIKV-GFP-R59Q while no signal was detected in GFP+cells containing CHIKV-GFP-B2 (Figure 12C). Thus, NoV B2-expressing saRNAs activate PKR autophosphorylation but prevents elF2a phosphorylation and the consequent general translational block.

[0160] Example 10

[0161] Previous studies have identified other mammalian viral proteins that display a VSR activity. The protein 3A from human enterovirus 71 (HEV71-3A) was demonstrated to suppress Dicer activity and thereby increase viral replication without affecting the IFN response. No PKR inhibitory activity has been previously described for HEV71-3A. Vaccinia virus E3 protein has the dual function of blocking Dicerand PKR activation. Furthermore, expression of E3 protein in trans has been shown to enhance vector gene delivery by inhibiting PKR activation and the IFN response. Thus, the NoV B2 encoding sequence in CHIKV-GFP-B2 was replaced with the genes encoding either HEV71-3A or Vaccinia E3. These viral proteins were then compared for their ability to enhance saRNA when expressed cis by assessing their efficiencies in both HeLa WT and PKR- / -cells (Figure 13). It was found that expression of HEV71-3A did not inhibit this restriction. However, E3L had a beneficial effect on the vector’s activity with higher GFP level compared to cells with NoV B2 R59Q (Figure 13). Yet, incontrast to vectors encoding NoV B2 WT, E3L-expressing saRNAs exhibited lower activity in WT cells relative to PKR- / -suggesting partial inhibition of PKR. Furthermore, E3L encoding saRNA presented significantly lower GFP expression than NoV B2 saRNA. Thus, compared to two other known VSRs, the cis-expression of NoV-B2 was the most efficient to subvert PKR restriction of saRNAs in HeLa cells.

[0162] Example 11

[0163] To get further insight into the mechanism by which B2 enhanced saRNA-mediated gene expression, B2 protein localisation was analysed by confocal microscopy. It was previously shown that an N-terminal IxSTrEP-tagged B2 protein was functional since it efficiently increased GFP expression from CHIKV-GFP-STOP when expressed in trans. To localise B2, HeLa cells were therefore transfected with saRNA-GFP encoding IxSTrEP tagged B2 or R59Q mutant. While GFP was distributed throughout the cell, STrEP-B2 localised at the periphery of the cell (Figure 15A(a)-(b)).

[0164] Since the viral RNA replication occurs at the plasma membrane during alphaviruses infection, visualisation of the replication complex (RC), as well as dsRNA, is prompted to compare their localisation relative to B2. The viral protein nsP1 is part of the RC and associates to the plasma membrane and thereby serves as a membrane anchor for the RC. It is also essential for the generation of spherules that are plasma membrane invaginations inside which the viral RNA synthesis and transcription occurs and hence protect the viral dsRNA intermediates from cytosolic innate immune systems. Staining of nsP1 in HeLa cells transfected with CHIKV-GFP-STrEP-B2 revealed a localisation at the cell periphery (Figure 15A(a)-(c)). Interestingly, NoV B2 did not colocalise with nsP1 but rather juxtaposed within a lower layer of the cell (Figure 15B(d)). The localisation of STrEP-tagged version of B2 R59Q was analysed next. As saRNAs encoding a nonfunctional NoV B2 are restricted in HeLa cells, the mutant distribution in HeLa PKR- / -cells was assessed. It was first verified that NoV B2-wt was also localised in the periphery of the cells in PKRAcells as observed in WT cells, showing that the lack of PKR did not affect B2 localisation (Figure 15B(a)-(b)). In contrast, it was found that NoV B2 R59Q was homogenously distributed in the cell and no longer accumulated in the periphery of the cell (Figure 15C(a)-(b)). These results were confirmed in Ifnar2- / -THP1 cells in which it was found that NoV B2-wt was present at the periphery of the cell, and hence showed no co-localisation with the diffuse distribution of GFP within the cells (Figure 16A). In contrast, NoV B2 R59Q was distributed within the cytoplasm and hence displayed some co-localisation with GFP (Figure 16B). These results indicate that NoV B2 localises in proximity with the vector’s RC at the periphery of the cell and that its ability to bind dsRNA is essential for this specific localisation.

[0165] Example 12

[0166] Given the importance of dsRNA-binding activity of NoV B2 for its localisation at the periphery of the cell, the distribution of dsRNA generated by saRNA encoding either NoV B2-wt or B2-R59Q mutant in HeLa PKR- / -cells was then explored. In the absence of a functional B2, dsRNA staining was distributed throughout the cell (Figure 15C(c)). This was observed at different Z-stacks, in whichorthogonal views revealed dsRNA present within the cytoplasm of B2-R59Q encoding vectors (Figure 15C(d)). Interestingly, upon transfection of saRNA encoding NoV B2-wt, a limited number of dsRNA foci were observed within the body of the cell, yet an enhanced accumulation of dsRNA at the cellular periphery (Figure 15B(c)) was also observed. Orthogonal views of STrEP and dsRNA staining did not show colocalization between NoV B2 and dsRNA. Instead, similar to nsP1 staining, NoV B2 proteins localised at the periphery of the cells, but form a layer at proximity yet underneath the dsRNA foci leaving the body of the cell devoid of dsRNA (Figure 15B(d)). Interestingly, when comparing the number of dsRNA particles in 13 different B2 expressing cells against R59Q expressing cells, the R59Q vector presented significantly higher number of dsRNA particles (Figure 15D). When segmenting all 13 cells into 10 different layers within the Z-axis, cells with saRNAs expressing NoV B2 R59Q exhibited more dsRNA foci at the lower layers (2nd, 3rdand 4th) when compared to cells with NoV B2 WT-encoding vectors (Figure 15E). Further towards the apical side of the cell, these differences were lost. These data suggest that, in the presence of functional NoV B2 proteins, dsRNA accumulation is reduced and remains in the periphery of the cell, thereby segregated from the body of the cell.

[0167] Example 13

[0168] It was next addressed whether NoV B2 effect on CHIKV-based saRNAs was also observed with vectors based on other alphavirus genome. The family of alphaviruses is widely distributed and consist of Old Word alphaviruses including CHIKV, SINV and SFV and New World alphaviruses that comprises Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV). As the mechanisms involved with the cellular environment and on the inhibition of host antiviral defend can vary between Old World and New World viruses, the impact of NoV B2 on VEEV-based saRNAs was tested, which are often used for vaccine studies.

[0169] VEEV-based vectors were generated harbouring, downstream of their subgenomic promoter, a GFP gene as well as the coding sequence of NoV B2 (WT or B2 R59Q) separated by P2A cleavage site. The CHlKV-based and VEEV-based B2 expressing vectors were compared in various cell types and it was found that VEEV-based vectors were more efficient and displayed higher percentage of GFP+cells in HEK293, HEK293T and THP1 cells compared to CHIKV-based vectors (Figure 18). HeLa cells were the least permissive cells for both vectors, suggesting that the restriction is the strongest in these cells. It was observed that, in these Hela cells displaying the strongest restriction, the cisexpression of NoV B2 from VEEV increased the MFI of the GFPhighpopulation, while this effect was not apparent in HEK 293T nor HEK 293 cells (Figure 17 A). This B2-mediated enhancement was lost in HeLa PKR cells, suggesting that VEEV-derived vectors, like CHIKV-based saRNAs, are restricted by PKR and expression of NoV B2 is able to alleviate this restriction that is stronger in Hela cells than in both HEK 293T and 293 cells (Figure 17B).

[0170] In THP1 cells, VEEV-R59Q vectors displayed significantly higher activity than CHIKV-R59Q, suggesting that VEEV is naturally less sensitive to PKR activity in THP1 cells (Figure 17C). Nevertheless, the c / s-expression of a functional NoV B2 caused an increase in VEEV-based vectoractivity compared to vector expressing NoV B2 R59Q (Figure 17C). The c / s-expression of NoV B2 from CHIKV and VEEV abolished the differences in GFP expression observed between both vectors (Figure 17C). This suggests that, while there is a difference in sensitivity to PKR between VEEV and CHlKV-based vector, the inhibition of PKR by B2 abolished this difference.

[0171] As previously observed with CHlKV-based saRNA, there was a similar activation of the IFN response in cells transfected with VEEV expressing NoV B2 WT or R59Q (Figure 17E), yet in contrast to CHIKV vectors, the B2-mediated enhancement of GFP in VEEV vectors was lost in / / har2 / THP1-2R (Figure 17D), suggesting that the boosted expression of PKR upon IFN signalling amplification is responsible for the restriction observed in VEEV vectors.

[0172] Example 14

[0173] A further aim was to understand the impact of protein B2 on the global host cell translation machinery. Indeed, PKR activation causes a general block in translation and thereby affect not only the SAM-derived transgene expression, but also most host cellular genes. To address this, the incorporation of a methionine analogue, L-homopropargylglycine (HPG), was assessed, which allows the measurement of nascent protein synthesis. HeLa cells were transfected with either VEEV-B2 or R59Q expressing vectors and 20 hours after transfection, cells were primed, stained for the incorporation of HPG and analysed by flow cytometry. A strong reduction of protein synthesis in the whole-cell population exposed to VEEV saRNA was found, regardless of the B2 version encoded. HPG incorporation was reduced in more than 50% of the bulk population reaching similar levels of staining as cells treated with cycloheximide, a widely used translation inhibitor. This could directly be caused by cell-intrinsic and extrinsic stress caused by saRNA activity following transfection. In order to distinguish transfected cells from non-transfected cells, HPG incorporation was measured for within the GFP+population. It was found that more than 95% of the cells containing B2-R59Q expressing vector showed a block of de novo protein synthesis, corresponding to 8.6 fold less than GFP- cells (Figure 17F). Interestingly, the B2-WT expressing vector was able to rescue the ability of cells to initiate translation with an increase of more than 20% of the GFP+cells incorporating HPG resulting in less than 2 fold difference with the GFP-population, reflecting a significant increase of nascent protein translation (Figure 17F). Therefore, the inclusion of B2-wt, not only enhances the expression of the desired transgene, but it also reduces the general host translational shutdown of cellular mRNAs.

[0174] Example 15

[0175] It was tested whether the B2-mediated enhancement of gene expression observed for both CHIKV and VEEV vectors occurs on a relevant antigen rather than GFP reporter. For this the GFP cassette in the generated constructs was replaced with the codon-optimised sequence encoding the prefusion form of SARS-CoV2 Spike protein or the surface glycoprotein hemagglutinin (HA) from Influenza virus. Supporting previous observations, CHIKV displayed enhanced Spike protein expression in HEK293T.17 cells transfected with vectors expressing NoV B2 WT compared to NoVB2 R59Q (Figure 17G). Interestingly, VEEV vectors presented an overall enhancement of Spike protein in HEK293T.17 even though FACS analysis of GFP did not seem to show a difference at the single cell level (Figure 17A). The same enhancement of B2 was observed in Hela cells transfected with VEEV-based vectors (Figure 17H), while the Spike protein could not be detected by Western blot in cells transfected with CHIKV-based vectors (Figure 17G). The B2-mediated enhancement of gene expression was also observed with both CHIKV and VEEV vectors expressing HA protein in HEK 293T.17 cells (Figure 171). Thus, NoV B2 has the ability to enhance in cis the expression of various genes of interest encoded by a saRNA based on CHIKV or VEEV-based genome.

[0176] Example 16

[0177] It was found that NoVB2 and FHVB2 share 47.81% identity in their DNA sequence and 24.64% identity in their amino acid sequence (amino acid sequence identity in the RNA-binding region of 38.1%). However, NoVB2 and FHVB2 share a very similar structure. This was discovered by superimposing the structures of NoVB2 and FHVB2 onto each other and then quantifying the similarity using the Root Mean Square Deviation (RMSD) value. RMSD is used as a quantitative measure of the similarity between two superimposed atomic coordinates. An RMSD value of less than 2 Angstrom (A) shows that two proteins are very similar in their structure. When superimposed, NoVB2 and FHVB2 have an RMSD value of 1.78 A. Accordingly, the conformational structures of the two proteins are highly similar.

[0178] Example 17

[0179] The modulation of protein expression by the B2 protein was measured by calculating the ratio of the Area Under the Curve for GFP expression over a 48-hour period with B2 in position 2 (downstream of GFP) (Figure 4B). This analysis revealed a 9.56-fold enhancement in GFP expression, corresponding to an 856% increase within the 48-hour window.

[0180] The increase in Spike protein expression was also analysed by western blot. This measurement reflects the actual protein levels in cell lysates after 24 hours. The band intensity for Spike expression in the presence of B2 protein was quantified and compared to the STOP vector, which served as the reference (set at 100%). With NoVB2 in position 2, Spike protein expression increased to 213% (2.13x), while with B2 in position 1 (upstream of Spike), the increase was 321% (3.21x) (Figure 19A-B).

[0181] Considering the 856% increase in GFP expression over 48 hours with B2 in position 2, and the same order resulted in a 2.13-fold increase in Spike expression at 24 hours, it stands to reason that a 3.21-fold increase in Spike expression with B2 in position 1 would correspond to a 14.9-fold increase over a 48-hour timeframe.

[0182] Example 18The saRNA constructs encoding GFP-STOP, GFP-B2, or GFP-R59Q were generated by in vitro transcription using the Invitrogen™ mMESSAGE mMACHINE™ SP6 Transcription Kit, as previously described. In parallel, messenger RNAs encoding the alphavirus capsid protein and envelope glycoproteins were separately generated by in vitro transcription using the same protocol. The plasmids used for expression of the capsid and envelope glycoproteins were first described in Glasker et al. 2013, Virol J. volume 10 article number 235, and correspond to pChikHelper-C and pChikHelper-E, respectively.

[0183] BHK-21 cells were electroporated with combinations of the saRNA construct and the capsid and envelope glycoprotein mRNAs to enable production of VRPs encapsidating the respective saRNA (Figure 20). Following electroporation, cells were incubated at 28 °C in Glasgow’s Minimum Essential Medium (GMEM) supplemented with 5% fetal bovine serum (FBS), 1% L-glutamine, 10% tryptose phosphate broth, 20 mM HEPES (pH 7.2), 100 U / ml penicillin, and 0.1 mg / ml streptomycin, under conditions permissive for VRP assembly and release. Culture supernatants containing VRPs were harvested approximately 48 hours post-electroporation, clarified by centrifugation for 30 minutes at 4,000 x g and 4 °C, and subsequently passed through a 0.45 pm filter.

[0184] VRP titres were determined by serial dilution on naive BHK-21 cells, followed by enumeration of GFP-positive cells using fluorescence microscopy (Figure 21 A). For functional evaluation, naive BHK-21 cells were transduced with the harvested VRPs at a multiplicity of infection of approximately 10 and incubated for approximately 24 hours prior to harvesting the cells and preparing them for flow cytometry analysis. Within the GFP+populations, self-replicating properties of the vectors were defined by gating on cells with mean fluorescent intensity (MFI) of 103or higher and the MFI of this group quantified (Figure 21 B).

[0185] The DNA sequences of the plasmids detailed in Table 1 are reproduced as follows.

[0186] The plasmid SP6-CHIKVRepl-ERR-GL-P2A-B2 may have the nucleic acid sequence represented by SEQ ID NO:3:

[0187] ATGGCTGCGTGAGACACACGTAGCCTACCAGTTTCTTACTGCTCTACTCTGCAAAGCAAGAGATTAATAACCCATCATGGA TCCTGTGTACGTGGACATAGACGCTGACAGCGCCTTTTTGAAGGCCCTGCAACGTGCGTACCCCATGTTTGAGGTGGAACC AAGGCAGGTCACACCGAATGACCATGCTAATGCTAGAGCGTTCTCGCATCTAGCTATAAAACTAATAGAGCAGGAAATTGA CCCCGACTCAACCATCCTGGATATCGGCAGTGCGCCAGCAAGGAGGATGATGTCGGACAGGAAGTACCACTGCGTCTGCCC GATGCGCAGTGCGGAAGATCCCGAGAGACTCGCCAATTATGCGAGAAAGCTAGCATCTGCCGCAGGAAAAGTCCTGGACAG AAACATCTCTGGAAAGATCGGGGACTTACAAGCAGTAATGGCCGTGCCAGACACGGAGACGCCAACATTCTGCTTACACAC AGACGTCTCATGTAGACAGAGAGCAGACGTCGCTATATACCAAGACGTCTATGCTGTACACGCACCCACGTCGCTATACCA CCAGGCGATTAAAGGGGTCCGAGTGGCGTACTGGGTTGGGTTCGACACAACCCCGTTCATGTACAATGCCATGGCGGGTGC CTACCCCTCATACTCGACAAACTGGGCAGATGAGCAGGTACTGAAGGCTAAGAACATAGGATTATGTTCAACAGACCTGAC GGAAGGTAGACGAGGCAAGTTGTCTATTATGAGAGGGAAAAAGCTAAAACCGTGCGACCGTGTGCTGTTCTCAGTAGGGTC AACGCTCTACCCGGAAAGCCGCAAGCTACTTAAGAGCTGGCACCTGCCATCGGTGTTCCATTTAAAGGGCAAACTCAGCTT CACATGCCGCTGTGATACAGTGGTTTCGTGTGAGGGCTACGTCGTTAAGAGAATAACGATGAGCCCAGGCCTTTATGGAAA AACCACAGGGTATGCGGTAACCCACCACGCAGACGGATTCCTGATGTGCAAGACTACCGACACGGTTGACGGCGAAAGAAT GTCATTCTCGGTGTGCACATACGTGCCGGCGACCATTTGTGATCAAATGACCGGCATCCTTGCTACAGAAGTCACGCCGGA GGATGCACAGAAGCTGTTGGTGGGGCTGAACCAGAGAATAGTGGTTAACGGCAGAACGCAACGGAATACGAACACCATGAA AAATTATCTGCTTCCCGTGGTCGCCCAAGCCTTCAGTAAGTGGGCAAAGGAGTGCCGGAAAGACATGGAAGATGAAAAACTCCTGGGGGTCAGAGAAAGAACACTGACCTGCTGCTGTCTATGGGCATTCAAGAAGCAGAAAACACACACGGTCTACAAGAG GCCTGATACCCAGTCAATTCAGAAGGTTCAGGCCGAGTTTGACAGCTTTGTGGTACCGAGTCTGTGGTCGTCCGGGTTGTC AATCCCTTTGAGGACTAGAATCAAATGGTTGTTAAGCAAGGTGCCAAAAACCGACCTGATCCCATACAGCGGAGACGCCCG AGAAGCCCGGGACGCAGAAAAAGAAGCAGAGGAAGAACGAGAAGCAGAACTGACTCGCGAAGCCCTACCACCTCTACAGGC AGCACAGGAAGATGTTCAGGTCGAAATCGACGTGGAACAGCTTGAGGACAGAGCGGGCGCAGGAATAATAGAGACTCCGAG AGGAGCTATCAAAGTTACTGCCCAACCAACAGACCACGTCGTGGGAGAGTACCTGGTACTCTCCCCGCAGACCGTACTACG TAGCCAGAAGCTCAGTCTGATTCACGCTTTGGCGGAGCAAGTGAAGACGTGCACGCACAACGGACGAGCAGGGAGGTATGC GGTCGAAGCGTACGACGGCCGAGTCCTAGTGCCCTCAGGCTATGCAATCTCGCCTGAAGACTTCCAGAGTCTAAGCGAAAG CGCAACGATGGTGTATAACGAAAGAGAGTTCGTAAACAGAAAGCTACACCATATTGCGATGCACGGACCAGCCCTGAACAC CGACGAAGAGTCGTATGAGCTGGTGAGGGCAGAGAGGACAGAACACGAGTACGTCTACGACGTGGATCAGAGAAGATGCTG TAAGAAGGAAGAAGCCGCAGGACTGGTACTGGTGGGCGACTTGACTAATCCGCCCTACCACGAATTCGCATATGAAGGGCT AAAAATCCGCCCTGCCTGCCCATACAAAATTGCAGTCATAGGAGTCTTCGGAGTACCGGGATCTGGCAAGTCAGCTATTAT CAAGAACCTAGTTACCAGGCAGGACCTGGTGACTAGCGGAAAGAAAGAAAACTGCCAAGAAATCACCACCGACGTGATGAG ACAGAGAGGTCTAGAGATATCTGCACGTACGGTTGACTCGCTGCTCTTGAATGGATGCAACAGACCAGTCGACGTGTTGTA CGTAGACGAGGCGTTTGCGTGCCACTCTGGAACGCTACTTGCTTTGATCGCCTTGGTGAGACCAAGGCAGAAAGTTGTACT TTGTGGTGACCCGAAGCAGTGCGGCTTCTTCAATATGATGCAGATGAAAGTCAACTATAATCACAACATCTGCACCCAAGT GTACCACAAAAGTATCTCCAGGCGGTGTACACTGCCTGTGACCGCCATTGTGTCATCGTTGCATTACGAAGGCAAAATGCG CACTACGAATGAGTACAACAAGCCGATTGTAGTGGACACTACAGGCTCAACAAAACCTGACCCTGGAGACCTCGTGTTAAC GTGCTTCAGAGGGTGGGTTAAACAACTGCAAATTGACTATCGTGGATACGAGGTCATGACAGCAGCCGCATCCCAAGGGTT AACCAGAAAAGGAGTTTACGCAGTTAGACAAAAAGTTAATGAAAACCCGCTCTATGCATCAACGTCAGAGCACGTCAACGT ACTCCTAACGCGTACGGAAGGTAAACTGGTATGGAAGACACTTTCCGGCGACCCGTGGATAAAGACGCTGCAGAACCCACC GAAAGGAAACTTCAAAGCAACTATTAAGGAGTGGGAGGTGGAGCATGCATCAATAATGGCGGGCATCTGCAGTCACCAAAT GACCTTCGATACATTCCAAAATAAAGCCAACGTTTGTTGGGCTAAGAGCTTGGTCCCTATCCTCGAAACAGCGGGGATAAA ACTAAATGATAGGCAGTGGTCTCAGATAATTCAAGCCTTCAAAGAAGACAAAGCATACTCACCTGAAGTAGCCCTGAATGA AATATGTACGCGCATGTATGGGGTGGATCTAGACAGCGGGCTATTTTCTAAACCGTTGGTGTCTGTGTATTACGCGGATAA CCACTGGGATAATAGGCCTGGAGGGAAAATGTTCGGATTTAACCCCGAGGCAGCATCCATTCTAGAAAGAAAGTATCCATT CACAAAAGGGAAGTGGAACATCAACAAGCAGATCTGCGTGACTACCAGGAGGATAGAAGACTTTAACCCTACCACCAACAT CATACCGGCCAACAGGAGACTACCACACTCATTAGTGGCCGAACACCGCCCAGTAAAAGGGGAAAGAATGGAATGGCTGGT TAACAAGATAAACGGCCACCACGTGCTCCTGGTCAGTGGCTATAACCTTGCACTGCCTACTAAGAGAGTCACTTGGGTAGC GCCGTTAGGTGTCCGCGGAGCGGACTACACATACAACCTAGAGTTGGGTCTGCCAGAGAGAAGGGGTAGGTATGACCTAGT GGTCATAAACATCCACACACCTTTTCGCATACACCATTACCAACAGTGCGTCGACCACGCAATGAAACTGCAAATGCTCGG GGGTGACTCATTGAGACTGCTCAAACCGGGCGGCTCTCTATTGATCAGAGCATATGGTTACGCAGATAGAACCAGTGAACG AGTCATCTGCGTATTGGGACGCAAGTTTAGATCGTCTAGAGCGTTGAAACCACCATGTGTCACCAGCAACACTGAGATGTT TTTCCTATTCAGCAACTTTGACAATGGCAGAAGGAATTTCACAACTCATGTCATGAACAATCAACTGAATGCAGCCTTCGT AGGACAGGTCACCCGAGCAGGATGTGCACCGTCGTACCGGGTAAAACGCATGGACATCGCGAAGAACGATGAAGAGTGCGT AGTCAACGCCGCTAACCCTCGCGGGTTACCGGGTGGCGGTGTTTGCAAGGCAGTATACAAAAAATGGCCGGAGTCCTTTAA GAACAGTGCAACACCAGTGGGAACCGCAAAAACAGTTATGTGCGGTACGTATCCAGTAATCCACGCTGTTGGACCAAACTT CTCTAATTATTCGGAGTCTGAAGGGGACCGGGAATTGGCAGCTGCCTATCGAGAAGTCGCAAAGGAAGTAACTAGGCTGGG AGTAAATAGTGTAGCTATACCTCTCCTCTCCACAGGTGTATACTCAGGAGGGAAAGACAGGCTGACCCAGTCACTGAACCA CCTCTTTACAGCCATGGACTCGACGGATGCAGACGTGGTCATCTACTGCCGCGACAAAGAATGGGAGAAGAAAATATCTGA GGCCATACAGATGCGGACCCAAGTAGAGCTGCTGGATGAGCACATCTCCATAGACTGCGATATTGTTCGCGTGCACCCTGA CAGCAGCTTGGCAGGCAGAAAAGGATACAGCACCACGGAAGGCGCACTGTACTCATATCTAGAAGGGACCCGTTTTCATCA GACGGCTGTGGATATGGCGGAGATACATACTATGTGGCCAAAGCAAACAGAGGCCAATGAGCAAGTCTGCCTATATGCCCT GGGGGAAAGTATTGAATCGATCAGGCAGAAATGCCCGGTGGATGATGCAGACGCATCATCTCCCCCCAAAACTGTCCCGTG CCTTTGCCGTTACGCTATGACTCCAGAACGCGTCACCCGGCTTCGCATGAACCACGTCACAAGCATAATTGTGTGTTCTTC GTTTCCCCTCCCAAAGTACAAAATAGAAGGAGTGCAAAAAGTCAAATGCTCTAAGGTAATGCTATTTGACCACAACGTGCC ATCGCGCGTAAGTCCAAGGGAATATAGATCTTCCCAGGAGTCTGCACAGGAGGCGAGTACAATCACGTCACTGACGCATAG TCAATTCGACCTAAGCGTTGATGGCGAGATACTGCCCGTCCCGTCAGACCTGGATGCTGACGCCCCAGCCCTAGAACCAGC ACTAGACGACGGGGCGACACACACGCTGCCATCCACAACCGGAAACCTTGCGGCCGTGTCTGATTGGGTAATGAGCACCGT ACCTGTCGCGCCGCCCAGAAGAAGGCGAGGGAGAAACCTGACTGTGACATGTGACGAGAGAGAAGGGAATATAACACCCAT GGCTAGCGTCCGATTCTTTAGGGCAGAGCTGTGTCCGGTCGTACAAGAAACAGCGGAGACGCGTGACACAGCAATGTCTCT TCAGGCACCACCGAGTACCGCCACGGAACCGAATCATCCGCCGATCTCCTTCGGAGCATCAAGCGAGACGTTCCCCATTAC ATTTGGGGACTTCAACGAAGGAGAAATCGAAAGCTTGTCTTCTGAGCTACTAACTTTCGGAGACTTCTTACCAGGAGAAGT GGATGACTTGACAGACAGCGACTGGTCCACGTGCTCAGACACGGACGACGAGTTAAGACTAGACAGGGCAGGTGGGTATAT ATTCTCGTCGGACACCGGTCCAGGTCATTTACAACAGAAGTCAGTACGCCAGTCAGTGCTGCCGGTGAACACCCTGGAGGA AGTCCACGAGGAGAAGTGTTACCCACCTAAGCTGGATGAAGCAAAGGAGCAACTATTACTTAAGAAACTCCAGGAGAGTGC ATCCATGGCCAACAGAAGCAGGTATCAGTCGCGCAAAGTAGAAAACATGAAAGCAGCAATCATCCAGAGACTAAAGAGAGG CTGTAGACTATACTTAATGTCAGAGACCCCAAAAGTCCCTACTTACCGGACTACATATCCGGCGCCTGTGTACTCGCCTCCGATCAACGTCCGATTGTCCAATCCCGAGTCCGCAGTGGCAGCATGCAATGAGTTCTTAGCTAGAAACTATCCAACTGTCTC ATCATACCAAATTACCGACGAGTATGATGCATATCTAGACATGGTGGACGGGTCGGAGAGTTGCCTGGACCGAGCGACATT CAATCCGTCAAAACTCAGGAGCTACCCGAAACAGCACGCTTACCACGCGCCCTCCATCAGAAGCGCTGTACCGTCCCCATT CCAGAACACACTACAGAATGTACTGGCAGCAGCCACGAAAAGAAACTGCAACGTCACACAGATGAGGGAATTACCCACTTT GGACTCAGCAGTATTCAACGTGGAGTGTTTCAAAAAATTCGCATGCAACCAAGAATACTGGGAAGAATTTGCTGCCAGCCC TATTAGGATAACAACTGAGAATTTAGCAACCTATGTTACTAAACTAAAAGGGCCAAAAGCAGCAGCGCTATTCGCAAAAAC CCATAATCTACTGCCACTACAGGAAGTACCAATGGATAGGTTCACAGTAGATATGAAAAGGGACGTAAAGGTGACTCCTGG TACAAAGCATACAGAGGAAAGACCTAAGGTGCAGGTTATACAGGCGGCTGAACCCTTGGCGACAGCATACCTATGTGGGAT TCACAGAGAGCTGGTTAGGAGGCTGAACGCCGTCCTCCTACCCAATGTACATACACTATTTGACATGTCTGCCGAGGATTT CGATGCCATCATAGCCGCACACTTTAAGCCAGGAGACACTGTTTTGGAAACGGACATAGCCTCCTTTGATAAGAGCCAAGA TGATTCACTTGCGCTTACTGCTTTGATGCTGTTAGAGGATTTAGGGGTGGATCACTCCCTGCTGGACTTGATAGAGGCTGC TTTCGGAGAGATTTCCAGCTGTCACCTACCGACAGGTACGCGCTTCAAGTTCGGCGCCATGATGAAATCAGGTATGTTCCT AACTCTGTTCGTCAACACATTGTTAAACATCACCATCGCCAGCCGAGTGCTGGAAGATCGTCTGACAAAATCCGCGTGCGC GGCCTTCATCGGCGACGACAACATAATACATGGAGTCGTCTCCGATGAATTGATGGCAGCCAGATGTGCCACTTGGATGAA CATGGAAGTGAAGATCATAGATGCAGTTGTATCCTTGAAAGCCCCTTACTTTTGTGGAGGGTTTATACTGCACGATACTGT GACAGGAACAGCTTGCAGAGTGGCAGACCCGCTAAAAAGGCTTTTTAAACTGGGCAAACCGCTAGCGGCAGGTGACGAACA AGATGAAGATAGAAGACGAGCGCTGGCTGACGAAGTGATCAGATGGCAACGAACAGGGCTAATTGATGAGCTGGAGAAAGC GGTATACTCTAGGTACGAAGTGCAGGGTATATCAGTTGTGGTAATGTCCATGGCCACCTTTGCAAGCTCCAGATCCAACTT CGAGAAGCTCAGAGGACCCGTCATAACTTTGTACGGCGGTCCTAAATAGGTACGCACTACAGCTACCTATTTTGCAGAAGC CGACAGCAAGTATCTAAACACTAATCAGCTACCTAGGAAGTTCGAATATAGGCGCGCCGCCACCATGGTGAGCAAGGGCGA GGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTCCGCGGCGA GGGCGAGGGCGATGCCACCAACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCAC CCTCGTGACCACCTTAGGCTACGGCGTGGCCTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTC CGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTCTTTCAAGGACGACGGTACCTACAAGACCCGCGCCGAGGTGAA GTTCGAGGGCGACACCCTGGTGAACCGCATCGTGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAA GCTGGAGTACAACTTCAACAGCCACAAGGTCTATATCACGGCCGACAAGCAGAAGAACGGCATCAAGGCTAACTTCAAGAC CCGCCACAACGTTGAGGACGGCGGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT GCTGCCCGACAACCACTACCTGAGCCATCAGTCCAAACTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGAA GGAGAGGGTGACCGCCGCCGGGATTACACATGACATGGACGAGCTGTACAAGgctagcggcagcggcgccaccaacttcag cctgctgaagcaggccggcgacgtggaggagaaccccGGGCCCgaattcATGACAAACATGTCATGCGCTTACGAGCTAAT CAAGTCACTTCCAGCCAAGCTGGAGCAGCTGGCTCAGGAGACGCAAGCAACGATCCAAACGCTCATGATCGCGGATCCCAA CGTCAACAAGGATCTGCGAGCGTTCTGCGAGTTCCTGACCGTGCAGCACCAGCGGGCGTATCGAGCGACGAACAGCCTGCT CATCAAACCGCGAGTCGCAGCAGCGCTTCGCGGGGAGGAGCTGGACCTGGGCGAGGCGGACGTCGCCGCCCGGGTCCGCCA GCTAAAACAACAGCTGGCGGAGCTCGAGATGGAAATCAAGCCAGGGCACCAACAAGTGGCCCAAGTAAGCGGCAGGCGGAA GGCCGCAGCCGCAGCTCCCGTGGCCCAGCTGGGTCGCGTGGGCGTGGTAAATGAGTGATAGTAGTTAATTAACTCGCGATC GCTAACTTGACAATTAAGTATGAAGGTATATGTGTCCCCTAAGAGACACACTGTACATAGCAAATAATCTATAGATCAAAG GGCTACGCAACCCCTGAATAGTAACAAAATACAAAATCACTAAAAATTATAAAAACAGAAAAATACATAAATAGGTATACG TGTCCCCTAAGAGACACATTGTATGTAGGTGATAAGTATAGATCAAAGGGCCGAATAACCCCTGAATAGTAACAAAATATG AAAATCAATAAAAATCATAAAATAGAAAAACCATAAACAGAAGTAGTTCAAAGGGCTATAAAACCCCTGAATAGTAACAAA ACATAAAATTAATAAAAATCAAATGAATACCATAATTGGCAAACGGAAGAGATGTAGGTACTTAAGCTTCCTAAAAGCAGC CGAACTCACTTTGAGAAGTAGGCATAGCATACCGAACTCTTCCACGATTCTCCGAACCCACAGGGACGTAGGAGATGTTAT TTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAGCGGCCGCTTCCGCTCACTGCCCGCTTTCCAGTCGGGAAA CCTGTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTCCTCGCTCACTGACTC GCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCC GCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAAC CCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACC GGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAG GTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTT GAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGC GGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAG CCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGC AAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAAC GAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGT TTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCG ATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGC CCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCC GAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCG CCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCG ATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCA TCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCT TGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGG CGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCT TTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAA TGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTT GAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATA TTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATA AATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGCGCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGG GAAGGGCGTTTCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGT AACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGATTTAAATACGatttaggtgacactataG [ SEQ ID NO: 3]

[0188] The plasmid VEEV-att-GL-NoV-B2 may have the nucleic acid sequence represented by SEQ ID NO: 4:

[0189] ATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAatgGAGAAAGTTCACGTTGACATCGAGGAAGACAGCC CATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATG CCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAAGTG CGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGT ATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTGGCCG CCGTCATGAGCGACCCTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCG CTGTTTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTCGCCTACT GGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACTCTACCAACTGGGCCGACG AAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGAGCGGTCACGTAGAGGGATGTCCATTCTTA GAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCTGTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGA GGAGCTGGCACCTGCCGTCTGTATTTCACTTACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCG ACGGGTACGTCGTTAAAAGAATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCG AGGGATTCTTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAGCTA CATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCTGGTTGGGCTCAACC AGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTACCTTTTGCCCGTAGTGGCCCAGGCAT TTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAAGGCCACTAGGACTACGAGATAGACAGTTAGTCATGG GGTGTTGTTGGGCTTTTAGAAGGCACAAGATAACATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACA GCGATTTCCACTCATTCGTGCTGCCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGT TAGAGGAGCACAAGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGG AGGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTGGAGGCAGACG TCGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAAAGGTTACCAGCTACgatGGCG AGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAAGAGTGAAAAATTATCTTGCATCCACCCTCTCG CTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAAGGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGC CAGAGGGACATGCAATACCCGTCCAGGACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCG TAAACAGGTACCTGCACCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCA GCGAGCACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAGGGCTCA CAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGCCGCTCCTTACCAAGTAC CAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAAAGCGCAGTCACCAAAAAAGATCTAGTGG TGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACGTCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTG TGGACTCAGTGCTCTTGAATGGATGCAAACACCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTA CTCTCAGAGCGCTCATAGCCATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACA TGATGTGCCTGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAAAT CTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACTAAGATTGTGATTG ACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAGGGTGGGTGAAGCAGTTGCAAATAG ATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCTGACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGG TGAATGAAAATCCTCTGTACGCACCCACCTCAGAACATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGA AAACACTAGCCGGCGACCCATGGATAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGC AAGCAGAGCATGATGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGT GTTGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTGTGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTTTGGACTCGATCTGGACTCCG GTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGGGATAACTCCCCGTCGCCTAACATGTACGGGC TGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACCCACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACA TGAACACTGGTACACTGCGCAATTATGATCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCC TCCACCATAATGAACACCCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGG AAAAGTTGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGATTTAG GCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATACCATCACTATCAGCAGT GTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATCTGAATCCCGGCGGAACCTGTGTCAGCA TAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGGTGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCA AACCGAAATCCTCACTTGAAGAGACGGAAGTTCTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATtctT ACAAGCTTTCATCAACCTTGACCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGG TGCGAGGGGATATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTGT GCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGACTGGTCAAAGGTG CAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGAAGGTGACAAACAGTTGGCAGAGG CTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCAGTAGCGATTCCACTGTTGTCCACCGGCATCTTTT CCGGGAACAAAGATCGACTAACCCAATCATTGAACCATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATAT ACTGCAGGGACAAGAAATGGGAAATGACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCG ACGACTCTTCAGTGACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACA GCACAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGAAATTAATG CCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGCATGAGCAGTATTAGGTCGA AATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTTGCTTGTGCATCCATGCCATGACTCCAGAAA GAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTACTGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTG GTGTGCAGAAGATCCAATGCTCCCAGCCTATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCG TGGAAACACCACCGGTAGACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCAC TTATAACCGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGTTTGC TGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTATCTAGCTCATCCTGGT CCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCTGGAGGGAGCTAGCGTGACCAGCGGGG CAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAGTTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTAT TCAGGAACCCTCCACATCCCGCTCCGCGCACAAGAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAG TTTCCACCCCGCCAGGCGTGAATAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGT CGGTCTCGAGAACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAG CACAACAACAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACAACAAAAATCAG TAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTATGCCCCGCGCCTCGACCAAGAAA AAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTAACAGAAGCAGATACCAGTCCAGGAAGGTGGAGA ACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGGCCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACC GAACCCTGCATCCTGTTCCTTTGTATTCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTA ACGCCATGTTGAAAGAGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTG ACGGAGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCCTATTTGG AACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAGCTGCCACAAAAAGAAATT GCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAATGTGGAATGCTTCAAGAAATATGCGTGTA ATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGGCTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAA AAGGACCAAAAGCTGCTGCTCTTTTTGCGAAGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGTTTGTAA TGGACTTAAAGAGAGACGTGAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTG CCGATCCGCTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGAACA TTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGGGGATTGTGTTCTGG AAACTGACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCGTTAATGATTCTGGAAGACTTAGGTG TGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAATTTCATCAATACATTTGCCCACTAAAACTAAATTTA AATTCGGAGCCATGATGAAATCTGGAATGTTCCTCACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAG TGTTGAGAGAACGGCTAACCGGATCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACA AATTAATGGCAGACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTT ATTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAAAGGCTGTTTA AGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGCATGAAGAGTCAACACGCTGGA ACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTATGAAACCGTAGGAACTTCCATCATAGTTATGG CCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGCTACCTGAGAGgggcccctataactctctacggctaacctgaat ggactacgacatagtctagtccgccaagttcgaatataggCGCGCCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCAC CGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTCCGCGGCGAGGGCGAGGGCGA TGCCACCAACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCAC CTTAGGCTACGGCGTGGCCTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTCTTTCAAGGACGACGGTACCTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGA CACCCTGGTGAACCGCATCGTGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAA CTTCAACAGCCACAAGGTCTATATCACGGCCGACAAGCAGAAGAACGGCATCAAGGCTAACTTCAAGACCCGCCACAACGT TGAGGACGGCGGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAA CCACTACCTGAGCCATCAGTCCAAACTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGAAGGAGAGGGTGAC CGCCGCCGGGATTACACATGACATGGACGAGCTGTACAAGgctagcggcagcggcgccaccaacttcagcctgctgaagca ggccggcgacgtggaggagaaccccGGGCCCgaattcATGACAAACATGTCATGCGCTTACGAGCTAATCAAGTCACTTCC AGCCAAGCTGGAGCAGCTGGCTCAGGAGACGCAAGCAACGATCCAAACGCTCATGATCGCGGATCCCAACGTCAACAAGGA TCTGCGAGCGTTCTGCGAGTTCCTGACCGTGCAGCACCAGCGGGCGTATCGAGCGACGAACAGCCTGCTCATCAAACCGCG AGTCGCAGCAGCGCTTCGCGGGGAGGAGCTGGACCTGGGCGAGGCGGACGTCGCCGCCCGGGTCCGCCAGCTAAAACAACA GCTGGCGGAGCTCGAGATGGAAATCAAGCCAGGGCACCAACAAGTGGCCCAAGTAAGCGGCAGGCGGAAGGCCGCAGCCGC AGCTCCCGTGGCCCAGCTGGGTCGCGTGGGCGTGGTAAATGAGTGATAGTAGTTAATtaactcgcgatcgctgaatacagc agcaattggcaagctgcttacatagaactcgcggcgattgGCATGCCGCCTTAAAATTTTTATTTTATTTTTCTTTTCTTT TCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAAACGCGTCGAGGGGAATTAATTCTTGAA GACGAAAGGGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATAT GTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCG TGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGC TGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGA AGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCA ACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCAT GACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGG ACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGA AGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACT ACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCT TCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGA TGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGA GATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCA TTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCA CTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAAC AAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG CAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTAC ATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACG ATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACAC CGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAG CGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCG CCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGAGCTC GATTTAGGTGACACTATAG

[0190] [ SEQ ID NO: 4 ]

[0191] The plasmid CHIKV-ERR-GL-FHV-B2 may have the nucleic acid sequence represented by SEQ ID NO: 5:

[0192] ATGGCTGCGTGAGACACACGTAGCCTACCAGTTTCTTACTGCTCTACTCTGCAAAGCAAGAGATTAATAACCCATCATGGA TCCTGTGTACGTGGACATAGACGCTGACAGCGCCTTTTTGAAGGCCCTGCAACGTGCGTACCCCATGTTTGAGGTGGAACC AAGGCAGGTCACACCGAATGACCATGCTAATGCTAGAGCGTTCTCGCATCTAGCTATAAAACTAATAGAGCAGGAAATTGA CCCCGACTCAACCATCCTGGATATCGGCAGTGCGCCAGCAAGGAGGATGATGTCGGACAGGAAGTACCACTGCGTCTGCCC GATGCGCAGTGCGGAAGATCCCGAGAGACTCGCCAATTATGCGAGAAAGCTAGCATCTGCCGCAGGAAAAGTCCTGGACAG AAACATCTCTGGAAAGATCGGGGACTTACAAGCAGTAATGGCCGTGCCAGACACGGAGACGCCAACATTCTGCTTACACAC AGACGTCTCATGTAGACAGAGAGCAGACGTCGCTATATACCAAGACGTCTATGCTGTACACGCACCCACGTCGCTATACCA CCAGGCGATTAAAGGGGTCCGAGTGGCGTACTGGGTTGGGTTCGACACAACCCCGTTCATGTACAATGCCATGGCGGGTGC CTACCCCTCATACTCGACAAACTGGGCAGATGAGCAGGTACTGAAGGCTAAGAACATAGGATTATGTTCAACAGACCTGAC GGAAGGTAGACGAGGCAAGTTGTCTATTATGAGAGGGAAAAAGCTAAAACCGTGCGACCGTGTGCTGTTCTCAGTAGGGTC AACGCTCTACCCGGAAAGCCGCAAGCTACTTAAGAGCTGGCACCTGCCATCGGTGTTCCATTTAAAGGGCAAACTCAGCTT CACATGCCGCTGTGATACAGTGGTTTCGTGTGAGGGCTACGTCGTTAAGAGAATAACGATGAGCCCAGGCCTTTATGGAAA AACCACAGGGTATGCGGTAACCCACCACGCAGACGGATTCCTGATGTGCAAGACTACCGACACGGTTGACGGCGAAAGAAT GTCATTCTCGGTGTGCACATACGTGCCGGCGACCATTTGTGATCAAATGACCGGCATCCTTGCTACAGAAGTCACGCCGGA GGATGCACAGAAGCTGTTGGTGGGGCTGAACCAGAGAATAGTGGTTAACGGCAGAACGCAACGGAATACGAACACCATGAAAAATTATCTGCTTCCCGTGGTCGCCCAAGCCTTCAGTAAGTGGGCAAAGGAGTGCCGGAAAGACATGGAAGATGAAAAACT CCTGGGGGTCAGAGAAAGAACACTGACCTGCTGCTGTCTATGGGCATTCAAGAAGCAGAAAACACACACGGTCTACAAGAG GCCTGATACCCAGTCAATTCAGAAGGTTCAGGCCGAGTTTGACAGCTTTGTGGTACCGAGTCTGTGGTCGTCCGGGTTGTC AATCCCTTTGAGGACTAGAATCAAATGGTTGTTAAGCAAGGTGCCAAAAACCGACCTGATCCCATACAGCGGAGACGCCCG AGAAGCCCGGGACGCAGAAAAAGAAGCAGAGGAAGAACGAGAAGCAGAACTGACTCGCGAAGCCCTACCACCTCTACAGGC AGCACAGGAAGATGTTCAGGTCGAAATCGACGTGGAACAGCTTGAGGACAGAGCGGGCGCAGGAATAATAGAGACTCCGAG AGGAGCTATCAAAGTTACTGCCCAACCAACAGACCACGTCGTGGGAGAGTACCTGGTACTCTCCCCGCAGACCGTACTACG TAGCCAGAAGCTCAGTCTGATTCACGCTTTGGCGGAGCAAGTGAAGACGTGCACGCACAACGGACGAGCAGGGAGGTATGC GGTCGAAGCGTACGACGGCCGAGTCCTAGTGCCCTCAGGCTATGCAATCTCGCCTGAAGACTTCCAGAGTCTAAGCGAAAG CGCAACGATGGTGTATAACGAAAGAGAGTTCGTAAACAGAAAGCTACACCATATTGCGATGCACGGACCAGCCCTGAACAC CGACGAAGAGTCGTATGAGCTGGTGAGGGCAGAGAGGACAGAACACGAGTACGTCTACGACGTGGATCAGAGAAGATGCTG TAAGAAGGAAGAAGCCGCAGGACTGGTACTGGTGGGCGACTTGACTAATCCGCCCTACCACGAATTCGCATATGAAGGGCT AAAAATCCGCCCTGCCTGCCCATACAAAATTGCAGTCATAGGAGTCTTCGGAGTACCGGGATCTGGCAAGTCAGCTATTAT CAAGAACCTAGTTACCAGGCAGGACCTGGTGACTAGCGGAAAGAAAGAAAACTGCCAAGAAATCACCACCGACGTGATGAG ACAGAGAGGTCTAGAGATATCTGCACGTACGGTTGACTCGCTGCTCTTGAATGGATGCAACAGACCAGTCGACGTGTTGTA CGTAGACGAGGCGTTTGCGTGCCACTCTGGAACGCTACTTGCTTTGATCGCCTTGGTGAGACCAAGGCAGAAAGTTGTACT TTGTGGTGACCCGAAGCAGTGCGGCTTCTTCAATATGATGCAGATGAAAGTCAACTATAATCACAACATCTGCACCCAAGT GTACCACAAAAGTATCTCCAGGCGGTGTACACTGCCTGTGACCGCCATTGTGTCATCGTTGCATTACGAAGGCAAAATGCG CACTACGAATGAGTACAACAAGCCGATTGTAGTGGACACTACAGGCTCAACAAAACCTGACCCTGGAGACCTCGTGTTAAC GTGCTTCAGAGGGTGGGTTAAACAACTGCAAATTGACTATCGTGGATACGAGGTCATGACAGCAGCCGCATCCCAAGGGTT AACCAGAAAAGGAGTTTACGCAGTTAGACAAAAAGTTAATGAAAACCCGCTCTATGCATCAACGTCAGAGCACGTCAACGT ACTCCTAACGCGTACGGAAGGTAAACTGGTATGGAAGACACTTTCCGGCGACCCGTGGATAAAGACGCTGCAGAACCCACC GAAAGGAAACTTCAAAGCAACTATTAAGGAGTGGGAGGTGGAGCATGCATCAATAATGGCGGGCATCTGCAGTCACCAAAT GACCTTCGATACATTCCAAAATAAAGCCAACGTTTGTTGGGCTAAGAGCTTGGTCCCTATCCTCGAAACAGCGGGGATAAA ACTAAATGATAGGCAGTGGTCTCAGATAATTCAAGCCTTCAAAGAAGACAAAGCATACTCACCTGAAGTAGCCCTGAATGA AATATGTACGCGCATGTATGGGGTGGATCTAGACAGCGGGCTATTTTCTAAACCGTTGGTGTCTGTGTATTACGCGGATAA CCACTGGGATAATAGGCCTGGAGGGAAAATGTTCGGATTTAACCCCGAGGCAGCATCCATTCTAGAAAGAAAGTATCCATT CACAAAAGGGAAGTGGAACATCAACAAGCAGATCTGCGTGACTACCAGGAGGATAGAAGACTTTAACCCTACCACCAACAT CATACCGGCCAACAGGAGACTACCACACTCATTAGTGGCCGAACACCGCCCAGTAAAAGGGGAAAGAATGGAATGGCTGGT TAACAAGATAAACGGCCACCACGTGCTCCTGGTCAGTGGCTATAACCTTGCACTGCCTACTAAGAGAGTCACTTGGGTAGC GCCGTTAGGTGTCCGCGGAGCGGACTACACATACAACCTAGAGTTGGGTCTGCCAGAGAGAAGGGGTAGGTATGACCTAGT GGTCATAAACATCCACACACCTTTTCGCATACACCATTACCAACAGTGCGTCGACCACGCAATGAAACTGCAAATGCTCGG GGGTGACTCATTGAGACTGCTCAAACCGGGCGGCTCTCTATTGATCAGAGCATATGGTTACGCAGATAGAACCAGTGAACG AGTCATCTGCGTATTGGGACGCAAGTTTAGATCGTCTAGAGCGTTGAAACCACCATGTGTCACCAGCAACACTGAGATGTT TTTCCTATTCAGCAACTTTGACAATGGCAGAAGGAATTTCACAACTCATGTCATGAACAATCAACTGAATGCAGCCTTCGT AGGACAGGTCACCCGAGCAGGATGTGCACCGTCGTACCGGGTAAAACGCATGGACATCGCGAAGAACGATGAAGAGTGCGT AGTCAACGCCGCTAACCCTCGCGGGTTACCGGGTGGCGGTGTTTGCAAGGCAGTATACAAAAAATGGCCGGAGTCCTTTAA GAACAGTGCAACACCAGTGGGAACCGCAAAAACAGTTATGTGCGGTACGTATCCAGTAATCCACGCTGTTGGACCAAACTT CTCTAATTATTCGGAGTCTGAAGGGGACCGGGAATTGGCAGCTGCCTATCGAGAAGTCGCAAAGGAAGTAACTAGGCTGGG AGTAAATAGTGTAGCTATACCTCTCCTCTCCACAGGTGTATACTCAGGAGGGAAAGACAGGCTGACCCAGTCACTGAACCA CCTCTTTACAGCCATGGACTCGACGGATGCAGACGTGGTCATCTACTGCCGCGACAAAGAATGGGAGAAGAAAATATCTGA GGCCATACAGATGCGGACCCAAGTAGAGCTGCTGGATGAGCACATCTCCATAGACTGCGATATTGTTCGCGTGCACCCTGA CAGCAGCTTGGCAGGCAGAAAAGGATACAGCACCACGGAAGGCGCACTGTACTCATATCTAGAAGGGACCCGTTTTCATCA GACGGCTGTGGATATGGCGGAGATACATACTATGTGGCCAAAGCAAACAGAGGCCAATGAGCAAGTCTGCCTATATGCCCT GGGGGAAAGTATTGAATCGATCAGGCAGAAATGCCCGGTGGATGATGCAGACGCATCATCTCCCCCCAAAACTGTCCCGTG CCTTTGCCGTTACGCTATGACTCCAGAACGCGTCACCCGGCTTCGCATGAACCACGTCACAAGCATAATTGTGTGTTCTTC GTTTCCCCTCCCAAAGTACAAAATAGAAGGAGTGCAAAAAGTCAAATGCTCTAAGGTAATGCTATTTGACCACAACGTGCC ATCGCGCGTAAGTCCAAGGGAATATAGATCTTCCCAGGAGTCTGCACAGGAGGCGAGTACAATCACGTCACTGACGCATAG TCAATTCGACCTAAGCGTTGATGGCGAGATACTGCCCGTCCCGTCAGACCTGGATGCTGACGCCCCAGCCCTAGAACCAGC ACTAGACGACGGGGCGACACACACGCTGCCATCCACAACCGGAAACCTTGCGGCCGTGTCTGATTGGGTAATGAGCACCGT ACCTGTCGCGCCGCCCAGAAGAAGGCGAGGGAGAAACCTGACTGTGACATGTGACGAGAGAGAAGGGAATATAACACCCAT GGCTAGCGTCCGATTCTTTAGGGCAGAGCTGTGTCCGGTCGTACAAGAAACAGCGGAGACGCGTGACACAGCAATGTCTCT TCAGGCACCACCGAGTACCGCCACGGAACCGAATCATCCGCCGATCTCCTTCGGAGCATCAAGCGAGACGTTCCCCATTAC ATTTGGGGACTTCAACGAAGGAGAAATCGAAAGCTTGTCTTCTGAGCTACTAACTTTCGGAGACTTCTTACCAGGAGAAGT GGATGACTTGACAGACAGCGACTGGTCCACGTGCTCAGACACGGACGACGAGTTAAGACTAGACAGGGCAGGTGGGTATAT ATTCTCGTCGGACACCGGTCCAGGTCATTTACAACAGAAGTCAGTACGCCAGTCAGTGCTGCCGGTGAACACCCTGGAGGA AGTCCACGAGGAGAAGTGTTACCCACCTAAGCTGGATGAAGCAAAGGAGCAACTATTACTTAAGAAACTCCAGGAGAGTGC ATCCATGGCCAACAGAAGCAGGTATCAGTCGCGCAAAGTAGAAAACATGAAAGCAGCAATCATCCAGAGACTAAAGAGAGGCTGTAGACTATACTTAATGTCAGAGACCCCAAAAGTCCCTACTTACCGGACTACATATCCGGCGCCTGTGTACTCGCCTCC GATCAACGTCCGATTGTCCAATCCCGAGTCCGCAGTGGCAGCATGCAATGAGTTCTTAGCTAGAAACTATCCAACTGTCTC ATCATACCAAATTACCGACGAGTATGATGCATATCTAGACATGGTGGACGGGTCGGAGAGTTGCCTGGACCGAGCGACATT CAATCCGTCAAAACTCAGGAGCTACCCGAAACAGCACGCTTACCACGCGCCCTCCATCAGAAGCGCTGTACCGTCCCCATT CCAGAACACACTACAGAATGTACTGGCAGCAGCCACGAAAAGAAACTGCAACGTCACACAGATGAGGGAATTACCCACTTT GGACTCAGCAGTATTCAACGTGGAGTGTTTCAAAAAATTCGCATGCAACCAAGAATACTGGGAAGAATTTGCTGCCAGCCC TATTAGGATAACAACTGAGAATTTAGCAACCTATGTTACTAAACTAAAAGGGCCAAAAGCAGCAGCGCTATTCGCAAAAAC CCATAATCTACTGCCACTACAGGAAGTACCAATGGATAGGTTCACAGTAGATATGAAAAGGGACGTAAAGGTGACTCCTGG TACAAAGCATACAGAGGAAAGACCTAAGGTGCAGGTTATACAGGCGGCTGAACCCTTGGCGACAGCATACCTATGTGGGAT TCACAGAGAGCTGGTTAGGAGGCTGAACGCCGTCCTCCTACCCAATGTACATACACTATTTGACATGTCTGCCGAGGATTT CGATGCCATCATAGCCGCACACTTTAAGCCAGGAGACACTGTTTTGGAAACGGACATAGCCTCCTTTGATAAGAGCCAAGA TGATTCACTTGCGCTTACTGCTTTGATGCTGTTAGAGGATTTAGGGGTGGATCACTCCCTGCTGGACTTGATAGAGGCTGC TTTCGGAGAGATTTCCAGCTGTCACCTACCGACAGGTACGCGCTTCAAGTTCGGCGCCATGATGAAATCAGGTATGTTCCT AACTCTGTTCGTCAACACATTGTTAAACATCACCATCGCCAGCCGAGTGCTGGAAGATCGTCTGACAAAATCCGCGTGCGC GGCCTTCATCGGCGACGACAACATAATACATGGAGTCGTCTCCGATGAATTGATGGCAGCCAGATGTGCCACTTGGATGAA CATGGAAGTGAAGATCATAGATGCAGTTGTATCCTTGAAAGCCCCTTACTTTTGTGGAGGGTTTATACTGCACGATACTGT GACAGGAACAGCTTGCAGAGTGGCAGACCCGCTAAAAAGGCTTTTTAAACTGGGCAAACCGCTAGCGGCAGGTGACGAACA AGATGAAGATAGAAGACGAGCGCTGGCTGACGAAGTGATCAGATGGCAACGAACAGGGCTAATTGATGAGCTGGAGAAAGC GGTATACTCTAGGTACGAAGTGCAGGGTATATCAGTTGTGGTAATGTCCATGGCCACCTTTGCAAGCTCCAGATCCAACTT CGAGAAGCTCAGAGGACCCGTCATAACTTTGTACGGCGGTCCTAAATAGGTACGCACTACAGCTACCTATTTTGCAGAAGC CGACAGCAAGTATCTAAACACTAATCAGCTACCTAGGAAGTTCGAATATAGGCGCGCCGCCACCATGGTGAGCAAGGGCGA GGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTCCGCGGCGA GGGCGAGGGCGATGCCACCAACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCAC CCTCGTGACCACCTTAGGCTACGGCGTGGCCTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTC CGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTCTTTCAAGGACGACGGTACCTACAAGACCCGCGCCGAGGTGAA GTTCGAGGGCGACACCCTGGTGAACCGCATCGTGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAA GCTGGAGTACAACTTCAACAGCCACAAGGTCTATATCACGGCCGACAAGCAGAAGAACGGCATCAAGGCTAACTTCAAGAC CCGCCACAACGTTGAGGACGGCGGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT GCTGCCCGACAACCACTACCTGAGCCATCAGTCCAAACTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGAA GGAGAGGGTGACCGCCGCCGGGATTACACATGACATGGACGAGCTGTACAAGgctagcggcagcggcgccaccaacttcag cctgctgaagcaggccggcgacgtggaggagaaccccGGGCCCgaattcatgtggtctcacccgcaatttgaaaaaggcgc tgcggctATGCCAAGCAAACTCGCGCTAATCCAGGAACTTCCCGACCGCATTCAAACGGCGGTGGAAGCAGCCATGGGAAT GAGCTACCAAGACGCACCGAACAACGTGCGCAGGGACCTCGACAACCTGCACGCTTGCCTAAACAAGGCAAAACTAACGGT AAGTCGGATGGTAACATCACTGCTGGAGAAACCCAGCGTGGTGGCATACCTAGAGGGAAAGGCCCCCGAGGAGGCAAAACC AACACTCGAAGAACGCCTCCGAAAGCTGGAGCTCAGCCACAGCCTTCCAACAACCGGAAGTGACCCCCCACCCGCAAAACT GTAGTAGTTAATTAACTCGCGATCGCTAACTTGACAATTAAGTATGAAGGTATATGTGTCCCCTAAGAGACACACTGTACA TAGCAAATAATCTATAGATCAAAGGGCTACGCAACCCCTGAATAGTAACAAAATACAAAATCACTAAAAATTATAAAAACA GAAAAATACATAAATAGGTATACGTGTCCCCTAAGAGACACATTGTATGTAGGTGATAAGTATAGATCAAAGGGCCGAATA AC CO CT GAATAGTAACAAAATAT GAAAAT CAATAAAAAT CATAAAATAGAAAAAC CATAAACAGAAGTAGT T CAAAG GG CT ATAAAACCCCTGAATAGTAACAAAACATAAAATTAATAAAAATCAAATGAATACCATAATTGGCAAACGGAAGAGATGTAG GTACTTAAGCTTCCTAAAAGCAGCCGAACTCACTTTGAGAAGTAGGCATAGCATACCGAACTCTTCCACGATTCTCCGAAC CCACAGGGACGTAGGAGATGTTATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAGCGGCCGCTTCCGCTC ACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCT CTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGCCAGCAA AAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGA CGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGC TGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGC GCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGG ATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTA TTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCT GGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCT ACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAG ATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTA ATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACG ATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCA ATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGC CGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAA GCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTG CATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAG TGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTC ATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGT GCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAA AAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTAT TGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTG CCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGG CCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGCGCTCCCATTCG CCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATG TGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGATTTAAATAC

[0193] GatttaggtgacactataG

[0194] [ SEQ ID NO: 5]

[0195] The plasmid SP6-VEEVatt-GL-1xST-fhvB2 may have the sequence represented by SEQ ID NO: 6: gtggcaatatcccctcgcaccacatgatatgagggtgcacatccggcttcgtggagtctggaacctgtataaatgttggtc aaggttgatgaaagcttgtaaggattgtgcgtacgggccttgcgatcgtacccaatgaatacaaacagaacttccgtctct tcaagtgaggatttcggtttgcatacccgggaaaacttgaactgccgcgctatagcaccaatgatgctttcgctggccctg tcagcgtaaccataacctatgctgacacaggttccgccgggattcagatgcagacaagctttcttggtcaacatgctaagc ttaatggcatggtcttcacactgctgatagtgatggtatttatatggggtcctcacattaacaaatattatgtcatatttg ggcacatcacctgggatgcctaaatccagccgagctctgaaggtagcctcaggccggtctgacaaccagtcaaccattttg cctgggacggacaacttttccccgaccaccaggacagttctgcccttcaatttgctgacgaatgaagaaaagtcactctgt gggtgttcattatggtggaggactaaagcatgaggcagtcttctgtttacaggtactaggtttatgcgcggatcataattg cgcagtgtaccagtgttcatgtcatagactcttccagtggcaactgcccgaggcagttgtgggtacctgcgagagagctga cggaccacttctttattcagcccgtacatgttaggcgacggggagttatcccagtgattattcctaatggataacggaaca gtgggtgcagaaaatagaccggagtccagatcgagtccaaagaacctcacgcatagttggttcaatactatctctgctgag tgagctttgtccgtttcaaaataatccacagtgttccattgttcagtggtcatgtctatgccagcggtcttcagcaccggc actaaagccttggcccaacacacgtttgccttattctggaagacgtcggtagggtccggtctctccaagatgtgcctcatg atggcatcatgctctgcttgccactcctctatcgtggcagtgaaattcccagggtacttggcagtcagtgtttttatccat gggtcgccggctagtgttttccacacgatgcggtcctccgtgcgggtcagtaggacgttcacatgttctgaggtgggtgcg tacagaggattttcattcaccttgtaccgaacggcatacacacctttacgggtcagcccttgagaggcagctgccgtcatt atttcgttgcctttgtaatctatttgcaactgcttcacccaccctctgaaacaagtgagaatgagatcgtcctgcttaggt ttggtactgccggtagtgtcaatcacaatcttagtctctttcggattcgtcgttctcatttttttgtcgtaaaacaaggtt gagacgaccgaagtcacagatttagtgcaacggcgagagatgcttttgtggaagacttgtgtgcaaatctcgtggttaaaa tgcactttcaggcacatcatgttaaaaaaaccgcactgtttgggatccccgcagagcactgcctttttaggtcttataatg gctatgagcgctctgagagtacctgcatgacaagcaaaagcttcgtcaatatacagggtctctacggggtgtttgcatcca ttcaagagcactgagtccacagttctggcattgacgtccagccctttcattttcttgacgtcccttataatttctgcacag ttttctttcttggcgctcaccactagatcttttttggtgactgcgcttttaatgatgccagacttgcctgatcctggcacg ccatacacccctatggttggtacttggtaaggagcggctggtcgtgttctcagactctcgtaggcgaattcatggaaggga ggatccaccagctcgcctgtgagccctagcccagtgactagttctttcttgacgcactgtttcctgtcgatgtcgtacagg tattcgccgtcgtgctcgctgggcttgacagttttgtaatattcttcatcagtgttcagcgctcctccatgtgtggcaata tggtgcaggtacctgtttacgaactcacgttcgttgtacacaatggtggcactttcactcagagcttgaaagtcctggacg ggtattgcatgtccctctggcaccactactttaccatggtatggttccacggcataacgcccttttcggccagagtgtgtt atcactatgacttgttcagcgagagggtggatgcaagataatttttcactcttgagtacagcctgcggagaaagcacagcg taagagccgatcttgtcctcgccagcgtagctggtaacctttatcaagccacgaggtgtctccactgagccggccccagcc tcttgtaacatcaagtcgacgtctgcctccagagtgggctcctcaacatcagctgccaaaggtggtagagctgcgcgcaac tcctcggcttcacgcacctccttagcctcatcggctgcgcacttagcttcttgtacgtcctcggcggtaatgagaggtgac ggctccttgtgctcctctaacattttcctgattcttgttctcagcccgatctccaatgtgttactgcctatcctgggcagc acgaatgagtggaaatcgctgttcactttgatgatggtttgggtatccgggcgcttataaatagatgttatcttgtgcctt ctaaaagcccaacaacaccccatgactaactgtctatctcgtagtcctagtggcctttcatcttcttgatcttccttatat tcctttgcccacctagcaaatgcctgggccactacgggcaaaaggtaatttttcatggtattggtgtttctctgggtgcga ccgttgacgactatacgctggttgagcccaaccagcagtttttgcgcgtcgtccgcactgacatctgttgccagtatgcca gtcatttggtcacacaatgtagctggcacatacgtgcacacgggaaaagagaccctctccccgttcaatgtgtctgtcact ttgcagcacaagaatccctcgcggtgcatcgtagcagcatagcctgaaggcttcccatacaggcctggactgatagctatt cttttaacgacgtacccgtcgcaactaactatagtctcacaccgacatgtgtaattttgcttgccacgtaagtgaaatacagacggcaggtgccagctcctcagtaagtccctcttctcgtggtagatggtcgagccaacagagaatagaacattgttggat ggtttcaaatacttctttctaagaatggacatccctctacgtgaccgctccataacgtcagagctgcataggcctatgtta cgagccgttaacacggtttcgtcggcccagttggtagagtatgatggatatgctccagccaagttcttaaacataaaaggg gtggtgtcaaagcctatccagtaggcgactctaactcccttattggcttggtgatagagacttgtcggtccgtcaaccgcg tatacatcctggtaaacagcgacttgcccttcgtagcgacacgactcgtcgtcgtggaggcacatagtctcagtttccagg tcagggtcgctcatgacggcggccagctccttcattttcttgtccaattccttatcagttatttccttacagtttttcttc agcttagttgcatacttatacaatctgtccggatcttccgcacatctcatcggacagatacaatgatacttgtgcttagaa tacattctgcgggcgggcgcacttccaatgtcaaggatcgtgtcggatgggtccacctccgtttcgatcagttttgaagcc agatgcgaaaacgctctggcattagcatggtcattatcagtgacctgcttggcttctacctcaaactgcgggaagctccgc tgcaaagctctgaggaatgggctgtcttcctcgatgtcaacgtgaactttctccattttgggtaggtaattggtctgggct tctctcatgcgccgcccatctatagtgtcacctaaatcgagctcgcgttgctggcgtttttccataggctccgcccccctg acgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctg gaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtgg cgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccc ccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactgg cagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacg gctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgat ccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaag aagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattat caaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggt ctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactcc ccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcac cggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctcca tccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgcta caggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgat cccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcac tcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaa ccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaacacgggataataccgcgccacata gcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatcca gttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacag gaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattatt gaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgc gcacatttccccgaaaagtgccacctggccctttcgtcttcaagaattaattcccctcgacgcgttttttttttttttttt tttttttgaaatattaaaaacaaaatccgattcggaaaagaaaagaaaaaataaaataaaaattttaaggcggcatgccaa tcgccgcgagttctatgtaagcagcttgccaattgctgctgtattcagcgatcgcgagttaattaactactacagttttgc gggtggggggtcacttccggttgttggaaggctgtggctgagctccagctttcggaggcgttcttcgagtgttggttttgc ctcctcgggggcctttccctctaggtatgccaccacgctgggtttctccagcagtgatgttaccatccgacttaccgttag ttttgccttgtttaggcaagcgtgcaggttgtcgaggtccctgcgcacgttgttcggtgcgtcttggtagctcattcccat ggctgcttccaccgccgtttgaatgcggtcgggaagttcctggattagcgcgagtttgcttggcatagccgcagcgccttt ttcaaattgcgggtgagaccacatgaattcgggcccggggttctcctccacgtcgccggcctgcttcagcaggctgaagtt ggtggcgccgctgccgctagccttgtacagctcgtccatgtcatgtgtaatcccggcggcggtcaccctctccttcaggac catgtgatcgcgcttctcgttggggtctttgctcagtttggactgatggctcaggtagtggttgtcgggcagcagcacggg gccgtcgccgatgggggtgttctgctggtagtggtcggcgagctgcacgccgccgtcctcaacgttgtggcgggtcttgaa gttagccttgatgccgttcttctgcttgtcggccgtgatatagaccttgtggctgttgaagttgtactccagcttgtgccc caggatgttgccgtcctccttgaagtcgatgcccttcagcacgatgcggttcaccagggtgtcgccctcgaacttcacctc ggcgcgggtcttgtaggtaccgtcgtccttgaaagagatggtgcgctcctggacgtagccttcgggcatggcggacttgaa gaagtcgtgctgcttcatgtggtcggggtagcgggcgaagcaggccacgccgtagcctaaggtggtcacgagggtgggcca gggcacgggcagcttgccggtggtgcagatgaacttcagggtcagcttgccgttggtggcatcgccctcgccctcgccgcg gacgctgaacttgtggccgtttacgtcgccgtccagctcgaccaggatgggcaccaccccggtgaacagctcctcgccctt gctcaccatggtggcggcgcgcctatattcgaacttggcggactagactatgtcgtagtccattcaggttagccgtagaga gttataggggcccctctcaggtagctgaatgatttaacactgctagctagagtagtcatggccataactatgatggaagtt cctacggtttcataccttgattctactgccttgcacagctctgaaagaatacccactcggttccagcgtgttgactcttca tgcaatgcccttctcctgtcatcatcatgttcatcgtctgctgccagaggtttgccaagcttaaacagcctttttaggggg tctgccacacggcacgctgtgccggtcacggagtcacacaaaataaaccctccacagaaataaggcgctttctcgcccacc acagcatctataatcttgacttccatattcaaccaggtggcgcacctgtctgccattaatttgtccgatttgactcctttc acgatattgtcatctccaatgaatgctgcacatggtgatccggttagccgttctctcaacactctgcttgcgattacaatg ttaatgactgtgttcacaaacagtgtgaggaacattccagatttcatcatggctccgaatttaaatttagttttagtgggc aaatgtattgatgaaatttcgccgaaagccgcctcaatcagcgtcaacagctctgcgtccacacctaagtcttccagaatcattaacgcggtcagagccatggcgtcgtcctcacttttatcaaacgacgcgatgtcagtttccagaacacaatccccaggc tggaagtgctcggctataatagcgtcaaagtcttcagccgacatatcaaacagtgtatgaatgttcggaagcaggaccgca tttaatctcctaaccagctctcggtggattccgcacagatacgctgttgctagcggatcggcagcctggatcacctgtacc ttgggccgttcttcagtatgttttgttcctggagtcactttcacgtctctctttaagtccattacaaacctgtccattggt atgtcctgcaacatattcaaattatgtgtcttcgcaaaaagagcagcagcttttggtccttttaatttggtaatgtaattt accacgttttcttcagtaagcctgatggggttttctttaaacgtttcccaatattcattattacacgcatatttcttgaag cattccacattaaaggccgccgaatccaatacgggcaattctctcatttgcgtgacattgcaatttctttttgtggcagct gccaggacgttctggagcgtgttctggatcgctgaaggcactgccgatcgtattgtgggttccaaataggagtgtttcttt ggaaagctgcgcagctttgcagggcaaaaactggcagtgtctaagcagcatgaagctccgtcaaccatgtccaaataggca tcgtactctggaataatacagtaagaagccacagtcggaaagttctctttcaacatggcgttacaggcttccactgcgacc ttggggcttgaaaaggcacggttcacactagatgaatacaaaggaacaggatgcagggttcggtagcactccacttttcct tctgccttcaaataatgccctaggccttgcagaatacgtctagctgttatggctttcatgttctccaccttcctggactgg tatctgcttctgttagcaggtgtgggatttaactgtaatttcttgcgtagtaattcttctttttcttggtcgaggcgcggg gcatacgaaatctccaattcggtcctctccaacaccacttcggatagcaccgtttgccttactgatttttgttgtaaatgc ccttgaccggtgtcggaggaaaagatgtatgcacccgcatcaaaccgtcattgttgttgtgctacgaacgcctcaaactcc tctcttgtaatcaccctatttacgcctggcgggttggagaccaggctggttctcgagaccgacctgctaggagtgcgtgac ggggtaagcgcctcgagctcctctctagtgatcaccctattcacgcctggcggggtggaaactaggctggttctcgagcag gccctgctgggtgcaagtgacggtgttcttgtgcgcggagcgggatgtggagggttcctgaatactgttcgaggcgcaggc accggtcgcgccagaaactccatactctttgcgaagtaagagttagtctcggctgacgttgccccgctggtcacgctagct ccctccagggtgtcaagtatggataaactgtccacatcaaagtcggatgcatgaggaatggaccaggatgagctagataca gagggcggcccgtgaatgtctgcctcgacttgcagcacctggtgggtcgggccatctgacagcaaacttatgctatcctct tcttcctcttcgatgatgatcggctcaggcgttctagtcctggtctcatcctcggttataagtggtggttgttcaggtgtc ccctctgtggattggttctctgccgatggctccggagtctcgtctaccggtggtgtttccacgagatacttccttggatga atatacgcaggcactttcggtgagaacaatataggctgggagcattggatcttctgcacaccagtgattctatacttcggc aatggaaaggatgagcacacagtaatttgttctggacgtgaggcttttaggcgctgtactctttctggagtcatggcatgg atgcacaagcaaggcagcgtgctaggtggtgtggaggcttccgactcttcgacggggcatttcgacctaatactgctcatg ctttctccgaggatatacatgcatacctgctcattggcctccgttgcaacgggccacatggcattaatttctgctatatcc ttggccgcctggtgaaacttggtcccttccaaatatgagaaagttttgccatcgcttgtgctgtagcccttccttccagcc aaagaactcttcggatgcaccctcaccagctctgcatcaggttctgtcactgaagagtcgtcggatatgcatatctcctcc actgcttctctcctagccactgcttccttgagagtcatttcccatttcttgtccctgcagtatatggctacatctgcatca gtggtgtctaaagctgtcagcaaatggttcaatgattgggttagtcgatctttgttcccggaaaagatgccggtggacaac agtggaatcgctactgacttgtaattgttatcgttgacaatcttagcgatggactcataagcctctgccaactgtttgtca ccttcaacctccgaaactttgttgaagtttggtcctacggcatgaatgatatgtttagctgcacctttgaccagtcgcgct tttcctacttcgatcggctgtaaatcgaagctttccgggaatttcttatacagcgctccgcacacccctccgccaggttgt cctttgctgttagcagcatttataatcactccttcggtggcc

[0196] [ SEQ ID NO: 6]

[0197] The plasmid 22_CHIKV-ERR-Spike-P2A-B2. annotations may have the nucleic acid sequence represented by SEQ ID NO: 7: ccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagg gaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtc tcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccac ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccga aatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtggccgctacagggcgctcccattcgccat tcaggctgcgcaactgttgggaagggcgtttcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgct gcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgatttaaatacgatt taggtgacactatagatggctgcgtgagacacacgtagcctaccagtttcttactgctctactctgcaaagcaagagatta ataacccatcatggatcctgtgtacgtggacatagacgctgacagcgcctttttgaaggccctgcaacgtgcgtaccccat gtttgaggtggaaccaaggcaggtcacaccgaatgaccatgctaatgctagagcgttctcgcatctagctataaaactaat agagcaggaaattgaccccgactcaaccatcctggatatcggcagtgcgccagcaaggaggatgatgtcggacaggaagta ccactgcgtctgcccgatgcgcagtgcggaagatcccgagagactcgccaattatgcgagaaagctagcatctgccgcagg aaaagtcctggacagaaacatctctggaaagatcggggacttacaagcagtaatggccgtgccagacacggagacgccaac attctgcttacacacagacgtctcatgtagacagagagcagacgtcgctatataccaagacgtctatgctgtacacgcacc cacgtcgctataccaccaggcgattaaaggggtccgagtggcgtactgggttgggttcgacacaaccccgttcatgtacaa tgccatggcgggtgcctacccctcatactcgacaaactgggcagatgagcaggtactgaaggctaagaacataggattatg ttcaacagacctgacggaaggtagacgaggcaagttgtctattatgagagggaaaaagctaaaaccgtgcgaccgtgtgctgttctcagtagggtcaacgctctacccggaaagccgcaagctacttaagagctggcacctgccatcggtgttccatttaaa gggcaaactcagcttcacatgccgctgtgatacagtggtttcgtgtgagggctacgtcgttaagagaataacgatgagccc aggcctttatggaaaaaccacagggtatgcggtaacccaccacgcagacggattcctgatgtgcaagactaccgacacggt tgacggcgaaagaatgtcattctcggtgtgcacatacgtgccggcgaccatttgtgatcaaatgaccggcatccttgctac agaagtcacgccggaggatgcacagaagctgttggtggggctgaaccagagaatagtggttaacggcagaacgcaacggaa tacgaacaccatgaaaaattatctgcttcccgtggtcgcccaagccttcagtaagtgggcaaaggagtgccggaaagacat ggaagatgaaaaactcctgggggtcagagaaagaacactgacctgctgctgtctatgggcattcaagaagcagaaaacaca cacggtctacaagaggcctgatacccagtcaattcagaaggttcaggccgagtttgacagctttgtggtaccgagtctgtg gtcgtccgggttgtcaatccctttgaggactagaatcaaatggttgttaagcaaggtgccaaaaaccgacctgatcccata cagcggagacgcccgagaagcccgggacgcagaaaaagaagcagaggaagaacgagaagcagaactgactcgcgaagccct accacctctacaggcagcacaggaagatgttcaggtcgaaatcgacgtggaacagcttgaggacagagcgggcgcaggaat aatagagactccgagaggagctatcaaagttactgcccaaccaacagaccacgtcgtgggagagtacctggtactctcccc gcagaccgtactacgtagccagaagctcagtctgattcacgctttggcggagcaagtgaagacgtgcacgcacaacggacg agcagggaggtatgcggtcgaagcgtacgacggccgagtcctagtgccctcaggctatgcaatctcgcctgaagacttcca gagtctaagcgaaagcgcaacgatggtgtataacgaaagagagttcgtaaacagaaagctacaccatattgcgatgcacgg accagccctgaacaccgacgaagagtcgtatgagctggtgagggcagagaggacagaacacgagtacgtctacgacgtgga tcagagaagatgctgtaagaaggaagaagccgcaggactggtactggtgggcgacttgactaatccgccctaccacgaatt cgcatatgaagggctaaaaatccgccctgcctgcccatacaaaattgcagtcataggagtcttcggagtaccgggatctgg caagtcagctattatcaagaacctagttaccaggcaggacctggtgactagcggaaagaaagaaaactgccaagaaatcac caccgacgtgatgagacagagaggtctagagatatctgcacgtacggttgactcgctgctcttgaatggatgcaacagacc agtcgacgtgttgtacgtagacgaggcgtttgcgtgccactctggaacgctacttgctttgatcgccttggtgagaccaag gcagaaagttgtactttgtggtgacccgaagcagtgcggcttcttcaatatgatgcagatgaaagtcaactataatcacaa catctgcacccaagtgtaccacaaaagtatctccaggcggtgtacactgcctgtgaccgccattgtgtcatcgttgcatta cgaaggcaaaatgcgcactacgaatgagtacaacaagccgattgtagtggacactacaggctcaacaaaacctgaccctgg agacctcgtgttaacgtgcttcagagggtgggttaaacaactgcaaattgactatcgtggatacgaggtcatgacagcagc cgcatcccaagggttaaccagaaaaggagtttacgcagttagacaaaaagttaatgaaaacccgctctatgcatcaacgtc agagcacgtcaacgtactcctaacgcgtacggaaggtaaactggtatggaagacactttccggcgacccgtggataaagac gctgcagaacccaccgaaaggaaacttcaaagcaactattaaggagtgggaggtggagcatgcatcaataatggcgggcat ctgcagtcaccaaatgaccttcgatacattccaaaataaagccaacgtttgttgggctaagagcttggtccctatcctcga aacagcggggataaaactaaatgataggcagtggtctcagataattcaagccttcaaagaagacaaagcatactcacctga agtagccctgaatgaaatatgtacgcgcatgtatggggtggatctagacagcgggctattttctaaaccgttggtgtctgt gtattacgcggataaccactgggataataggcctggagggaaaatgttcggatttaaccccgaggcagcatccattctaga aagaaagtatccattcacaaaagggaagtggaacatcaacaagcagatctgcgtgactaccaggaggatagaagactttaa ccctaccaccaacatcataccggccaacaggagactaccacactcattagtggccgaacaccgcccagtaaaaggggaaag aatggaatggctggttaacaagataaacggccaccacgtgctcctggtcagtggctataaccttgcactgcctactaagag agtcacttgggtagcgccgttaggtgtccgcggagcggactacacatacaacctagagttgggtctgccagagagaagggg taggtatgacctagtggtcataaacatccacacaccttttcgcatacaccattaccaacagtgcgtcgaccacgcaatgaa actgcaaatgctcgggggtgactcattgagactgctcaaaccgggcggctctctattgatcagagcatatggttacgcaga tagaaccagtgaacgagtcatctgcgtattgggacgcaagtttagatcgtctagagcgttgaaaccaccatgtgtcaccag caacactgagatgtttttcctattcagcaactttgacaatggcagaaggaatttcacaactcatgtcatgaacaatcaact gaatgcagccttcgtaggacaggtcacccgagcaggatgtgcaccgtcgtaccgggtaaaacgcatggacatcgcgaagaa cgatgaagagtgcgtagtcaacgccgctaaccctcgcgggttaccgggtggcggtgtttgcaaggcagtatacaaaaaatg gccggagtcctttaagaacagtgcaacaccagtgggaaccgcaaaaacagttatgtgcggtacgtatccagtaatccacgc tgttggaccaaacttctctaattattcggagtctgaaggggaccgggaattggcagctgcctatcgagaagtcgcaaagga agtaactaggctgggagtaaatagtgtagctatacctctcctctccacaggtgtatactcaggagggaaagacaggctgac ccagtcactgaaccacctctttacagccatggactcgacggatgcagacgtggtcatctactgccgcgacaaagaatggga gaagaaaatatctgaggccatacagatgcggacccaagtagagctgctggatgagcacatctccatagactgcgatattgt tcgcgtgcaccctgacagcagcttggcaggcagaaaaggatacagcaccacggaaggcgcactgtactcatatctagaagg gacccgttttcatcagacggctgtggatatggcggagatacatactatgtggccaaagcaaacagaggccaatgagcaagt ctgcctatatgccctgggggaaagtattgaatcgatcaggcagaaatgcccggtggatgatgcagacgcatcatctccccc caaaactgtcccgtgcctttgccgttacgctatgactccagaacgcgtcacccggcttcgcatgaaccacgtcacaagcat aattgtgtgttcttcgtttcccctcccaaagtacaaaatagaaggagtgcaaaaagtcaaatgctctaaggtaatgctatt tgaccacaacgtgccatcgcgcgtaagtccaagggaatatagatcttcccaggagtctgcacaggaggcgagtacaatcac gtcactgacgcatagtcaattcgacctaagcgttgatggcgagatactgcccgtcccgtcagacctggatgctgacgcccc agccctagaaccagcactagacgacggggcgacacacacgctgccatccacaaccggaaaccttgcggccgtgtctgattg ggtaatgagcaccgtacctgtcgcgccgcccagaagaaggcgagggagaaacctgactgtgacatgtgacgagagagaagg gaatataacacccatggctagcgtccgattctttagggcagagctgtgtccggtcgtacaagaaacagcggagacgcgtga cacagcaatgtctcttcaggcaccaccgagtaccgccacggaaccgaatcatccgccgatctccttcggagcatcaagcgagacgttccccattacatttggggacttcaacgaaggagaaatcgaaagcttgtcttctgagctactaactttcggagactt cttaccaggagaagtggatgacttgacagacagcgactggtccacgtgctcagacacggacgacgagttaagactagacag ggcaggtgggtatatattctcgtcggacaccggtccaggtcatttacaacagaagtcagtacgccagtcagtgctgccggt gaacaccctggaggaagtccacgaggagaagtgttacccacctaagctggatgaagcaaaggagcaactattacttaagaa actccaggagagtgcatccatggccaacagaagcaggtatcagtcgcgcaaagtagaaaacatgaaagcagcaatcatcca gagactaaagagaggctgtagactatacttaatgtcagagaccccaaaagtccctacttaccggactacatatccggcgcc tgtgtactcgcctccgatcaacgtccgattgtccaatcccgagtccgcagtggcagcatgcaatgagttcttagctagaaa ctatccaactgtctcatcataccaaattaccgacgagtatgatgcatatctagacatggtggacgggtcggagagttgcct ggaccgagcgacattcaatccgtcaaaactcaggagctacccgaaacagcacgcttaccacgcgccctccatcagaagcgc tgtaccgtccccattccagaacacactacagaatgtactggcagcagccacgaaaagaaactgcaacgtcacacagatgag ggaattacccactttggactcagcagtattcaacgtggagtgtttcaaaaaattcgcatgcaaccaagaatactgggaaga atttgctgccagccctattaggataacaactgagaatttagcaacctatgttactaaactaaaagggccaaaagcagcagc gctattcgcaaaaacccataatctactgccactacaggaagtaccaatggataggttcacagtagatatgaaaagggacgt aaaggtgactcctggtacaaagcatacagaggaaagacctaaggtgcaggttatacaggcggctgaacccttggcgacagc atacctatgtgggattcacagagagctggttaggaggctgaacgccgtcctcctacccaatgtacatacactatttgacat gtctgccgaggatttcgatgccatcatagccgcacactttaagccaggagacactgttttggaaacggacatagcctcctt tgataagagccaagatgattcacttgcgcttactgctttgatgctgttagaggatttaggggtggatcactccctgctgga cttgatagaggctgctttcggagagatttccagctgtcacctaccgacaggtacgcgcttcaagttcggcgccatgatgaa atcaggtatgttcctaactctgttcgtcaacacattgttaaacatcaccatcgccagccgagtgctggaagatcgtctgac aaaatccgcgtgcgcggccttcatcggcgacgacaacataatacatggagtcgtctccgatgaattgatggcagccagatg tgccacttggatgaacatggaagtgaagatcatagatgcagttgtatccttgaaagccccttacttttgtggagggtttat actgcacgatactgtgacaggaacagcttgcagagtggcagacccgctaaaaaggctttttaaactgggcaaaccgctagc ggcaggtgacgaacaagatgaagatagaagacgagcgctggctgacgaagtgatcagatggcaacgaacagggctaattga tgagctggagaaagcggtatactctaggtacgaagtgcagggtatatcagttgtggtaatgtccatggccacctttgcaag ctccagatccaacttcgagaagctcagaggacccgtcataactttgtacggcggtcctaaataggtacgcactacagctac ctattttgcagaagccgacagcaagtatctaaacactaatcagctacctaggaagttcgaatataggcgcgccgccaccat gttcgtgtttctggtgctgctgcctctggtgtccagccagtgtgtgaacctgaccaccagaacacagctgcctccagccta caccaacagctttaccagaggcgtgtactaccccgacaaggtgttcagatccagcgtgctgcactctacccaggacctgtt cctgcctttcttcagcaacgtgacctggttccacgccatccacgtgtccggcaccaatggcaccaagagattcgacaaccc cgtgctgcccttcaacgacggggtgtactttgccagcaccgagaagtccaacatcatcagaggctggatcttcggcaccac actggacagcaagacccagagcctgctgatcgtgaacaacgccaccaacgtggtcatcaaagtgtgcgagttccagttctg caacgaccccttcctgggcgtctactaccacaagaacaacaagagctggatggaaagcgagttccgggtgtacagcagcgc caacaactgcaccttcgagtacgtgtcccagcctttcctgatggacctggaaggcaagcagggcaacttcaagaacctgcg cgagttcgtgttcaagaacatcgacggctacttcaagatctacagcaagcacacccctatcaacctcgtgcgggatctgcc tcagggcttctctgctctggaacccctggtggatctgcccatcggcatcaacatcacccggtttcagacactgctggccct gcacagaagctacctgacacctggcgatagcagcagcggatggacagctggtgccgccgcttactatgtgggctacctgca gcctagaaccttcctgctgaagtacaacgagaacggcaccatcaccgacgccgtggattgtgcccttgatcctctgagcga gacaaagtgcaccctgaagtccttcaccgtggaaaagggcatctaccagaccagcaacttccgggtgcagcccaccgaatc catcgtgcggttccccaatatcaccaatctgtgccccttcggcgaggtgttcaatgccaccagattcgcctctgtgtacgc ctggaaccggaagcggatcagcaattgcgtggccgactactccgtgctgtacaactccgccagcttcagcaccttcaagtg ctacggcgtgtcccctaccaagctgaacgacctgtgcttcacaaacgtgtacgccgacagcttcgtgatccggggagatga agtgcggcagattgcccctggacagacaggcaagatcgccgactacaactacaagctgcccgacgacttcaccggctgtgt gattgcctggaacagcaacaacctggactccaaagtcggcggcaactacaattacctgtaccggctgttccggaagtccaa tctgaagcccttcgagcgggacatctccaccgagatctatcaggccggcagcaccccttgtaacggcgtggaaggcttcaa ctgctacttcccactgcagtcctacggctttcagcccacaaatggcgtgggctatcagccctacagagtggtggtgctgag cttcgaactgctgcatgcccctgccacagtgtgcggccctaagaaaagcaccaatctcgtgaagaacaaatgcgtgaactt caacttcaacggcctgaccggcacaggcgtgctgacagagagcaacaagaagttcctgccattccagcagtttggccggga tatcgccgataccacagacgccgttagagatccccagacactggaaatcctggacatcaccccttgcagcttcggcggagt gtctgtgatcacccctggcaccaacaccagcaatcaggtggcagtgctgtaccaggacgtgaactgtaccgaagtgcccgt ggccattcacgccgatcagctgacacctacatggcgggtgtactccaccggcagcaatgtgtttcagaccagagccggctg tctgatcggagccgagcacgtgaacaatagctacgagtgcgacatccccatcggcgctggcatctgtgccagctaccagac acagacaaacagccccagacgggccagatctgtggccagccagagcatcattgcctacacaatgtctctgggcgccgagaa cagcgtggcctactccaacaactctatcgctatccccaccaacttcaccatcagcgtgaccacagagatcctgcctgtgtc catgaccaagaccagcgtggactgcaccatgtacatctgcggcgattccaccgagtgctccaacctgctgctgcagtacgg cagcttctgcacccagctgaatagagccctgacagggatcgccgtggaacaggacaagaacacccaagaggtgttcgccca agtgaagcagatctacaagacccctcctatcaaggacttcggcggcttcaatttcagccagattctgcccgatcctagcaa gcccagcaagcggagcttcatcgaggacctgctgttcaacaaagtgacactggccgacgccggcttcatcaagcagtatgg cgattgtctgggcgacattgccgccagggatctgatttgcgcccagaagtttaacggactgacagtgctgcctcctctgctgaccgatgagatgatcgcccagtacacatctgccctgctggccggcacaatcacaagcggctggacatttggagctggcgc cgctctgcagatcccctttgctatgcagatggcctacagattcaacggcatcggagtgacccagaatgtgctgtacgagaa ccagaagctgatcgccaaccagttcaacagcgccatcggcaagatccaggacagcctgagcagcacagcaagcgccctggg aaagctgcaggacgtggtcaaccagaatgcccaggcactgaacaccctggtcaagcagctgtcctccaacttcggcgccat cagctctgtgctgaacgatatcctgagcagactggaccctcctgaggccgaggtgcagatcgacagactgatcacaggcag actgcagagcctccagacatacgtgacccagcagctgatcagagccgccgagattagagcctctgccaatctggccgccac caagatgtctgagtgtgtgctgggccagagcaagagagtggacttttgcggcaagggctaccacctgatgagcttccctca gtctgcccctcacggcgtggtgtttctgcacgtgacatacgttcccgctcaagagaagaatttcaccaccgctccagccat ctgccacgacggcaaagcccactttcctagagaaggcgtgttcgtgtccaacggcacccattggttcgtgacacagcggaa cttctacgagccccagatcatcaccaccgacaacaccttcgtgtctggcaactgcgacgtcgtgatcggcattgtgaacaa taccgtgtacgaccctctgcagcccgagctggacagcttcaaagaggaactggacaagtactttaagaaccacacaagccc cgacgtggacctgggcgatatcagcggaatcaatgccagcgtcgtgaacatccagaaagagatcgaccggctgaacgaggt ggccaagaatctgaacgagagcctgatcgacctgcaagaactggggaagtacgagcagtacatcaagtggccctggtacat ctggctgggctttatcgccggactgattgccatcgtgatggtcacaatcatgctgtgttgcatgaccagctgctgtagctg cctgaagggctgttgtagctgtggcagctgctgcaagttcgacgaggacgattctgaggctagcggcagcggcgccaccaa cttcagcctgctgaagcaggccggcgacgtggaggagaaccccgggcccgaattcatgacaaacatgtcatgcgcttacga gctaatcaagtcacttccagccaagctggagcagctggctcaggagacgcaagcaacgatccaaacgctcatgatcgcgga tcccaacgtcaacaaggatctgcgagcgttctgcgagttcctgaccgtgcagcaccagcgggcgtatcgagcgacgaacag cctgctcatcaaaccgcgagtcgcagcagcgcttcgcggggaggagctggacctgggcgaggcggacgtcgccgcccgggt ccgccagctaaaacaacagctggcggagctcgagatggaaatcaagccagggcaccaacaagtggcccaagtaagcggcag gcggaaggccgcagccgcagctcccgtggcccagctgggtcgcgtgggcgtggtaaatgagtgatagtagttaattaactc gcgatcgctaacttgacaattaagtatgaaggtatatgtgtcccctaagagacacactgtacatagcaaataatctataga tcaaagggctacgcaacccctgaatagtaacaaaatacaaaatcactaaaaattataaaaacagaaaaatacataaatagg tatacgtgtcccctaagagacacattgtatgtaggtgataagtatagatcaaagggccgaataacccctgaatagtaacaa aatatgaaaatcaataaaaatcataaaatagaaaaaccataaacagaagtagttcaaagggctataaaacccctgaatagt aacaaaacataaaattaataaaaatcaaatgaataccataattggcaaacggaagagatgtaggtacttaagcttcctaaa agcagccgaactcactttgagaagtaggcatagcataccgaactcttccacgattctccgaacccacagggacgtaggaga tgttattttgtttttaatatttcaaaaaaaaaaaaaaaaaaaaaaagcggccgcttccgctcactgcccgctttccagtcg ggaaacctgtcgtgccagctgcattaacatggtcatagctgtttccttgcgtattgggcgctctccgcttcctcgctcact gactcgctgcgctcggtcgttcgggtaaagcctggggtgcctaatgagcaaaaggccagcaaaaggccaggaaccgtaaaa aggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggc gaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgc ttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcgg tgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatc gtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatg taggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgc tgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttg tttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagt ggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaat gaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatct cagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccat ctggccccagtgctgcaatgataccgcgagaaccacgctcaccggctccagatttatcagcaataaaccagccagccggaa gggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagta gttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggctt cattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtc ctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtca tgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagtt gctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttctt cggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcac

[0198] [ SEQ ID NO: 7 ]

[0199] The plasmid 24_VEEVatt-Spike-P2A-B2. annotations may have the nucleic acid sequence represented by SEQ ID NO: 8: tattgccacggccaccgaaggagtgattataaatgctgctaacagcaaaggacaacctggcggaggggtgtgcggagcgct gtataagaaattcccggaaagcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaaca tatcattcatgccgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggcagaggcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattccactgttgtccaccggcatcttttccgggaacaa agatcgactaacccaatcattgaaccatttgctgacagctttagacaccactgatgcagatgtagccatatactgcaggga caagaaatgggaaatgactctcaaggaagcagtggctaggagagaagcagtggaggagatatgcatatccgacgactcttc agtgacagaacctgatgcagagctggtgagggtgcatccgaagagttctttggctggaaggaagggctacagcacaagcga tggcaaaactttctcatatttggaagggaccaagtttcaccaggcggccaaggatatagcagaaattaatgccatgtggcc cgttgcaacggaggccaatgagcaggtatgcatgtatatcctcggagaaagcatgagcagtattaggtcgaaatgccccgt cgaagagtcggaagcctccacaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagtacagcg cctaaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaagtatagaatcactggtgtgcagaa gatccaatgctcccagcctatattgttctcaccgaaagtgcctgcgtatattcatccaaggaagtatctcgtggaaacacc accggtagacgagactccggagccatcggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccga ggatgagaccaggactagaacgcctgagccgatcatcatcgaagaggaagaagaggatagcataagtttgctgtcagatgg cccgacccaccaggtgctgcaagtcgaggcagacattcacgggccgccctctgtatctagctcatcctggtccattcctca tgcatccgactttgatgtggacagtttatccatacttgacaccctggagggagctagcgtgaccagcggggcaacgtcagc cgagactaactcttacttcgcaaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccc tccacatcccgctccgcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagcctagtttccacccc gccaggcgtgaatagggtgatcactagagaggagctcgaggcgcttaccccgtcacgcactcctagcaggtcggtctcgag aaccagcctggtctccaacccgccaggcgtaaatagggtgattacaagagaggagtttgaggcgttcgtagcacaacaaca atgacggtttgatgcgggtgcatacatcttttcctccgacaccggtcaagggcatttacaacaaaaatcagtaaggcaaac ggtgctatccgaagtggtgttggagaggaccgaattggagatttcgtatgccccgcgcctcgaccaagaaaaagaagaatt actacgcaagaaattacagttaaatcccacacctgctaacagaagcagataccagtccaggaaggtggagaacatgaaagc cataacagctagacgtattctgcaaggcctagggcattatttgaaggcagaaggaaaagtggagtgctaccgaaccctgca tcctgttcctttgtattcatctagtgtgaaccgtgccttttcaagccccaaggtcgcagtggaagcctgtaacgccatgtt gaaagagaactttccgactgtggcttcttactgtattattccagagtacgatgcctatttggacatggttgacggagcttc atgctgcttagacactgccagtttttgccctgcaaagctgcgcagctttccaaagaaacactcctatttggaacccacaat acgatcggcagtgccttcagcgatccagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaatgtcac gcaaatgagagaattgcccgtattggattcggcggcctttaatgtggaatgcttcaagaaatatgcgtgtaataatgaata ttgggaaacgtttaaagaaaaccccatcaggcttactgaagaaaacgtggtaaattacattaccaaattaaaaggaccaaa agctgctgctctttttgcgaagacacataatttgaatatgttgcaggacataccaatggacaggtttgtaatggacttaaa gagagacgtgaaagtgactccaggaacaaaacatactgaagaacggcccaaggtacaggtgatccaggctgccgatccgct agcaacagcgtatctgtgcggaatccaccgagagctggttaggagattaaatgcggtcctgcttccgaacattcatacact gtttgatatgtcggctgaagactttgacgctattatagccgagcacttccagcctggggattgtgttctggaaactgacat cgcgtcgtttgataaaagtgaggacgacgccatggctctgaccgcgttaatgattctggaagacttaggtgtggacgcaga gctgttgacgctgattgaggcggctttcggcgaaatttcatcaatacatttgcccactaaaactaaatttaaattcggagc catgatgaaatctggaatgttcctcacactgtttgtgaacacagtcattaacattgtaatcgcaagcagagtgttgagaga acggctaaccggatcaccatgtgcagcattcattggagatgacaatatcgtgaaaggagtcaaatcggacaaattaatggc agacaggtgcgccacctggttgaatatggaagtcaagattatagatgctgtggtgggcgagaaagcgccttatttctgtgg agggtttattttgtgtgactccgtgaccggcacagcgtgccgtgtggcagaccccctaaaaaggctgtttaagcttggcaa acctctggcagcagacgatgaacatgatgatgacaggagaagggcattgcatgaagagtcaacacgctggaaccgagtggg tattctttcagagctgtgcaaggcagtagaatcaaggtatgaaaccgtaggaacttccatcatagttatggccatgactac tctagctagcagtgttaaatcattcagctacctgagaggggcccctataactctctacggctaacctgaatggactacgac atagtctagtccgccaagttcgaatataggcgcgccgccaccatgttcgtgtttctggtgctgctgcctctggtgtccagc cagtgtgtgaacctgaccaccagaacacagctgcctccagcctacaccaacagctttaccagaggcgtgtactaccccgac aaggtgttcagatccagcgtgctgcactctacccaggacctgttcctgcctttcttcagcaacgtgacctggttccacgcc atccacgtgtccggcaccaatggcaccaagagattcgacaaccccgtgctgcccttcaacgacggggtgtactttgccagc accgagaagtccaacatcatcagaggctggatcttcggcaccacactggacagcaagacccagagcctgctgatcgtgaac aacgccaccaacgtggtcatcaaagtgtgcgagttccagttctgcaacgaccccttcctgggcgtctactaccacaagaac aacaagagctggatggaaagcgagttccgggtgtacagcagcgccaacaactgcaccttcgagtacgtgtcccagcctttc ctgatggacctggaaggcaagcagggcaacttcaagaacctgcgcgagttcgtgttcaagaacatcgacggctacttcaag atctacagcaagcacacccctatcaacctcgtgcgggatctgcctcagggcttctctgctctggaacccctggtggatctg cccatcggcatcaacatcacccggtttcagacactgctggccctgcacagaagctacctgacacctggcgatagcagcagc ggatggacagctggtgccgccgcttactatgtgggctacctgcagcctagaaccttcctgctgaagtacaacgagaacggc accatcaccgacgccgtggattgtgcccttgatcctctgagcgagacaaagtgcaccctgaagtccttcaccgtggaaaag ggcatctaccagaccagcaacttccgggtgcagcccaccgaatccatcgtgcggttccccaatatcaccaatctgtgcccc ttcggcgaggtgttcaatgccaccagattcgcctctgtgtacgcctggaaccggaagcggatcagcaattgcgtggccgac tactccgtgctgtacaactccgccagcttcagcaccttcaagtgctacggcgtgtcccctaccaagctgaacgacctgtgc ttcacaaacgtgtacgccgacagcttcgtgatccggggagatgaagtgcggcagattgcccctggacagacaggcaagatc gccgactacaactacaagctgcccgacgacttcaccggctgtgtgattgcctggaacagcaacaacctggactccaaagtc ggcggcaactacaattacctgtaccggctgttccggaagtccaatctgaagcccttcgagcgggacatctccaccgagatctatcaggccggcagcaccccttgtaacggcgtggaaggcttcaactgctacttcccactgcagtcctacggctttcagccc acaaatggcgtgggctatcagccctacagagtggtggtgctgagcttcgaactgctgcatgcccctgccacagtgtgcggc cctaagaaaagcaccaatctcgtgaagaacaaatgcgtgaacttcaacttcaacggcctgaccggcacaggcgtgctgaca gagagcaacaagaagttcctgccattccagcagtttggccgggatatcgccgataccacagacgccgttagagatccccag acactggaaatcctggacatcaccccttgcagcttcggcggagtgtctgtgatcacccctggcaccaacaccagcaatcag gtggcagtgctgtaccaggacgtgaactgtaccgaagtgcccgtggccattcacgccgatcagctgacacctacatggcgg gtgtactccaccggcagcaatgtgtttcagaccagagccggctgtctgatcggagccgagcacgtgaacaatagctacgag tgcgacatccccatcggcgctggcatctgtgccagctaccagacacagacaaacagccccagacgggccagatctgtggcc agccagagcatcattgcctacacaatgtctctgggcgccgagaacagcgtggcctactccaacaactctatcgctatcccc accaacttcaccatcagcgtgaccacagagatcctgcctgtgtccatgaccaagaccagcgtggactgcaccatgtacatc tgcggcgattccaccgagtgctccaacctgctgctgcagtacggcagcttctgcacccagctgaatagagccctgacaggg atcgccgtggaacaggacaagaacacccaagaggtgttcgcccaagtgaagcagatctacaagacccctcctatcaaggac ttcggcggcttcaatttcagccagattctgcccgatcctagcaagcccagcaagcggagcttcatcgaggacctgctgttc aacaaagtgacactggccgacgccggcttcatcaagcagtatggcgattgtctgggcgacattgccgccagggatctgatt tgcgcccagaagtttaacggactgacagtgctgcctcctctgctgaccgatgagatgatcgcccagtacacatctgccctg ctggccggcacaatcacaagcggctggacatttggagctggcgccgctctgcagatcccctttgctatgcagatggcctac agattcaacggcatcggagtgacccagaatgtgctgtacgagaaccagaagctgatcgccaaccagttcaacagcgccatc ggcaagatccaggacagcctgagcagcacagcaagcgccctgggaaagctgcaggacgtggtcaaccagaatgcccaggca ctgaacaccctggtcaagcagctgtcctccaacttcggcgccatcagctctgtgctgaacgatatcctgagcagactggac cctcctgaggccgaggtgcagatcgacagactgatcacaggcagactgcagagcctccagacatacgtgacccagcagctg atcagagccgccgagattagagcctctgccaatctggccgccaccaagatgtctgagtgtgtgctgggccagagcaagaga gtggacttttgcggcaagggctaccacctgatgagcttccctcagtctgcccctcacggcgtggtgtttctgcacgtgaca tacgttcccgctcaagagaagaatttcaccaccgctccagccatctgccacgacggcaaagcccactttcctagagaaggc gtgttcgtgtccaacggcacccattggttcgtgacacagcggaacttctacgagccccagatcatcaccaccgacaacacc ttcgtgtctggcaactgcgacgtcgtgatcggcattgtgaacaataccgtgtacgaccctctgcagcccgagctggacagc ttcaaagaggaactggacaagtactttaagaaccacacaagccccgacgtggacctgggcgatatcagcggaatcaatgcc agcgtcgtgaacatccagaaagagatcgaccggctgaacgaggtggccaagaatctgaacgagagcctgatcgacctgcaa gaactggggaagtacgagcagtacatcaagtggccctggtacatctggctgggctttatcgccggactgattgccatcgtg atggtcacaatcatgctgtgttgcatgaccagctgctgtagctgcctgaagggctgttgtagctgtggcagctgctgcaag ttcgacgaggacgattctgaggctagcggcagcggcgccaccaacttcagcctgctgaagcaggccggcgacgtggaggag aaccccgggcccgaattcatgacaaacatgtcatgcgcttacgagctaatcaagtcacttccagccaagctggagcagctg gctcaggagacgcaagcaacgatccaaacgctcatgatcgcggatcccaacgtcaacaaggatctgcgagcgttctgcgag ttcctgaccgtgcagcaccagcgggcgtatcgagcgacgaacagcctgctcatcaaaccgcgagtcgcagcagcgcttcgc ggggaggagctggacctgggcgaggcggacgtcgccgcccgggtccgccagctaaaacaacagctggcggagctcgagatg gaaatcaagccagggcaccaacaagtggcccaagtaagcggcaggcggaaggccgcagccgcagctcccgtggcccagctg ggtcgcgtgggcgtggtaaatgagtgatagtagttaatcgctgaatacagcagcaattggcaagctgcttacatagaactc gcggcgattggcatgccgccttaaaatttttattttattttttcttttcttttccgaatcggattttgtttttaatatttc aaaaaaaaaaaaaaaaaaaaaaaaacgcgtcgaggggaattaattcttgaagacgaaagggccaggtggcacttttcgggg aaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgata aatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcatt ttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggtta catcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaa agttctgctatgtggcgcggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaa tgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccat aaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaa catgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccac gatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaat agactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatc tggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatcta cacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggta actgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagat cctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaa aggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttg tttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttct agtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagt ggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcggg ctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatg agaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtg atgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcgagctcgatttaggtgacactatagatgggcggcgc atgagagaagcccagaccaattacctacccaaaatggagaaagttcacgttgacatcgaggaagacagcccattcctcaga gctttgcagcggagcttcccgcagtttgaggtagaagccaagcaggtcactgataatgaccatgctaatgccagagcgttt tcgcatctggcttcaaaactgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgc agaatgtattctaagcacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacagattgtataagtatgca actaagctgaagaaaaactgtaaggaaataactgataaggaattggacaagaaaatgaaggagctggccgccgtcatgagc gaccctgacctggaaactgagactatgtgcctccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccag gatgtatacgcggttgacggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggcttt gacaccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaactgggccgacgaaaccgtgtta acggctcgtaacataggcctatgcagctctgacgttatggagcggtcacgtagagggatgtccattcttagaaagaagtat ttgaaaccatccaacaatgttctattctctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcac ctgccgtctgtatttcacttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacgggtacgtc gttaaaagaatagctatcagtccaggcctgtatgggaagccttcaggctatgctgctacgatgcaccgcgagggattcttg tgctgcaaagtgacagacacattgaacggggagagggtctcttttcccgtgtgcacgtatgtgccagctacattgtgtgac caaatgactggcatactggcaacagatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtc gtcaacggtcgcacccagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatttgctaggtgg gcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacgagatagacagttagtcatggggtgttgttgg gcttttagaaggcacaagataacatctatttataagcgcccggatacccaaaccatcatcaaagtgaacagcgatttccac tcattcgtgctgcccaggataggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcac aaggagccgtcacctctcattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggctaaggaggtgcgtgaa gccgaggagttgcgcgcagctctaccacctttggcagctgatgttgaggagcccactctggaggcagacgtcgacttgatg ttacaagaggctggggccggctcagtggagacacctcgtggcttgataaaggttaccagctacgctggcgaggacaagatc ggctcttacgctgtgctttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagtc atagtgataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagtagtggtgccagagggacat gcaatacccgtccaggactttcaagctctgagtgaaagtgccaccattgtgtacaacgaacgtgagttcgtaaacaggtac ctgcaccatattgccacacatggaggagcgctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgac ggcgaatacctgtacgacatcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggcgagctg gtggatcctcccttccatgaattcgcctacgagagtctgagaacacgaccagccgctccttaccaagtaccaaccataggg gtgtatggcgtgccaggatcaggcaagtctggcatcattaaaagcgcagtcaccaaaaaagatctagtggtgagcgccaag aaagaaaactgtgcagaaattataagggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtg ctcttgaatggatgcaaacaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtactctcagagcg ctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaacagtgcggtttttttaacatgatgtgcctg aaagtgcattttaaccacgagatttgcacacaagtcttccacaaaagcatctctcgccgttgcactaaatctgtgacttcg gtcgtctcaaccttgttttacgacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggc agtaccaaacctaagcaggacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaatagattacaaaggc aacgaaataatgacggcagctgcctctcaagggctgacccgtaaaggtgtgtatgccgttcggtacaaggtgaatgaaaat cctctgtacgcacccacctcagaacatgtgaacgtcctactgacccgcacggaggaccgcatcgtgtggaaaacactagcc gg cgacccat ggataaaaacactgactgccaa gtacc ct gggaatttca ct gccacgatagaggagtggcaagca gagcat gatgccatcatgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggcaaacgtgtgttgggccaag gctttagtgccggtgctgaagaccgctggcatagacatgaccactgaacaatggaacactgtggattattttgaaacggac aaagctcactcagcagagatagtattgaaccaactatgcgtgaggttctttggactcgatctggactccggtctattttct gcacccactgttccgttatccattaggaataatcactgggataactccccgtcgcctaacatgtacgggctgaataaagaa gtggtccgtcagctctctcgcaggtacccacaactgcctcgggcagttgccactggaagagtctatgacatgaacactggt acactgcgcaattatgatccgcgcataaacctagtacctgtaaacagaagactgcctcatgctttagtcctccaccataat gaacacccacagagtgacttttcttcattcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtcc gtcccaggcaaaatggttgactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggcatcccaggt gatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaataccatcactatcagcagtgtgaagaccat gccattaagcttagcatgttgaccaagaaagcttgtctgcatctgaatcccggcggaacctgtgtcagcataggttatggt tacgctgacagggccagcgaaagcatcattggtgctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcc tcacttgaagagacggaagttctgtttgtattcattgggtacgatcgcaaggcccgtacgcacaatccttacaagctttca tcaaccttgaccaacatttatacaggttccagactccacgaagccggatgtgcaccctcatatcatgtggtgcgagggga [ SEQ ID NO: 8 ]

[0200] The plasmid 26_CHIKV-ERR-RBD-P2A-B2. annotations may have the nucleic acid sequence represented by SEQ ID NO: 9:cgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgca aaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggt tattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaa gtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaat aggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtggccgctacagggcgctcccat tcgccattcaggctgcgcaactgttgggaagggcgtttcggtgcgggcctcttcgctattacgccagctggcgaaaggggg atgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgatttaaa tacgatttaggtgacactatagatggctgcgtgagacacacgtagcctaccagtttcttactgctctactctgcaaagcaa gagattaataacccatcatggatcctgtgtacgtggacatagacgctgacagcgcctttttgaaggccctgcaacgtgcgt accccatgtttgaggtggaaccaaggcaggtcacaccgaatgaccatgctaatgctagagcgttctcgcatctagctataa aactaatagagcaggaaattgaccccgactcaaccatcctggatatcggcagtgcgccagcaaggaggatgatgtcggaca ggaagtaccactgcgtctgcccgatgcgcagtgcggaagatcccgagagactcgccaattatgcgagaaagctagcatctg ccgcaggaaaagtcctggacagaaacatctctggaaagatcggggacttacaagcagtaatggccgtgccagacacggaga cgccaacattctgcttacacacagacgtctcatgtagacagagagcagacgtcgctatataccaagacgtctatgctgtac acgcacccacgtcgctataccaccaggcgattaaaggggtccgagtggcgtactgggttgggttcgacacaaccccgttca tgtacaatgccatggcgggtgcctacccctcatactcgacaaactgggcagatgagcaggtactgaaggctaagaacatag gattatgttcaacagacctgacggaaggtagacgaggcaagttgtctattatgagagggaaaaagctaaaaccgtgcgacc gtgtgctgttctcagtagggtcaacgctctacccggaaagccgcaagctacttaagagctggcacctgccatcggtgttcc atttaaagggcaaactcagcttcacatgccgctgtgatacagtggtttcgtgtgagggctacgtcgttaagagaataacga tgagcccaggcctttatggaaaaaccacagggtatgcggtaacccaccacgcagacggattcctgatgtgcaagactaccg acacggttgacggcgaaagaatgtcattctcggtgtgcacatacgtgccggcgaccatttgtgatcaaatgaccggcatcc ttgctacagaagtcacgccggaggatgcacagaagctgttggtggggctgaaccagagaatagtggttaacggcagaacgc aacggaatacgaacaccatgaaaaattatctgcttcccgtggtcgcccaagccttcagtaagtgggcaaaggagtgccgga aagacatggaagatgaaaaactcctgggggtcagagaaagaacactgacctgctgctgtctatgggcattcaagaagcaga aaacacacacggtctacaagaggcctgatacccagtcaattcagaaggttcaggccgagtttgacagctttgtggtaccga gtctgtggtcgtccgggttgtcaatccctttgaggactagaatcaaatggttgttaagcaaggtgccaaaaaccgacctga tcccatacagcggagacgcccgagaagcccgggacgcagaaaaagaagcagaggaagaacgagaagcagaactgactcgcg aagccctaccacctctacaggcagcacaggaagatgttcaggtcgaaatcgacgtggaacagcttgaggacagagcgggcg caggaataatagagactccgagaggagctatcaaagttactgcccaaccaacagaccacgtcgtgggagagtacctggtac tctccccgcagaccgtactacgtagccagaagctcagtctgattcacgctttggcggagcaagtgaagacgtgcacgcaca acggacgagcagggaggtatgcggtcgaagcgtacgacggccgagtcctagtgccctcaggctatgcaatctcgcctgaag acttccagagtctaagcgaaagcgcaacgatggtgtataacgaaagagagttcgtaaacagaaagctacaccatattgcga tgcacggaccagccctgaacaccgacgaagagtcgtatgagctggtgagggcagagaggacagaacacgagtacgtctacg acgtggatcagagaagatgctgtaagaaggaagaagccgcaggactggtactggtgggcgacttgactaatccgccctacc acgaattcgcatatgaagggctaaaaatccgccctgcctgcccatacaaaattgcagtcataggagtcttcggagtaccgg gatctggcaagtcagctattatcaagaacctagttaccaggcaggacctggtgactagcggaaagaaagaaaactgccaag aaatcaccaccgacgtgatgagacagagaggtctagagatatctgcacgtacggttgactcgctgctcttgaatggatgca acagaccagtcgacgtgttgtacgtagacgaggcgtttgcgtgccactctggaacgctacttgctttgatcgccttggtga ga ccaaggcagaaagttgta ct ttgtggtgacccgaagcagtgcggctt ct tcaatatgatgcagatgaaagtca aetata atcacaacatctgcacccaagtgtaccacaaaagtatctccaggcggtgtacactgcctgtgaccgccattgtgtcatcgt tgcattacgaaggcaaaatgcgcactacgaatgagtacaacaagccgattgtagtggacactacaggctcaacaaaacctg accctggagacctcgtgttaacgtgcttcagagggtgggttaaacaactgcaaattgactatcgtggatacgaggtcatga cagcagccgcatcccaagggttaaccagaaaaggagtttacgcagttagacaaaaagttaatgaaaacccgctctatgcat caacgtcagagcacgtcaacgtactcctaacgcgtacggaaggtaaactggtatggaagacactttccggcgacccgtgga taaagacgctgcagaacccaccgaaaggaaacttcaaagcaactattaaggagtgggaggtggagcatgcatcaataatgg cgggcatctgcagtcaccaaatgaccttcgatacattccaaaataaagccaacgtttgttgggctaagagcttggtcccta tcctcgaaacagcggggataaaactaaatgataggcagtggtctcagataattcaagccttcaaagaagacaaagcatact cacctgaagtagccctgaatgaaatatgtacgcgcatgtatggggtggatctagacagcgggctattttctaaaccgttgg tgtctgtgtattacgcggataaccactgggataataggcctggagggaaaatgttcggatttaaccccgaggcagcatcca ttctagaaagaaagtatccattcacaaaagggaagtggaacatcaacaagcagatctgcgtgactaccaggaggatagaag actttaaccctaccaccaacatcataccggccaacaggagactaccacactcattagtggccgaacaccgcccagtaaaag gggaaagaatggaatggctggttaacaagataaacggccaccacgtgctcctggtcagtggctataaccttgcactgccta ctaagagagtcacttgggtagcgccgttaggtgtccgcggagcggactacacatacaacctagagttgggtctgccagaga gaaggggtaggtatgacctagtggtcataaacatccacacaccttttcgcatacaccattaccaacagtgcgtcgaccacg caatgaaactgcaaatgctcgggggtgactcattgagactgctcaaaccgggcggctctctattgatcagagcatatggtt acgcagatagaaccagtgaa egagteat ctgcgtattggga eg caagtttagatcgtctagag eg ttgaaaccaccatgtg tcaccagcaacactgagatgtttttcctattcagcaactttgacaatggcagaaggaatttcacaactcatgtcatgaaca atcaactgaatgcagccttcgtaggacaggtcacccgagcaggatgtgcaccgtcgtaccgggtaaaacgcatggacatcgcgaagaacgatgaagagtgcgtagtcaacgccgctaaccctcgcgggttaccgggtggcggtgtttgcaaggcagtataca aaaaatggccggagtcctttaagaacagtgcaacaccagtgggaaccgcaaaaacagttatgtgcggtacgtatccagtaa tccacgctgttggaccaaacttctctaattattcggagtctgaaggggaccgggaattggcagctgcctatcgagaagtcg caaaggaagtaactaggctgggagtaaatagtgtagctatacctctcctctccacaggtgtatactcaggagggaaagaca ggctgacccagtca ct gaaccacctcttta cagecat ggactcgacggatgcagacgtggt cat eta ct gccgcgacaaag aatgggagaagaaaatatctgaggccatacagatgcggacccaagtagagctgctggatgagcacatctccatagactgcg atattgttcgcgtgcaccctgacagcagcttggcaggcagaaaaggatacagcaccacggaaggcgcactgtactcatatc tagaagggacccgttttcatcagacggctgtggatatggcggagatacatactatgtggccaaagcaaacagaggccaatg agcaagtctgcctatatgccctgggggaaagtattgaatcgatcaggcagaaatgcccggtggatgatgcagacgcatcat ctccccccaaaactgtcccgtgcctttgccgttacgctatgactccagaacgcgtcacccggcttcgcatgaaccacgtca caagcataattgtgtgttcttcgtttcccctcccaaagtacaaaatagaaggagtgcaaaaagtcaaatgctctaaggtaa tgctatttgaccacaacgtgccatcgcgcgtaagtccaagggaatatagatcttcccaggagtctgcacaggaggcgagta caatcacgtcactgacgcatagtcaattcgacctaagcgttgatggcgagatactgcccgtcccgtcagacctggatgctg acgccccagccctagaaccagcactagacgacggggcgacacacacgctgccatccacaaccggaaaccttgcggccgtgt ctgattgggtaatgagcaccgtacctgtcgcgccgcccagaagaaggcgagggagaaacctgactgtgacatgtgacgaga gagaagggaatataacacccatggctagcgtccgattctttagggcagagctgtgtccggtcgtacaagaaacagcggaga cgcgtgacacagcaatgtctcttcaggcaccaccgagtaccgccacggaaccgaatcatccgccgatctccttcggagcat caagcgagacgttccccattacatttggggacttcaacgaaggagaaatcgaaagcttgtcttctgagctactaactttcg gagacttcttaccaggagaagtggatgacttgacagacagcgactggtccacgtgctcagacacggacgacgagttaagac tagacagggcaggtgggtatatattctcgtcggacaccggtccaggtcatttacaacagaagtcagtacgccagtcagtgc tgccggtgaacaccctggaggaagtccacgaggagaagtgttacccacctaagctggatgaagcaaaggagcaactattac ttaagaaactccaggagagtgcatccatggccaacagaagcaggtatcagtcgcgcaaagtagaaaacatgaaagcagcaa tcatccagagactaaagagaggctgtagactatacttaatgtcagagaccccaaaagtccctacttaccggactacatatc cggcgcctgtgtactcgcctccgatcaacgtccgattgtccaatcccgagtccgcagtggcagcatgcaatgagttcttag ctagaaactatccaactgtctcatcataccaaattaccgacgagtatgatgcatatctagacatggtggacgggtcggaga gttgcctggaccgagcgacattcaatccgtcaaaactcaggagctacccgaaacagcacgcttaccacgcgccctccatca gaagcgctgtaccgtccccattccagaacacactacagaatgtactggcagcagccacgaaaagaaactgcaacgtcacac agatgagggaattacccactttggactcagcagtattcaacgtggagtgtttcaaaaaattcgcatgcaaccaagaatact gggaagaatttgctgccagccctattaggataacaactgagaatttagcaacctatgttactaaactaaaagggccaaaag cagcagcgctattcgcaaaaacccataatctactgccactacaggaagtaccaatggataggttcacagtagatatgaaaa gggacgtaaaggtgactcctggtacaaagcatacagaggaaagacctaaggtgcaggttatacaggcggctgaacccttgg cgacagcatacctatgtgggattcacagagagctggttaggaggctgaacgccgtcctcctacccaatgtacatacactat ttgacatgtctgccgaggatttcgatgccatcatagccgcacactttaagccaggagacactgttttggaaacggacatag cctcctttgataagagccaagatgattcacttgcgcttactgctttgatgctgttagaggatttaggggtggatcactccc tgctggacttgatagaggctgctttcggagagatttccagctgtcacctaccgacaggtacgcgcttcaagttcggcgcca tgat ga aatcaggt at gt tc ct aa ct ct gt tegt caa cacattgttaaacatcaccatcgc cage cgagtgctggaa gate gtctgacaaaatccgcgtgcgcggccttcatcggcgacgacaacataatacatggagtcgtctccgatgaattgatggcag ccagatgtgccacttggatgaacatggaagtgaagatcatagatgcagttgtatccttgaaagccccttacttttgtggag ggtttatactgcacgatactgtgacaggaacagcttgcagagtggcagacccgctaaaaaggctttttaaactgggcaaac cgctagcggcaggtgacgaacaagatgaagatagaagacgagcgctggctgacgaagtgatcagatggcaacgaacagggc taattgatgagctggagaaagcggtatactctaggtacgaagtgcagggtatatcagttgtggtaatgtccatggccacct ttgcaagctccagatccaactt cgagaagctcagaggacccgtcataactttgta eggegg tcctaaataggtacgcacta cagctacctattttgcagaagccgacagcaagtatctaaacactaatcagctacctaggaagttcgaatataggcgcgccg ccaccatgcggttccccaatatcaccaatctgtgccccttcggcgaggtgttcaatgccaccagattcgcctctgtgtacg cctggaaccggaagcggatcagcaattgcgtggccgactactccgtgctgtacaactccgccagcttcagcaccttcaagt geta eggegt gtcccctaccaagctgaa cgacctgtgcttcacaaacgtgtacgc cgacagct tegt gate cggggagatg aagtgcggcagattgcccctggacagacaggcaagatcgccgactacaactacaagctgcccgacgacttcaccggctgtg tgattgcctgga acagcaacaacctggactccaaagt eggegg ca acta caattacctgtaccggctgttccggaagtcca atctgaagcccttcgagcgggacatctccaccgagatctatcaggccggcagcaccccttgtaacggcgtggaaggcttca actgctacttcccactgcagtcctacggctttcagcccacaaatggcgtgggctatcagccctacagagtggtggtgctga gcttcgaactgctgcatgcccctgccacagtgtgcggccctaagaaaagcaccgctagcggcagcggcgccaccaacttca gc ct gctgaagcaggc eggega eg tggaggagaaccc egggee cgaattcatgacaaacatgtcatg eg etta egag ctaa tcaagtcacttccagccaagctggagcagctggctcaggagacgcaagcaacgatccaaacgctcatgatcgcggatccca acgtcaacaaggatctgcgagcgttctgcgagttcctgaccgtgcagcaccagcgggcgtatcgagcgacgaacagcctgc tcatcaaaccgcgagtcgcagcagcgcttcgcggggaggagctggacctgggcgaggcggacgtcgccgcccgggtccgcc agctaaaacaacagctggcggagctcgagatggaaatcaagccagggcaccaacaagtggcccaagtaagcggcaggcgga aggccgcagccgcagctcccgtggcccagctgggtcgcgtgggcgtggtaaatgagtgatagtagttaattaactcgcgat cgctaacttgacaattaagtatgaaggtatatgtgtcccctaagagacacactgtacatagcaaataatctatagatcaaagggctacgcaacccctgaatagtaacaaaatacaaaatcactaaaaattataaaaacagaaaaatacataaataggtatac gtgtcccctaagagacacattgtatgtaggtgataagtatagatcaaagggccgaataacccctgaatagtaacaaaatat gaaaatcaataaaaatcataaaatagaaaaaccataaacagaagtagttcaaagggctataaaacccctgaatagtaacaa aacataaaattaataaaaatcaaatgaataccataattggcaaacggaagagatgtaggtacttaagcttcctaaaagcag ccgaactcactttgagaagtaggcatagcataccgaactcttccacgattctccgaacccacagggacgtaggagatgtta ttttgtttttaatatttcaaaaaaaaaaaaaaaaaaaaaaaagcggccgcttccgctcactgcccgctttccagtcgggaa acctgtcgtgccagctgcattaacatggtcatagctgtttccttgcgtattgggcgctctccgcttcctcgctcactgact cgctgcgctcggtcgttcgggtaaagcctggggtgcctaatgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggc cgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaa cccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttac cggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgta ggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtct tgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtagg cggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaa gccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttg caagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaa cgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaag ttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagc gatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctgg ccccagtgctgcaatgataccgcgagaaccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggc cgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttc gccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcatt cagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctcc gatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgcc atccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctc ttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggg gcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccact

[0201] [ SEQ ID NO: 9]

[0202] The plasmid 30_CHIKV-ERR-B2-P2A-RBD. annotations may have the nucleic acid sequence represented by SEQ ID NO: 10: tcactccctgctggacttgatagaggctgctttcggagagatttccagctgtcacctaccgacaggtacgcgcttcaagtt cggcgccatgatgaaatcaggtatgttcctaactctgttcgtcaacacattgttaaacatcaccatcgccagccgagtgct ggaagatcgtctgacaaaatccgcgtgcgcggccttcatcggcgacgacaacataatacatggagtcgtctccgatgaatt gatggcagccagatgtgccacttggatgaacatggaagtgaagatcatagatgcagttgtatccttgaaagccccttactt ttgtggagggtttatactgcacgatactgtgacaggaacagcttgcagagtggcagacccgctaaaaaggctttttaaact gggcaaaccgctagcggcaggtgacgaacaagatgaagatagaagacgagcgctggctgacgaagtgatcagatggcaacg aacagggctaattgatgagctggagaaagcggtatactctaggtacgaagtgcagggtatatcagttgtggtaatgtccat ggccacctttgcaagctccagatccaacttcgagaagctcagaggacccgtcataactttgtacggcggtcctaaataggt acgcactacagctacctattttgcagaagccgacagcaagtatctaaacactaatcagctacctaggaagttcgaatatag gcgcgccgccaccatgacaaacatgtcatgcgcttacgagctaatcaagtcacttccagccaagctggagcagctggctca ggagacgcaagcaacgatccaaacgctcatgatcgcggatcccaacgtcaacaaggatctgcgagcgttctgcgagttcct gaccgtgcagcaccagcgggcgtatcgagcgacgaacagcctgctcatcaaaccgcgagtcgcagcagcgcttcgcgggga ggagctggacctgggcgaggcggacgtcgccgcccgggtccgccagctaaaacaacagctggcggagctcgagatggaaat caagccagggcaccaacaagtggcccaagtaagcggcaggcggaaggccgcagccgcagctcccgtggcccagctgggtcg cgtgggcgtggtaaatgaggctagcggcagcggcgccaccaacttcagcctgctgaagcaggccggcgacgtggaggagaa ccccgggcccgaattcatgcggttccccaatatcaccaatctgtgccccttcggcgaggtgttcaatgccaccagattcgc ctctgtgtacgcctggaaccggaagcggatcagcaattgcgtggccgactactccgtgctgtacaactccgccagcttcag caccttcaagtgctacggcgtgtcccctaccaagctgaacgacctgtgcttcacaaacgtgtacgccgacagcttcgtgat ccggggagatgaagtgcggcagattgcccctggacagacaggcaagatcgccgactacaactacaagctgcccgacgactt caccggctgtgtgattgcctggaacagcaacaacctggactccaaagtcggcggcaactacaattacctgtaccggctgtt ccggaagtccaatctgaagcccttcgagcgggacatctccaccgagatctatcaggccggcagcaccccttgtaacggcgt ggaaggcttcaactgctacttcccactgcagtcctacggctttcagcccacaaatggcgtgggctatcagccctacagagt ggtggtgctgagcttcgaactgctgcatgcccctgccacagtgtgcggccctaagaaaagcacctgatagtagttaattaa ctcgcgatcgctaacttgacaattaagtatgaaggtatatgtgtcccctaagagacacactgtacatagcaaataatctat agatcaaagggctacgcaacccctgaatagtaacaaaatacaaaatcactaaaaattataaaaacagaaaaatacataaataggtatacgtgtcccctaagagacacattgtatgtaggtgataagtatagatcaaagggccgaataacccctgaatagtaa caaaatatgaaaatcaataaaaatcataaaatagaaaaaccataaacagaagtagttcaaagggctataaaacccctgaat agtaacaaaacataaaattaataaaaatcaaatgaataccataattggcaaacggaagagatgtaggtacttaagcttcct aaaagcagccgaactcactttgagaagtaggcatagcataccgaactcttccacgattctccgaacccacagggacgtagg agatgttattttgtttttaatatttcaaaaaaaaaaaaaaaaaaaaagcggccgcttccgctcactgcccgctttccagtc gggaaacctgtcgtgccagctgcattaacatggtcatagctgtttccttgcgtattgggcgctctccgcttcctcgctcac tgactcgctgcgctcggtcgttcgggtaaagcctggggtgcctaatgagcaaaaggccagcaaaaggccaggaaccgtaaa aaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtgg cgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccg cttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcg gtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactat cgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtat gtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctg ctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggttttttt gtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcag tggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaa tgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatc tcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttacca tctggccc cagtgctgca at gataccgcgagaaccacgctcaccggctccagatttatcagcaataaac cage cage cgga agggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagt agttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggct tcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggt cctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtc atgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagt tgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttct tcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttca gcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgaca cggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatac atatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctaaattgtaagcg ttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcc cttataaatcaaaagaatagaccgagatagggttgagtggccgctacagggcgctcccattcgccattcaggctgcgcaac tgttgggaagggcgtttcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaag ttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgatttaaatacgatttaggtgacactata gatggctgcgtgagacacacgtagcctaccagtttcttactgctctactctgcaaagcaagagattaataacccatcatgg atcctgtgtacgtggacatagacgctgacagcgcctttttgaaggccctgcaacgtgcgtaccccatgtttgaggtggaac caaggcaggtcacaccgaatgaccatgctaatgctagagcgttctcgcatctagctataaaactaatagagcaggaaattg accccgactcaaccatcctggatatcggcagtgcgccagcaaggaggatgatgtcggacaggaagtaccactgcgtctgcc cgatgcgcagtgcggaagatcccgagagactcgccaattatgcgagaaagctagcatctgccgcaggaaaagtcctggaca gaaacatctctggaaagatcggggacttacaagcagtaatggccgtgccagacacggagacgccaacattctgcttacaca cagacgtctcatgtagacagagagcagacgtcgctatataccaagacgtctatgctgtacacgcacccacgtcgctatacc accaggcgattaaaggggtccgagtggcgtactgggttgggttcgacacaaccccgttcatgtacaatgccatggcgggtg cctacccctcatactcgacaaactgggcagatgagcaggtactgaaggctaagaacataggattatgttcaacagacctga cggaaggtagacgaggcaagttgtctattatgagagggaaaaagctaaaaccgtgcgaccgtgtgctgttctcagtagggt caacgctctacccggaaagccgcaagctacttaagagctggcacctgccatcggtgttccatttaaagggcaaactcagct tcacatgccgctgtgatacagtggtttcgtgtgagggctacgtcgttaagagaataacgatgagcccaggcctttatggaa aaaccacagggtatgcggtaacccaccacgcagacggattcctgatgtgcaagactaccgacacggttgacggcgaaagaa tgtcattctcggtgtgcacatacgtgccggcgaccatttgtgatcaaatgaccggcatccttgctacagaagtcacgccgg aggatgcacagaagctgttggtggggctgaaccagagaatagtggttaacggcagaacgcaacggaatacgaacaccatga aaaattatctgcttcccgtggtcgcccaagccttcagtaagtgggcaaaggagtgccggaaagacatggaagatgaaaaac tcctgggggtcagagaaagaacactgacctgctgctgtctatgggcattcaagaagcagaaaacacacacggtctacaaga ggcctgatacccagtcaattcagaaggttcaggccgagtttgacagctttgtggtaccgagtctgtggtcgtccgggttgt caatccctttgaggactagaatcaaatggttgttaagcaaggtgccaaaaaccgacctgatcccatacagcggagacgccc gagaagcccgggacgcagaaaaagaagcagaggaagaacgagaagcagaactgactcgcgaagccctaccacctctacagg cagcacaggaagatgttcaggtcgaaatcgacgtggaacagcttgaggacagagcgggcgcaggaataatagagactccga gaggagctatcaaagttactgcccaaccaacagaccacgtcgtgggagagtacctggtactctccccgcagaccgtactac gtagccagaagctcagtctgattcacgctttggcggagcaagtgaagacgtgcacgcacaa cgga egag cagggaggtatg cggtcgaagcgtacgacggccgagtcctagtgccctcaggctatgcaatctcgcctgaagacttccagagtctaagcgaaa gcgcaacgatggtgtataacgaaagagagttcgtaaacagaaagctacaccatattgcgatgcacggaccagccctgaacaccgacgaagagtcgtatgagctggtgagggcagagaggacagaacacgagtacgtctacgacgtggatcagagaagatgct gtaagaaggaagaagccgcaggactggtactggtgggcgacttgactaatccgccctaccacgaattcgcatatgaagggc taaaaatccgccctgcctgcccatacaaaattgcagtcataggagtcttcggagtaccgggatctggcaagtcagctatta tcaagaacctagttaccaggcaggacctggtgactagcggaaagaaagaaaactgccaagaaatcaccaccgacgtgatga gacagagaggtctagagatatctgcacgtacggttgactcgctgctcttgaatggatgcaacagaccagtcgacgtgttgt acgtagacgaggcgtttgcgtgccactctggaacgctacttgctttgatcgccttggtgagaccaaggcagaaagttgtac tttgtggtgacccgaagcagtgcggcttcttcaatatgatgcagatgaaagtcaactataatcacaacatctgcacccaag tgtaccacaaaagtatctccaggcggtgtacactgcctgtgaccgccattgtgtcatcgttgcattacgaaggcaaaatgc gcactacgaatgagtacaacaagccgattgtagtggacactacaggctcaacaaaacctgaccctggagacctcgtgttaa cgtgcttcagagggtgggttaaacaactgcaaattgactatcgtggatacgaggtcatgacagcagccgcatcccaagggt taaccagaaaaggagtttacgcagttagacaaaaagttaatgaaaacccgctctatgcatcaacgtcagagcacgtcaacg tactcctaacgcgtacggaaggtaaactggtatggaagacactttccggcgacccgtggataaagacgctgcagaacccac cgaaaggaaacttcaaagcaactattaaggagtgggaggtggagcatgcatcaataatggcgggcatctgcagtcaccaaa tgaccttcgatacattccaaaataaagccaacgtttgttgggctaagagcttggtccctatcctcgaaacagcggggataa aactaaatgataggcagtggtctcagataattcaagccttcaaagaagacaaagcatactcacctgaagtagccctgaatg aaatatgtacgcgcatgtatggggtggatctagacagcgggctattttctaaaccgttggtgtctgtgtattacgcggata accactgggataataggcctggagggaaaatgttcggatttaaccccgaggcagcatccattctagaaagaaagtatccat tcacaaaagggaagtggaacatcaacaagcagatctgcgtgactaccaggaggatagaagactttaaccctaccaccaaca tcataccggccaacaggagactaccacactcattagtggccgaacaccgcccagtaaaaggggaaagaatggaatggctgg ttaacaagataaacggccaccacgtgctcctggtcagtggctataaccttgcactgcctactaagagagtcacttgggtag cgccgttaggtgtccgcggagcggactacacatacaacctagagttgggtctgccagagagaaggggtaggtatgacctag tggtcataaacatccacacaccttttcgcatacaccattaccaacagtgcgtcgaccacgcaatgaaactgcaaatgctcg ggggtgactcattgagactgctcaaaccgggcggctctctattgatcagagcatatggttacgcagatagaaccagtgaac gagtcatctgcgtattgggacgcaagtttagatcgtctagagcgttgaaaccaccatgtgtcaccagcaacactgagatgt ttttcctattcagcaactttgacaatggcagaaggaatttcacaactcatgtcatgaacaatcaactgaatgcagccttcg taggacaggtcacccgagcaggatgtgcaccgtcgtaccgggtaaaacgcatggacatcgcgaagaacgatgaagagtgcg tagtcaacgccgctaaccctcgcgggttaccgggtggcggtgtttgcaaggcagtatacaaaaaatggccggagtccttta agaacagtgcaacaccagtgggaaccgcaaaaacagttatgtgcggtacgtatccagtaatccacgctgttggaccaaact tctctaattattcggagtctgaaggggaccgggaattggcagctgcctatcgagaagtcgcaaaggaagtaactaggctgg gagtaaatagtgtagctatacctctcctctccacaggtgtatactcaggagggaaagacaggctgacccagtcactgaacc acctctttacagccatggactcgacggatgcagacgtggtcatctactgccgcgacaaagaatgggagaagaaaatatctg aggccatacagatgcggacccaagtagagctgctggatgagcacatctccatagactgcgatattgttcgcgtgcaccctg acagcagcttggcaggcagaaaaggatacagcaccacggaaggcgcactgtactcatatctagaagggacccgttttcatc agacggctgtggatatggcggagatacatactatgtggccaaagcaaacagaggccaatgagcaagtctgcctatatgccc tgggggaaagtattgaatcgatcaggcagaaatgcccggtggatgatgcagacgcatcatctccccccaaaactgtcccgt gcctttgccgttacgctatgactccagaacgcgtcacccggcttcgcatgaaccacgtcacaagcataattgtgtgttctt cgtttcccctcccaaagtacaaaatagaaggagtgcaaaaagtcaaatgctctaaggtaatgctatttgaccacaacgtgc catcgcgcgtaagtccaagggaatatagatcttcccaggagtctgcacaggaggcgagtacaatcacgtcactgacgcata gtcaattcgacctaagcgttgatggcgagatactgcccgtcccgtcagacctggatgctgacgccccagccctagaaccag cactagacgacggggcgacacacacgctgccatccacaaccggaaaccttgcggccgtgtctgattgggtaatgagcaccg tacctgtcgcgccgcccagaagaaggcgagggagaaacctgactgtgacatgtgacgagagagaagggaatataacaccca tggctagcgtccgattctttagggcagagctgtgtccggtcgtacaagaaacagcggagacgcgtgacacagcaatgtctc ttcaggcaccaccgagtaccgccacggaaccgaatcatccgccgatctccttcggagcatcaagcgagacgttccccatta catttggggacttcaacgaaggagaaatcgaaagcttgtcttctgagctactaactttcggagacttcttaccaggagaag tggatgacttgacagacagcgactggtccacgtgctcagacacggacgacgagttaagactagacagggcaggtgggtata tattctcgtcggacaccggtccaggtcatttacaacagaagtcagtacgccagtcagtgctgccggtgaacaccctggagg aagtccacgaggagaagtgttacccacctaagctggatgaagcaaaggagcaactattacttaagaaactccaggagagtg catccatggccaacagaagcaggtatcagtcgcgcaaagtagaaaacatgaaagcagcaatcatccagagactaaagagag gctgtagactatacttaatgtcagagaccccaaaagtccctacttaccggactacatatccggcgcctgtgtactcgcctc cgatcaacgtccgattgtccaatcccgagtccgcagtggcagcatgcaatgagttcttagctagaaactatccaactgtct catcataccaaattaccgacgagtatgatgcatatctagacatggtggacgggtcggagagttgcctggaccgagcgacat tcaatccgtcaaaactcaggagctacccgaaacagcacgcttaccacgcgccctccatcagaagcgctgtaccgtccccat tccagaacacactacagaatgtactggcagcagccacgaaaagaaactgcaacgtcacacagatgagggaattacccactt tggactcagcagtattcaacgtggagtgtttcaaaaaattcgcatgcaaccaagaatactgggaagaatttgctgccagcc ctattaggataacaactgagaatttagcaacctatgttactaaactaaaagggccaaaagcagcagcgctattcgcaaaaa cccataatctactgccactacaggaagtaccaatggataggttcacagtagatatgaaaagggacgtaaaggtgactcctg gtacaaagcatacagaggaaagacctaaggtgcaggttatacaggcggctgaacccttggcgacagcatacctatgtggga ttcacagagagctggttaggaggctgaacgccgtcctcctacccaatgtacatacactatttgacatgtctgccgaggatttcgatgccatcatagccgcacactttaagccaggagacactgttttggaaacggacatagcctcctttgataagagccaag atgattcacttgcgcttactgctttgatgctgttagaggatttaggggtgga

[0203] [ SEQ ID NO: 10]

[0204] The plasmid 33_CHIKV-ERR-B2-P2A-Spike. annotations may have the nucleic acid sequence represented by SEQ ID NO: 11: agctacttaagagctggcacctgccatcggtgttccatttaaagggcaaactcagcttcacatgccgctgtgatacagtgg tttcgtgtgagggctacgtcgttaagagaataacgatgagcccaggcctttatggaaaaaccacagggtatgcggtaaccc accacgcagacggattcctgatgtgcaagactaccgacacggttgacggcgaaagaatgtcattctcggtgtgcacatacg tgccggcgaccatttgtgatcaaatgaccggcatccttgctacagaagtcacgccggaggatgcacagaagctgttggtgg ggctgaaccagagaatagtggttaacggcagaacgcaacggaatacgaacaccatgaaaaattatctgcttcccgtggtcg cccaagccttcagtaagtgggcaaaggagtgccggaaagacatggaagatgaaaaactcctgggggtcagagaaagaacac tgacctgctgctgtctatgggcattcaagaagcagaaaacacacacggtctacaagaggcctgatacccagtcaattcaga aggttcaggccgagtttgacagctttgtggtaccgagtctgtggtcgtccgggttgtcaatccctttgaggactagaatca aatggttgttaagcaaggtgccaaaaaccgacctgatcccatacagcggagacgcccgagaagcccgggacgcagaaaaag aagcagaggaagaacgagaagcagaactgactcgcgaagccctaccacctctacaggcagcacaggaagatgttcaggtcg aaatcgacgtggaacagcttgaggacagagcgggcgcaggaataatagagactccgagaggagctatcaaagttactgccc aaccaacagaccacgtcgtgggagagtacctggtactctccccgcagaccgtactacgtagccagaagctcagtctgattc acgctttggcggagcaagtgaagacgtgcacgcacaacggacgagcagggaggtatgcggtcgaagcgtacgacggccgag tcctagtgccctcaggctatgcaatctcgcctgaagacttccagagtctaagcgaaagcgcaacgatggtgtataacgaaa gagagttcgtaaacagaaagctacaccatattgcgatgcacggaccagccctgaacaccgacgaagagtcgtatgagctgg tgagggcagagaggacagaacacgagtacgtctacgacgtggatcagagaagatgctgtaagaaggaagaagccgcaggac tggtactggtgggcgacttgactaatccgccctaccacgaattcgcatatgaagggctaaaaatccgccctgcctgcccat acaaaattgcagtcataggagtcttcggagtaccgggatctggcaagtcagctattatcaagaacctagttaccaggcagg acctggtgactagcggaaagaaagaaaactgccaagaaatcaccaccgacgtgatgagacagagaggtctagagatatctg cacgtacggttgactcgctgctcttgaatggatgcaacagaccagtcgacgtgttgtacgtagacgaggcgtttgcgtgcc actctggaacgctacttgctttgatcgccttggtgagaccaaggcagaaagttgtactttgtggtgacccgaagcagtgcg gcttcttcaatatgatgcagatgaaagtcaactataatcacaacatctgcacccaagtgtaccacaaaagtatctccaggc ggtgtacactgcctgtgaccgccattgtgtcatcgttgcattacgaaggcaaaatgcgcactacgaatgagtacaacaagc cgattgtagtggacactacaggctcaacaaaacctgaccctggagacctcgtgttaacgtgcttcagagggtgggttaaac aactgcaaattgactatcgtggatacgaggtcatgacagcagccgcatcccaagggttaaccagaaaaggagtttacgcag ttagacaaaaagttaatgaaaacccgctctatgcatcaacgtcagagcacgtcaacgtactcctaacgcgtacggaaggta aactggtatggaagacactttccggcgacccgtggataaagacgctgcagaacccaccgaaaggaaacttcaaagcaacta ttaaggagtgggaggtggagcatgcatcaataatggcgggcatctgcagtcaccaaatgaccttcgatacattccaaaata aagccaacgtttgttgggctaagagcttggtccctatcctcgaaacagcggggataaaactaaatgataggcagtggtctc agataattcaagccttcaaagaagacaaagcatactcacctgaagtagccctgaatgaaatatgtacgcgcatgtatgggg tggatctagacagcgggctattttctaaaccgttggtgtctgtgtattacgcggataaccactgggataataggcctggag ggaaaatgttcggatttaaccccgaggcagcatccattctagaaagaaagtatccattcacaaaagggaagtggaacatca acaagcagatctgcgtgactaccaggaggatagaagactttaaccctaccaccaacatcataccggccaacaggagactac cacactcattagtggccgaacaccgcccagtaaaaggggaaagaatggaatggctggttaacaagataaacggccaccacg tgctcctggtcagtggctataaccttgcactgcctactaagagagtcacttgggtagcgccgttaggtgtccgcggagcgg actacacatacaacctagagttgggtctgccagagagaaggggtaggtatgacctagtggtcataaacatccacacacctt ttcgcatacaccattaccaacagtgcgtcgaccacgcaatgaaactgcaaatgctcgggggtgactcattgagactgctca aaccgggcggctctctattgatcagagcatatggttacgcagatagaaccagtgaacgagtcatctgcgtattgggacgca agtttagatcgtctagagcgttgaaaccaccatgtgtcaccagcaacactgagatgtttttcctattcagcaactttgaca atggcagaaggaatttcacaactcatgtcatgaacaatcaactgaatgcagccttcgtaggacaggtcacccgagcaggat gtgcaccgtcgtaccgggtaaaacgcatggacatcgcgaagaacgatgaagagtgcgtagtcaacgccgctaaccctcgcg ggttaccgggtggcggtgtttgcaaggcagtatacaaaaaatggccggagtcctttaagaacagtgcaacaccagtgggaa ccgcaaaaacagttatgtgcggtacgtatccagtaatccacgctgttggaccaaacttctctaattattcggagtctgaag gggaccgggaattggcagctgcctatcgagaagtcgcaaaggaagtaactaggctgggagtaaatagtgtagctatacctc tcctctccacaggtgtatactcaggagggaaagacaggctgacccagtcactgaaccacctctttacagccatggactcga cggatgcagacgtggtcatctactgccgcgacaaagaatgggagaagaaaatatctgaggccatacagatgcggacccaag tagagctgctggatgagcacatctccatagactgcgatattgttcgcgtgcaccctgacagcagcttggcaggcagaaaag gatacagcaccacggaaggcgcactgtactcatatctagaagggacccgttttcatcagacggctgtggatatggcggaga tacatactatgtggccaaagcaaacagaggccaatgagcaagtctgcctatatgccctgggggaaagtattgaatcgatca ggcagaaatgcccggtggatgatgcagacgcatcatctccccccaaaactgtcccgtgcctttgccgttacgctatgactccagaacgcgtcacccggcttcgcatgaaccacgtcacaagcataattgtgtgttcttcgtttcccctcccaaagtacaaaa tagaaggagtgcaaaaagtcaaatgctctaaggtaatgctatttgaccacaacgtgccatcgcgcgtaagtccaagggaat atagatcttcccaggagtctgcacaggaggcgagtacaatcacgtcactgacgcatagtcaattcgacctaagcgttgatg gcgagatactgcccgtcccgtcagacctggatgctgacgccccagccctagaaccagcactagacgacggggcgacacaca cgctgccatccacaaccggaaaccttgcggccgtgtctgattgggtaatgagcaccgtacctgtcgcgccgcccagaagaa ggcgagggagaaacctgactgtgacatgtgacgagagagaagggaatataacacccatggctagcgtccgattctttaggg cagagctgtgtccggtcgtacaagaaacagcggagacgcgtgacacagcaatgtctcttcaggcaccaccgagtaccgcca cggaaccgaatcatccgccgatctccttcggagcatcaagcgagacgttccccattacatttggggacttcaacgaaggag aaatcgaaagcttgtcttctgagctactaactttcggagacttcttaccaggagaagtggatgacttgacagacagcgact ggtccacgtgctcagacacggacgacgagttaagactagacagggcaggtgggtatatattctcgtcggacaccggtccag gtcatttacaacagaagtcagtacgccagtcagtgctgccggtgaacaccctggaggaagtccacgaggagaagtgttacc cacctaagctggatgaagcaaaggagcaactattacttaagaaactccaggagagtgcatccatggccaacagaagcaggt atcagtcgcgcaaagtagaaaacatgaaagcagcaatcatccagagactaaagagaggctgtagactatacttaatgtcag agaccccaaaagtccctacttaccggactacatatccggcgcctgtgtactcgcctccgatcaacgtccgattgtccaatc ccgagtccgcagtggcagcatgcaatgagttcttagctagaaactatccaactgtctcatcataccaaattaccgacgagt atgatgcatatctagacatggtggacgggtcggagagttgcctggaccgagcgacattcaatccgtcaaaactcaggagct acccgaaacagcacgcttaccacgcgccctccatcagaagcgctgtaccgtccccattccagaacacactacagaatgtac tggcagcagccacgaaaagaaactgcaacgtcacacagatgagggaattacccactttggactcagcagtattcaacgtgg agtgtttcaaaaaattcgcatgcaaccaagaatactgggaagaatttgctgccagccctattaggataacaactgagaatt tagcaacctatgttactaaactaaaagggccaaaagcagcagcgctattcgcaaaaacccataatctactgccactacagg aagtaccaatggataggttcacagtagatatgaaaagggacgtaaaggtgactcctggtacaaagcatacagaggaaagac ctaaggtgcaggttatacaggcggctgaacccttggcgacagcatacctatgtgggattcacagagagctggttaggaggc tgaacgccgtcctcctacccaatgtacatacactatttgacatgtctgccgaggatttcgatgccatcatagccgcacact ttaagccaggagacactgttttggaaacggacatagcctcctttgataagagccaagatgattcacttgcgcttactgctt tgatgctgttagaggatttaggggtggatcactccctgctggacttgatagaggctgctttcggagagatttccagctgtc acctaccgacaggtacgcgcttcaagttcggcgccatgatgaaatcaggtatgttcctaactctgttcgtcaacacattgt taaacatcaccatcgccagccgagtgctggaagatcgtctgacaaaatccgcgtgcgcggccttcatcggcgacgacaaca taatacatggagtcgtctccgatgaattgatggcagccagatgtgccacttggatgaacatggaagtgaagatcatagatg cagttgtatccttgaaagccccttacttttgtggagggtttatactgcacgatactgtgacaggaacagcttgcagagtgg cagacccgctaaaaaggctttttaaactgggcaaaccgctagcggcaggtgacgaacaagatgaagatagaagacgagcgc tggctgacgaagtgatcagatggcaacgaacagggctaattgatgagctggagaaagcggtatactctaggtacgaagtgc agggtatatcagttgtggtaatgtccatggccacctttgcaagctccagatccaacttcgagaagctcagaggacccgtca taactttgtacggcggtcctaaataggtacgcactacagctacctattttgcagaagccgacagcaagtatctaaacacta atcagctacctaggaagttcgaatataggcgcgccgccaccatgacaaacatgtcatgcgcttacgagctaatcaagtcac ttccagccaagctggagcagctggctcaggagacgcaagcaacgatccaaacgctcatgatcgcggatcccaacgtcaaca aggatctgcgagcgttctgcgagttcctgaccgtgcagcaccagcgggcgtatcgagcgacgaacagcctgctcatcaaac cgcgagtcgcagcagcgcttcgcggggaggagctggacctgggcgaggcggacgtcgccgcccgggtccgccagctaaaac aacagctggcggagctcgagatggaaatcaagccagggcaccaacaagtggcccaagtaagcggcaggcggaaggccgcag ccgcagctcccgtggcccagctgggtcgcgtgggcgtggtaaatgaggctagcggcagcggcgccaccaacttcagcctgc tgaagcaggccggcgacgtggaggagaaccccgggcccgaattcatgttcgtgtttctggtgctgctgcctctggtgtcca gccagtgtgtgaacctgaccaccagaacacagctgcctccagcctacaccaacagctttaccagaggcgtgtactaccccg acaaggtgttcagatccagcgtgctgcactctacccaggacctgttcctgcctttcttcagcaacgtgacctggttccacg ccatccacgtgtccggcaccaatggcaccaagagattcgacaaccccgtgctgcccttcaacgacggggtgtactttgcca gcaccgagaagtccaacatcatcagaggctggatcttcggcaccacactggacagcaagacccagagcctgctgatcgtga acaacgccaccaacgtggtcatcaaagtgtgcgagttccagttctgcaacgaccccttcctgggcgtctactaccacaaga acaacaagagctggatggaaagcgagttccgggtgtacagcagcgccaacaactgcaccttcgagtacgtgtcccagcctt tcctgatggacctggaaggcaagcagggcaacttcaagaacctgcgcgagttcgtgttcaagaacatcgacggctacttca agatctacagcaagcacacccctatcaacctcgtgcgggatctgcctcagggcttctctgctctggaacccctggtggatc tgcccatcggcatcaacatcacccggtttcagacactgctggccctgcacagaagctacctgacacctggcgatagcagca gcggatggacagctggtgccgccgcttactatgtgggctacctgcagcctagaaccttcctgctgaagtacaacgagaacg gcaccatcaccgacgccgtggattgtgcccttgatcctctgagcgagacaaagtgcaccctgaagtccttcaccgtggaaa agggcatctaccagaccagcaacttccgggtgcagcccaccgaatccatcgtgcggttccccaatatcaccaatctgtgcc ccttcggcgaggtgttcaatgccaccagattcgcctctgtgtacgcctggaaccggaagcggatcagcaattgcgtggccg actactccgtgctgtacaactccgccagcttcagcaccttcaagtgctacggcgtgtcccctaccaagctgaacgacctgt gcttcacaaacgtgtacgccgacagcttcgtgatccggggagatgaagtgcggcagattgcccctggacagacaggcaaga tcgccgactacaactacaagctgcccgacgacttcaccggctgtgtgattgcctggaacagcaacaacctggactccaaag tcggcggcaactacaattacctgtaccggctgttccggaagtccaatctgaagcccttcgagcgggacatctccaccgaga tctatcaggccggcagcaccccttgtaacggcgtggaaggcttcaactgctacttcccactgcagtcctacggctttcagcccacaaatggcgtgggctatcagccctacagagtggtggtgctgagcttcgaactgctgcatgcccctgccacagtgtgcg gccctaagaaaagcaccaatctcgtgaagaacaaatgcgtgaacttcaacttcaacggcctgaccggcacaggcgtgctga cagagagcaacaagaagttcctgccattccagcagtttggccgggatatcgccgataccacagacgccgttagagatcccc agacactggaaatcctggacatcaccccttgcagcttcggcggagtgtctgtgatcacccctggcaccaacaccagcaatc aggtggcagtgctgtaccaggacgtgaactgtaccgaagtgcccgtggccattcacgccgatcagctgacacctacatggc gggtgtactccaccggcagcaatgtgtttcagaccagagccggctgtctgatcggagccgagcacgtgaacaatagctacg agtgcgacatccccatcggcgctggcatctgtgccagctaccagacacagacaaacagccccagacgggccagatctgtgg ccagccagagcatcattgcctacacaatgtctctgggcgccgagaacagcgtggcctactccaacaactctatcgctatcc ccaccaacttcaccatcagcgtgaccacagagatcctgcctgtgtccatgaccaagaccagcgtggactgcaccatgtaca tctgcggcgattccaccgagtgctccaacctgctgctgcagtacggcagcttctgcacccagctgaatagagccctgacag ggatcgccgtggaacaggacaagaacacccaagaggtgttcgcccaagtgaagcagatctacaagacccctcctatcaagg acttcggcggcttcaatttcagccagattctgcccgatcctagcaagcccagcaagcggagcttcatcgaggacctgctgt tcaacaaagtgacactggccgacgccggcttcatcaagcagtatggcgattgtctgggcgacattgccgccagggatctga tttgcgcccagaagtttaacggactgacagtgctgcctcctctgctgaccgatgagatgatcgcccagtacacatctgccc tgctggccggcacaatcacaagcggctggacatttggagctggcgccgctctgcagatcccctttgctatgcagatggcct acagattcaacggcatcggagtgacccagaatgtgctgtacgagaaccagaagctgatcgccaaccagttcaacagcgcca tcggcaagatccaggacagcctgagcagcacagcaagcgccctgggaaagctgcaggacgtggtcaaccagaatgcccagg cactgaacaccctggtcaagcagctgtcctccaacttcggcgccatcagctctgtgctgaacgatatcctgagcagactgg accctcctgaggccgaggtgcagatcgacagactgatcacaggcagactgcagagcctccagacatacgtgacccagcagc tgatcagagccgccgagattagagcctctgccaatctggccgccaccaagatgtctgagtgtgtgctgggccagagcaaga gagtggacttttgcggcaagggctaccacctgatgagcttccctcagtctgcccctcacggcgtggtgtttctgcacgtga catacgttcccgctcaagagaagaatttcaccaccgctccagccatctgccacgacggcaaagcccactttcctagagaag gcgtgttcgtgtccaacggcacccattggttcgtgacacagcggaacttctacgagccccagatcatcaccaccgacaaca ccttcgtgtctggcaactgcgacgtcgtgatcggcattgtgaacaataccgtgtacgaccctctgcagcccgagctggaca gcttcaaagaggaactggacaagtactttaagaaccacacaagccccgacgtggacctgggcgatatcagcggaatcaatg ccagcgtcgtgaacatccagaaagagatcgaccggctgaacgaggtggccaagaatctgaacgagagcctgatcgacctgc aagaactggggaagtacgagcagtacatcaagtggccctggtacatctggctgggctttatcgccggactgattgccatcg tgatggtcacaatcatgctgtgttgcatgaccagctgctgtagctgcctgaagggctgttgtagctgtggcagctgctgca agttcgacgaggacgattctgagtgatagtagttaattaactcgcgatcgctaacttgacaattaagtatgaaggtatatg tgtcccctaagagacacactgtacatagcaaataatctatagatcaaagggctacgcaacccctgaatagtaacaaaatac aaaatcactaaaaattataaaaacagaaaaatacataaataggtatacgtgtcccctaagagacacattgtatgtaggtga taagtatagatcaaagggccgaataacccctgaatagtaacaaaatatgaaaatcaataaaaatcataaaatagaaaaacc ataaacagaagtagttcaaagggctataaaacccctgaatagtaacaaaacataaaattaataaaaatcaaatgaatacca taattggcaaacggaagagatgtaggtacttaagcttcctaaaagcagccgaactcactttgagaagtaggcatagcatac cgaactcttccacgattctccgaacccacagggacgtaggagatgttattttgtttttaatatttcaaaaaaaaaaaaaaa aaaaaagcggccgcttccgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaacatggtcatagct gtttccttgcgtattgggcgctctccgcttcctcgctcactgactcgctgcgctcggtcgttcgggtaaagcctggggtgc ctaatgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccc tgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccc tggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgt ggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacc ccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccact ggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaacta cggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttg atccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctca agaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatt atcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttg gtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgact ccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagaaccacgctc accggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctc catccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgc tacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatg atcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatc actcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactc aaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccaca tagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatc cagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaac aggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttcc gcgcacatttccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatc agctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtggc cgctacagggcgctcccattcgccattcaggctgcgcaactgttgggaagggcgtttcggtgcgggcctcttcgctattac gccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaa cgacggccagtgatttaaatacgatttaggtgacactatagatggctgcgtgagacacacgtagcctaccagtttcttact gctctactctgcaaagcaagagattaataacccatcatggatcctgtgtacgtggacatagacgctgacagcgcctttttg aaggccctgcaacgtgcgtaccccatgtttgaggtggaaccaaggcaggtcacaccgaatgaccatgctaatgctagagcg ttctcgcatctagctataaaactaatagagcaggaaattgaccccgactcaaccatcctggatatcggcagtgcgccagca aggaggatgatgtcggacaggaagtaccactgcgtctgcccgatgcgcagtgcggaagatcccgagagactcgccaattat gcgagaaagctagcatctgccgcaggaaaagtcctggacagaaacatctctggaaagatcggggacttacaagcagtaatg gccgtgccagacacggagacgccaacattctgcttacacacagacgtctcatgtagacagagagcagacgtcgctatatac caagacgtctatgctgtacacgcacccacgtcgctataccaccaggcgattaaaggggtccgagtggcgtactgggttggg ttcgacacaaccccgttcatgtacaatgccatggcgggtgcctacccctcatactcgacaaactgggcagatgagcaggta ctgaaggctaagaacataggattatgttcaacagacctgacggaaggtagacgaggcaagttgtctattatgagagggaaa aagctaaaaccgtgcgaccgtgtgctgttctcagtagggtcaacgctctacccggaaagccgca

[0205] [ SEQ ID NO: 11]

[0206] The plasmid 37_VEEVatt-B2-P2A-Spike-610578301.annotations may have the nucleic acid sequence represented by SEQ ID NO: 12: tgaactgtaccgaagtgcccgtggccattcacgccgatcagctgacacctacatggcgggtgtactccaccggcagcaatg tgtttcagaccagagccggctgtctgatcggagccgagcacgtgaacaatagctacgagtgcgacatccccatcggcgctg gcatctgtgccagctaccagacacagacaaacagccccagacgggccagatctgtggccagccagagcatcattgcctaca caatgtctctgggcgccgagaacagcgtggcctactccaacaactctatcgctatccccaccaacttcaccatcagcgtga ccacagagatcctgcctgtgtccatgaccaagaccagcgtggactgcaccatgtacatctgcggcgattccaccgagtgct ccaacctgctgctgcagtacggcagcttctgcacccagctgaatagagccctgacagggatcgccgtggaacaggacaaga acacccaagaggtgttcgcccaagtgaagcagatctacaagacccctcctatcaaggacttcggcggcttcaatttcagcc agattctgcccgatcctagcaagcccagcaagcggagcttcatcgaggacctgctgttcaacaaagtgacactggccgacg ccggcttcatcaagcagtatggcgattgtctgggcgacattgccgccagggatctgatttgcgcccagaagtttaacggac tgacagtgctgcctcctctgctgaccgatgagatgatcgcccagtacacatctgccctgctggccggcacaatcacaagcg gctggacatttggagctggcgccgctctgcagatcccctttgctatgcagatggcctacagattcaacggcatcggagtga cccagaatgtgctgtacgagaaccagaagctgatcgccaaccagttcaacagcgccatcggcaagatccaggacagcctga gcagcacagcaagcgccctgggaaagctgcaggacgtggtcaaccagaatgcccaggcactgaacaccctggtcaagcagc tgtcctccaacttcggcgccatcagctctgtgctgaacgatatcctgagcagactggaccctcctgaggccgaggtgcaga tcgacagactgatcacaggcagactgcagagcctccagacatacgtgacccagcagctgatcagagccgccgagattagag cctctgccaatctggccgccaccaagatgtctgagtgtgtgctgggccagagcaagagagtggacttttgcggcaagggct accacctgatgagcttccctcagtctgcccctcacggcgtggtgtttctgcacgtgacatacgttcccgctcaagagaaga atttcaccaccgctccagccatctgccacgacggcaaagcccactttcctagagaaggcgtgttcgtgtccaacggcaccc attggttcgtgacacagcggaacttctacgagccccagatcatcaccaccgacaacaccttcgtgtctggcaactgcgacg tcgtgatcggcattgtgaacaataccgtgtacgaccctctgcagcccgagctggacagcttcaaagaggaactggacaagt actttaagaaccacacaagccccgacgtggacctgggcgatatcagcggaatcaatgccagcgtcgtgaacatccagaaag agatcgaccggctgaacgaggtggccaagaatctgaacgagagcctgatcgacctgcaagaactggggaagtacgagcagt acatcaagtggccctggtacatctggctgggctttatcgccggactgattgccatcgtgatggtcacaatcatgctgtgtt gcatgaccagctgctgtagctgcctgaagggctgttgtagctgtggcagctgctgcaagttcgacgaggacgattctgagt gatagtagttaattaactcgcgatcgctgaatacagcagcaattggcaagctgcttacatagaactcgcggcgattggcat gccgccttaaaatttttattttattttttcttttcttttccgaatcggattttgtttttaatatttcaaaaaaaaaaaaaa aaaaaaacgcgtcgaggggaattaattcttgaagacgaaagggccaggtggcacttttcggggaaatgtgcgcggaacccc tatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattg aaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgc tcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaa cagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgc ggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactc accagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacac tgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaac tcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggc aacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgg gtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggc aactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagttta ctcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcat gaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcc tttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagct accaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttagg ccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcga taagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtg cacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcc cgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaa cgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcg gagcctatggaaaaacgccagcaacgcgagctcgatttaggtgacactatagatgggcggcgcatgagagaagcccagacc aattacctacccaaaatggagaaagttcacgttgacatcgaggaagacagcccattcctcagagctttgcagcggagcttc ccgcagtttgaggtagaagccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttcaaaa ctgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgcagaatgtattctaagcac aagtatcattgtatctgtccgatgagatgtgcggaagatccggacagattgtataagtatgcaactaagctgaagaaaaac tgtaaggaaataactgataaggaattggacaagaaaatgaaggagctggccgccgtcatgagcgaccctgacctggaaact gagactatgtgcctccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggttgac ggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggctttgacaccaccccttttatg tttaagaacttggctggagcatatccatcatactctaccaactgggccgacgaaaccgtgttaacggctcgtaacataggc ctatgcagctctgacgttatggagcggtcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaacaat gttctattctctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcacctgccgtctgtatttcac ttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacgggtacgtcgttaaaagaatagctatc agtccaggcctgtatgggaagccttcaggctatgctgctacgatgcaccgcgagggattcttgtgctgcaaagtgacagac acattgaacggggagagggtctcttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcatactg gcaacagatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacggtcgcacccag agaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatttgctaggtgggcaaaggaatataaggaa gatcaagaagatgaaaggccactaggactacgagatagacagttagtcatggggtgttgttgggcttttagaaggcacaag ataacatctatttataagcgcccggatacccaaaccatcatcaaagtgaacagcgatttccactcattcgtgctgcccagg ataggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcacaaggagccgtcacctctc attaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggctaaggaggtgcgtgaagccgaggagttgcgcgca gctctaccacctttggcagctgatgttgaggagcccactctggaggcagacgtcgacttgatgttacaagaggctggggcc ggctcagtggagacacctcgtggcttgataaaggttaccagctacgctggcgaggacaagatcggctcttacgctgtgctt tctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagtcatagtgataacacactct ggccgaaaagggcgttatgccgtggaaccataccatggtaaagtagtggtgccagagggacatgcaatacccgtccaggac tttcaagctctgagtgaaagtgccaccattgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccaca catggaggagcgctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgacggcgaatacctgtacgac atcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggcgagctggtggatcctcccttccat gaattcgcctacgagagtctgagaacacgaccagccgctccttaccaagtaccaaccataggggtgtatggcgtgccagga tcaggcaagtctggcatcattaaaagcgcagtcaccaaaaaagatctagtggtgagcgccaagaaagaaaactgtgcagaa attataagggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctcttgaatggatgcaaa caccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtactctcagagcgctcatagccattataaga cctaaaaaggcagtgctctgcggggatcccaaacagtgcggtttttttaacatgatgtgcctgaaagtgcattttaaccac gagatttgcacacaagtcttccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaaccttgttt tacgacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggcagtaccaaacctaagcag gacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaatagattacaaaggcaacgaaataatgacggca gctgcctctcaagggctgacccgtaaaggtgtgtatgccgttcggtacaaggtgaatgaaaatcctctgtacgcacccacc tcagaacatgtgaacgtcctactgacccgcacggaggaccgcatcgtgtggaaaacactagccggcgacccatggataaaa acactgactgccaagtaccctgggaatttcactgccacgatagaggagtggcaagcagagcatgatgccatcatgaggcac atcttggagagaccggaccctaccgacgtcttccagaataaggcaaacgtgtgttgggccaaggctttagtgccggtgctg aagaccgctggcatagacatgaccactgaacaatggaacactgtggattattttgaaacggacaaagctcactcagcagag atagtattgaaccaactatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgttccgtta tccattaggaataatcactgggataactccccgtcgcctaacatgtacgggctgaataaagaagtggtccgtcagctctct cgcaggtacccacaactgcctcgggcagttgccactggaagagtctatgacatgaacactggtacactgcgcaattatgat ccgcgcataaacctagtacctgtaaacagaagactgcctcatgctttagtcctccaccataatgaacacccacagagtgac ttttcttcattcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccgtcccaggcaaaatggtt gactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggcatcccaggtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaataccatcactatcagcagtgtgaagaccatgccattaagcttagcatg ttgaccaagaaagcttgtctgcatctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggccagc gaaagcatcattggtgctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcctcacttgaagagacggaa gttctgtttgtattcattgggtacgatcgcaaggcccgtacgcacaatccttacaagctttcatcaaccttgaccaacatt tatacaggttccagactccacgaagccggatgtgcaccctcatatcatgtggtgcgaggggatattgccacggccaccgaa ggagtgattataaatgctgctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaattcccggaa agcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacatatcattcatgccgtagga ccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggcagaggcttatgagtccatcgctaagattgtcaac gataacaattacaagtcagtagcgattccactgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatca ttgaaccatttgctgacagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatgggaaatgact ctcaaggaagcagtggctaggagagaagcagtggaggagatatgcatatccgacgactcttcagtgacagaacctgatgca gagctggtgagggtgcatccgaagagttctttggctggaaggaagggctacagcacaagcgatggcaaaactttctcatat ttggaagggaccaagtttcaccaggcggccaaggatatagcagaaattaatgccatgtggcccgttgcaacggaggccaat gagcaggtatgcatgtatatcctcggagaaagcatgagcagtattaggtcgaaatgccccgtcgaagagtcggaagcctcc acaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagtacagcgcctaaaagcctcacgtcca gaacaaattactgtgtgctcatcctttccattgccgaagtatagaatcactggtgtgcagaagatccaatgctcccagcct atattgttctcaccgaaagtgcctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgagactccg gagccatcggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccgaggatgagaccaggactaga acgc ct gage cgat catcat cgaagaggaagaagaggatagcataagtttg ct gtcagatggcccgacccaccaggtgctg caagtcgaggcagacattcacgggccgccctctgtatctagctcatcctggtccattcctcatgcatccgactttgatgtg gacagtttatccatacttgacaccctggagggagctagcgtgaccagcggggcaacgtcagccgagactaactcttacttc gcaaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctccacatcccgctccgcgc acaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagcctagtttccaccccgccaggcgtgaatagggtg atcactagagaggagctcgaggcgcttaccccgtcacgcactcctagcaggtcggtctcgagaaccagcctggtctccaac ccgccaggcgtaaatagggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatgacggtttgatgcgggt gcatacatcttttcctccgacaccggtcaagggcatttacaacaaaaatcagtaaggcaaacggtgctatccgaagtggtg ttggagaggaccgaattggagatttcgtatgccccgcgcctcgaccaagaaaaagaagaattactacgcaagaaattacag ttaaatcccacacctgctaacagaagcagataccagtccaggaaggtggagaacatgaaagccataacagctagacgtatt ctgcaaggcctagggcattatttgaaggcagaaggaaaagtggagtgctaccgaaccctgcatcctgttcctttgtattca tctagtgtgaaccgtgccttttcaagccccaaggtcgcagtggaagcctgtaacgccatgttgaaagagaactttccgact gtggcttcttactgtattattccagagtacgatgcctatttggacatggttgacggagcttcatgctgcttagacactgcc agtttttgccctgcaaagctgcgcagctttccaaagaaacactcctatttggaacccacaatacgatcggcagtgccttca gcgatccagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaatgtcacgcaaatgagagaattgccc gtattggatt eggegg cctttaatgtggaatgcttcaagaaatatgcgtgtaataatgaatattgggaaacgtttaaagaa aaccccatcaggcttactgaagaaaacgtggtaaattacattaccaaattaaaaggaccaaaagctgctgctctttttgcg aagacacataatttgaatatgttgcaggacataccaatggacaggtttgtaatggacttaaagagagacgtgaaagtgact ccaggaacaaaacatactgaagaacggcccaaggtacaggtgatccaggctgccgatccgctagcaacagcgtatctgtgc ggaatccaccgagagctggttaggagattaaatgcggtcctgcttccgaacattcatacactgtttgatatgtcggctgaa gactttgacgctattatagccgagcacttccagcctggggattgtgttctggaaactgacatcgcgtcgtttgataaaagt gaggacgacgccatggctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttgacgctgattgag gcggctttcggcgaaatttcatcaatacatttgcccactaaaactaaatttaaattcggagccatgatgaaatctggaatg ttcctcacactgtttgtgaacacagtcattaacattgtaatcgcaagcagagtgttgagagaacggctaaccggatcacca tgtgcagcattcattggagatgacaatatcgtgaaaggagtcaaatcggacaaattaatggcagacaggtgcgccacctgg ttgaatatggaagtcaagattatagatgctgtggtgggcgagaaagcgccttatttctgtggagggtttattttgtgtgac tccgtgaccggcacagcgtgccgtgtggcagaccccctaaaaaggctgtttaagcttggcaaacctctggcagcagacgat gaacatgatgatgacaggagaagggcattgcatgaagagtcaacacgctggaaccgagtgggtattctttcagagctgtgc aaggcagtagaatcaaggtatgaaaccgtaggaacttccatcatagttatggccatgactactctagctagcagtgttaaa tcattcagctacctgagaggggcccctataactctctacggctaacctgaatggactacgacatagtctagtccgccaagt tcgaatataggcgcgccgccaccatgacaaacatgtcatgcgcttacgagctaatcaagtcacttccagccaagctggagc agctggctcaggagacgcaagcaacgatccaaacgctcatgatcgcggatcccaacgtcaacaaggatctgcgagcgttct gcgagttcctgaccgtgcagcaccagcgggcgtatcgagcgacgaacagcctgctcatcaaaccgcgagtcgcagcagcgc ttcgcggggaggagctggacctgggcgaggcggacgtcgccgcccgggtccgccagctaaaacaacagctggcggagctcg agatggaaatcaagccagggcaccaacaagtggcccaagtaagcggcaggcggaaggccgcagccgcagctcccgtggccc agctgggtcgcgtgggcgtggtaaatgaggctagcggcagcggcgccaccaacttcagcctgctgaagcaggccggcgacg tggaggagaaccccgggcccgaattcatgttcgtgtttctggtgctgctgcctctggtgtccagccagtgtgtgaacctga ccaccagaacacagctgcctccagcctacaccaacagctttaccagaggcgtgtactaccccgacaaggtgttcagatcca gcgtgctgcactctacccaggacctgttcctgcctttcttcagcaacgtgacctggttccacgccatccacgtgtccggca ccaatggcaccaagagattcgacaaccccgtgctgcccttcaacgacggggtgtactttgccagcaccgagaagtccaacatcatcagaggctggatcttcggcaccacactggacagcaagacccagagcctgctgatcgtgaacaacgccaccaacgtgg tcatcaaagtgtgcgagttccagttctgcaacgaccccttcctgggcgtctactaccacaagaacaacaagagctggatgg aaagcgagttccgggtgtacagcagcgccaacaactgcaccttcgagtacgtgtcccagcctttcctgatggacctggaag gcaagcagggcaacttcaagaacctgcgcgagttcgtgttcaagaacatcgacggctacttcaagatctacagcaagcaca cccctatcaacctcgtgcgggatctgcctcagggcttctctgctctggaacccctggtggatctgcccatcggcatcaaca tcacccggtttcagacactgctggccctgcacagaagctacctgacacctggcgatagcagcagcggatggacagctggtg ccgccgcttactatgtgggctacctgcagcctagaaccttcctgctgaagtacaacgagaacggcaccatcaccgacgccg tggattgtgcccttgatcctctgagcgagacaaagtgcaccctgaagtccttcaccgtggaaaagggcatctaccagacca gcaacttccgggtgcagcccaccgaatccatcgtgcggttccccaatatcaccaatctgtgccccttcggcgaggtgttca atgccaccagattcgcctctgtgtacgcctggaaccggaagcggatcagcaattgcgtggccgactactccgtgctgtaca actccgccagcttcagcaccttcaagtgctacggcgtgtcccctaccaagctgaacgacctgtgcttcacaaacgtgtacg ccgacagcttcgtgatccggggagatgaagtgcggcagattgcccctggacagacaggcaagatcgccgactacaactaca agctgcccgacgacttcaccggctgtgtgattgcctggaacagcaacaacctggactccaaagtcggcggcaactacaatt acctgtaccggctgttccggaagtccaatctgaagcccttcgagcgggacatctccaccgagatctatcaggccggcagca ccccttgtaacggcgtggaaggcttcaactgctacttcccactgcagtcctacggctttcagcccacaaatggcgtgggct atcagccctacagagtggtggtgctgagcttcgaactgctgcatgcccctgccacagtgtgcggccctaagaaaagcacca atctcgtgaagaacaaatgcgtgaacttcaacttcaacggcctgaccggcacaggcgtgctgacagagagcaacaagaagt tcctgccattccagcagtttggccgggatatcgccgataccacagacgccgttagagatccccagacactggaaatcctgg acatcaccccttgcagcttcggcggagtgtctgtgatcacccctggcaccaacaccagcaatcaggtggcagtgctgtacc aggacg

[0207] [ SEQ ID NO: 12 ]

[0208] The plasmid 39_VEEVatt-RBD-P2A-B2_610578301.annotations may have the nucleic acid sequence represented by SEQ ID NO: 13: ccaccgaaggagtgattataaatgctgctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaat tcccggaaagcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacatatcattcatg ccgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggcagaggcttatgagtccatcgctaaga ttgtcaacgataacaattacaagtcagtagcgattccactgttgtccaccggcatcttttccgggaacaaagatcgactaa cccaatcattgaaccatttgctgacagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatggg aaatgactctcaaggaagcagtggctaggagagaagcagtggaggagatatgcatatccgacgactcttcagtgacagaac ctgatgcagagctggtgagggtgcatccgaagagttctttggctggaaggaagggctacagcacaagcgatggcaaaactt tctcatatttggaagggaccaagtttcaccaggcggccaaggatatagcagaaattaatgccatgtggcccgttgcaacgg aggccaatgagcaggtatgcatgtatatcctcggagaaagcatgagcagtattaggtcgaaatgccccgtcgaagagtcgg aagcctccacaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagtacagcgcctaaaagcct cacgtccagaacaaattactgtgtgctcatcctttccattgccgaagtatagaatcactggtgtgcagaagatccaatgct cccagcctatattgttctcaccgaaagtgcctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacg agactccggagccatcggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccgaggatgagacca ggactagaacgcctgagccgatcatcatcgaagaggaagaagaggatagcataagtttgctgtcagatggcccgacccacc aggtgctgcaagtcgaggcagacattcacgggccgccctctgtatctagctcatcctggtccattcctcatgcatccgact ttgatgtggacagtttatccatacttgacaccctggagggagctagcgtgaccagcggggcaacgtcagccgagactaact cttacttcgcaaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctccacatcccg ctccgcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagcctagtttccaccccgccaggcgtga atagggtgatcactagagaggagctcgaggcgcttaccccgtcacgcactcctagcaggtcggtctcgagaaccagcctgg tctccaacccgccaggcgtaaatagggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatgacggtttg atgcgggtgcatacatcttttcctccgacaccggtcaagggcatttacaacaaaaatcagtaaggcaaacggtgctatccg aagtggtgttggagaggaccgaattggagatttcgtatgccccgcgcctcgaccaagaaaaagaagaattactacgcaaga aattacagttaaatcccacacctgctaacagaagcagataccagtccaggaaggtggagaacatgaaagccataacagcta gacgtattctgcaaggcctagggcattatttgaaggcagaaggaaaagtggagtgctaccgaaccctgcatcctgttcctt tgtattcatctagtgtgaaccgtgccttttcaagccccaaggtcgcagtggaagcctgtaacgccatgttgaaagagaact ttccgactgtggcttcttactgtattattccagagtacgatgcctatttggacatggttgacggagcttcatgctgcttag acactgccagtttttgccctgcaaagctgcgcagctttccaaagaaacactcctatttggaacccacaatacgatcggcag tgccttcagcgatccagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaatgtcacgcaaatgagag aattgcccgtattggattcggcggcctttaatgtggaatgcttcaagaaatatgcgtgtaataatgaatattgggaaacgt ttaaagaaaaccccatcaggcttactgaagaaaacgtggtaaattacattaccaaattaaaaggaccaaaagctgctgctc tttttgcgaagacacataatttgaatatgttgcaggacataccaatggacaggtttgtaatggacttaaagagagacgtga aagtgactccaggaacaaaacatactgaagaacggcccaaggtacaggtgatccaggctgccgatccgctagcaacagcgtatctgtgcggaatccaccgagagctggttaggagattaaatgcggtcctgcttccgaacattcatacactgtttgatatgt cggctgaagactttgacgctattatagccgagcacttccagcctggggattgtgttctggaaactgacatcgcgtcgtttg ataaaagtgaggacgacgccatggctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttgacgc tgattgaggcggctttcggcgaaatttcatcaatacatttgcccactaaaactaaatttaaattcggagccatgatgaaat ctggaatgttcctcacactgtttgtgaacacagtcattaacattgtaatcgcaagcagagtgttgagagaacggctaaccg gatcaccatgtgcagcattcattggagatgacaatatcgtgaaaggagtcaaatcggacaaattaatggcagacaggtgcg ccacctggttgaatatggaagtcaagattatagatgctgtggtgggcgagaaagcgccttatttctgtggagggtttattt tgtgtgactccgtgaccggcacagcgtgccgtgtggcagaccccctaaaaaggctgtttaagcttggcaaacctctggcag cagacgatgaacatgatgatgacaggagaagggcattgcatgaagagtcaacacgctggaaccgagtgggtattctttcag agctgtgcaaggcagtagaatcaaggtatgaaaccgtaggaacttccatcatagttatggccatgactactctagctagca gtgttaaatcattcagctacctgagaggggcccctataactctctacggctaacctgaatggactacgacatagtctagtc cgccaagttcgaatataggcgcgccgccaccatgcggttccccaatatcaccaatctgtgccccttcggcgaggtgttcaa tgccaccagattcgcctctgtgtacgcctggaaccggaagcggatcagcaattgcgtggccgactactccgtgctgtacaa ctccgccagcttcagcaccttcaagtgctacggcgtgtcccctaccaagctgaacgacctgtgcttcacaaacgtgtacgc cgacagcttcgtgatccggggagatgaagtgcggcagattgcccctggacagacaggcaagatcgccgactacaactacaa gctgcccgacgacttcaccggctgtgtgattgcctggaacagcaacaacctggactccaaagtcggcggcaactacaatta cctgtaccggctgttccggaagtccaatctgaagcccttcgagcgggacatctccaccgagatctatcaggccggcagcac cccttgtaacggcgtggaaggcttcaactgctacttcccactgcagtcctacggctttcagcccacaaatggcgtgggcta tcagccctacagagtggtggtgctgagcttcgaactgctgcatgcccctgccacagtgtgcggccctaagaaaagcaccgc tagcggcagcggcgccaccaacttcagcctgctgaagcaggccggcgacgtggaggagaaccccgggcccgaattcatgac aaacatgtcatgcgcttacgagctaatcaagtcacttccagccaagctggagcagctggctcaggagacgcaagcaacgat ccaaacgctcatgatcgcggatcccaacgtcaacaaggatctgcgagcgttctgcgagttcctgaccgtgcagcaccagcg ggcgtatcgagcgacgaacagcctgctcatcaaaccgcgagtcgcagcagcgcttcgcggggaggagctggacctgggcga ggcggacgtcgccgcccgggtccgccagctaaaacaacagctggcggagctcgagatggaaatcaagccagggcaccaaca agtggcccaagtaagcggcaggcggaaggccgcagccgcagctcccgtggcccagctgggtcgcgtgggcgtggtaaatga gtgatagtagttaattaactcgcgatcgctgaatacagcagcaattggcaagctgcttacatagaactcgcggcgattggc atgccgccttaaaatttttattttattttttcttttcttttccgaatcggattttgtttttaatatttcaaaaaaaaaaaa aaaaaaaaaaacgcgtcgaggggaattaattcttgaagacgaaagggccaggtggcacttttcggggaaatgtgcgcggaa cccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataat attgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgttt ttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatc tcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtg gcgcggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagt actcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgata acactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatg taactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaa tggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggagg cggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagc gtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtc aggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaag tttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatc tcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgag atcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaag agctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagt taggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtg gcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggtt cgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgc ttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggg gaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagggg ggcggagcctatggaaaaacgccagcaacgcgagctcgatttaggtgacactatagatgggcggcgcatgagagaagccca gaccaattacctacccaaaatggagaaagttcacgttgacatcgaggaagacagcccattcctcagagctttgcagcggag cttcccgcagtttgaggtagaagccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttc aaaactgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgcagaatgtattctaa gcacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacagattgtataagtatgcaactaagctgaagaa aaactgtaaggaaataactgataaggaattggacaagaaaatgaaggagctggccgccgtcatgagcgaccctgacctgga aactgagactatgtgcctccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggt tgacggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggctttgacaccaccccttt tatgtttaagaacttggctggagcatatccatcatactctaccaactgggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgttatggagcggtcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaa caatgttctattctctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcacctgccgtctgtatt tcacttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacgggtacgtcgttaaaagaatagc tatcagtccaggcctgtatgggaagccttcaggctatgctgctacgatgcaccgcgagggattcttgtgctgcaaagtgac agacacattgaacggggagagggtctcttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcat actggcaacagatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacggtcgcac ccagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatttgctaggtgggcaaaggaatataa ggaagatcaagaagatgaaaggccactaggactacgagatagacagttagtcatggggtgttgttgggcttttagaaggca caagataacatctatttataagcgcccggatacccaaaccatcatcaaagtgaacagcgatttccactcattcgtgctgcc caggataggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcacaaggagccgtcacc tctcattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggctaaggaggtgcgtgaagccgaggagttgcg cgcagctctaccacctttggcagctgatgttgaggagcccactctggaggcagacgtcgacttgatgttacaagaggctgg ggccggctcagtggagacacctcgtggcttgataaaggttaccagctacgctggcgaggacaagatcggctcttacgctgt gctttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagtcatagtgataacaca ctctggccgaaaagggcgttatgccgtggaaccataccatggtaaagtagtggtgccagagggacatgcaatacccgtcca ggactttcaagctctgagtgaaagtgccaccattgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgc cacacatggaggagcgctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgacggcgaatacctgta cgacatcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggcgagctggtggatcctccctt ccatgaattcgcctacgagagtctgagaacacgaccagccgctccttaccaagtaccaaccataggggtgtatggcgtgcc aggatcaggcaagtctggcatcattaaaagcgcagtcaccaaaaaagatctagtggtgagcgccaagaaagaaaactgtgc agaaattataagggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctcttgaatggatg caaacaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtactctcagagcgctcatagccattat aagacctaaaaaggcagtgctctgcggggatcccaaacagtgcggtttttttaacatgatgtgcctgaaagtgcattttaa ccacgagatttgcacacaagtcttccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaacctt gttttacgacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggcagtaccaaacctaa gcaggacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaatagattacaaaggcaacgaaataatgac ggcagctgcctctcaagggctgacccgtaaaggtgtgtatgccgttcggtacaaggtgaatgaaaatcctctgtacgcacc cacctcagaacatgtgaacgtcctactgacccgcacggaggaccgcatcgtgtggaaaacactagccggcgacccatggat aaaaacactgactgccaagtaccctgggaatttca ct gccacgatagaggagtggcaagcagagcatgatgccat catgag gcacatcttggagagaccggaccctaccgacgtcttccagaataaggcaaacgtgtgttgggccaaggctttagtgccggt gctgaagaccgctggcatagacatgaccactgaacaatggaacactgtggattattttgaaacggacaaagctcactcagc agagatagtattgaaccaactatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgttcc gttatccattaggaataatcactgggataactccccgtcgcctaacatgtacgggctgaataaagaagtggtccgtcagct ctctcgcaggtacccacaactgcctcgggcagttgccactggaagagtctatgacatgaacactggtacactgcgcaatta tgatccgcgcataaacctagtacctgtaaacagaagactgcctcatgctttagtcctccaccataatgaacacccacagag tgacttttcttcattcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccgtcccaggcaaaat ggttgactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggcatcccaggtgatgtgcccaaata tgacataatatttgttaatgtgaggaccccatataaataccatcactatcagcagtgtgaagaccatgccattaagcttag catgttgaccaagaaagcttgtctgcatctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggc cagcgaaagcatcattggtgctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcctcacttgaagagac ggaagttctgtttgtattcattgggtacgatcgcaaggcccgtacgcacaatccttacaagctttcatcaaccttgaccaa catttatacaggttccagactccacgaagccggatgtgcaccctcatatcatgtggtgcgaggggatattgccacgg [ SEQ ID NO: 13]

[0209] The plasmid 41_VEEVatt-B2-P2A-RBD_610578301.annotations may have the nucleic acid sequence represented by SEQ ID NO: 14: caccgaaggagtgattataaatgctgctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaatt cccggaaagcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacatatcattcatgc cgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggcagaggcttatgagtccatcgctaagat tgtcaacgataacaattacaagtcagtagcgattccactgttgtccaccggcatcttttccgggaacaaagatcgactaac ccaatcattgaaccatttgctgacagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatggga aatgactctcaaggaagcagtggctaggagagaagcagtggaggagatatgcatatccgacgactcttcagtgacagaacc tgatgcagagctggtgagggtgcatccgaagagttctttggctggaaggaagggctacagcacaagcgatggcaaaacttt ctcatatttggaagggaccaagtttcaccaggcggccaaggatatagcagaaattaatgccatgtggcccgttgcaacgga ggccaatgagcaggtatgcatgtatatcctcggagaaagcatgagcagtattaggtcgaaatgccccgtcgaagagtcgga agcctccacaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagtacagcgcctaaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaagtatagaatcactggtgtgcagaagatccaatgctc ccagcctatattgttctcaccgaaagtgcctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacga gactccggagccatcggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccgaggatgagaccag gactagaacgcctgagccgatcatcatcgaagaggaagaagaggatagcataagtttgctgtcagatggcccgacccacca ggtgctgcaagtcgaggcagacattcacgggccgccctctgtatctagctcatcctggtccattcctcatgcatccgactt tgatgtggacagtttatccatacttgacaccctggagggagctagcgtgaccagcggggcaacgtcagccgagactaactc ttacttcgcaaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctccacatcccgc tccgcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagcctagtttccaccccgccaggcgtgaa tagggtgatcactagagaggagctcgaggcgcttaccccgtcacgcactcctagcaggtcggtctcgagaaccagcctggt ctccaacccgccaggcgtaaatagggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatgacggtttga tgcgggtgcatacatcttttcctccgacaccggtcaagggcatttacaacaaaaatcagtaaggcaaacggtgctatccga agtggtgttggagaggaccgaattggagatttcgtatgccccgcgcctcgaccaagaaaaagaagaattactacgcaagaa attacagttaaatcccacacctgctaacagaagcagataccagtccaggaaggtggagaacatgaaagccataacagctag acgtattctgcaaggcctagggcattatttgaaggcagaaggaaaagtggagtgctaccgaaccctgcatcctgttccttt gtattcatctagtgtgaaccgtgccttttcaagccccaaggtcgcagtggaagcctgtaacgccatgttgaaagagaactt tccgactgtggcttcttactgtattattccagagtacgatgcctatttggacatggttgacggagcttcatgctgcttaga cactgccagtttttgccctgcaaagctgcgcagctttccaaagaaacactcctatttggaacccacaatacgatcggcagt gccttcagcgatccagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaatgtcacgcaaatgagaga attgcccgtattggattcggcggcctttaatgtggaatgcttcaagaaatatgcgtgtaataatgaatattgggaaacgtt taaagaaaaccccatcaggcttactgaagaaaacgtggtaaattacattaccaaattaaaaggaccaaaagctgctgctct ttttgcgaagacacataatttgaatatgttgcaggacataccaatggacaggtttgtaatggacttaaagagagacgtgaa agtgactccaggaacaaaacatactgaagaacggcccaaggtacaggtgatccaggctgccgatccgctagcaacagcgta tctgtgcggaatccaccgagagctggttaggagattaaatgcggtcctgcttccgaacattcatacactgtttgatatgtc ggctgaagactttgacgctattatagccgagcacttccagcctggggattgtgttctggaaactgacatcgcgtcgtttga taaaagtgaggacgacgccatggctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttgacgct gattgaggcggctttcggcgaaatttcatcaatacatttgcccactaaaactaaatttaaattcggagccatgatgaaatc tggaatgttcctcacactgtttgtgaacacagtcattaacattgtaatcgcaagcagagtgttgagagaacggctaaccgg atcaccatgtgcagcattcattggagatgacaatatcgtgaaaggagtcaaatcggacaaattaatggcagacaggtgcgc cacctggttgaatatggaagtcaagattatagatgctgtggtgggcgagaaagcgccttatttctgtggagggtttatttt gtgtgactccgtgaccggcacagcgtgccgtgtggcagaccccctaaaaaggctgtttaagcttggcaaacctctggcagc agacgatgaacatgatgatgacaggagaagggcattgcatgaagagtcaacacgctggaaccgagtgggtattctttcaga gctgtgcaaggcagtagaatcaaggtatgaaaccgtaggaacttccatcatagttatggccatgactactctagctagcag tgttaaatcattcagctacctgagaggggcccctataactctctacggctaacctgaatggactacgacatagtctagtcc gccaagttcgaatataggcgcgccgccaccatgacaaacatgtcatgcgcttacgagctaatcaagtcacttccagccaag ctggagcagctggctcaggagacgcaagcaacgatccaaacgctcatgatcgcggatcccaacgtcaacaaggatctgcga gcgttctgcgagttcctgaccgtgcagcaccagcgggcgtatcgagcgacgaacagcctgctcatcaaaccgcgagtcgca gcagcgcttcgcggggaggagctggacctgggcgaggcggacgtcgccgcccgggtccgccagctaaaacaacagctggcg gagctcgagatggaaatcaagccagggcaccaacaagtggcccaagtaagcggcaggcggaaggccgcagccgcagctccc gtggcccagctgggtcgcgtgggcgtggtaaatgaggctagcggcagcggcgccaccaacttcagcctgctgaagcaggcc ggcgacgtggaggagaaccccgggcccgaattcatgcggttccccaatatcaccaatctgtgccccttcggcgaggtgttc aatgccaccagattcgcctctgtgtacgcctggaaccggaagcggatcagcaattgcgtggccgactactccgtgctgtac aactccgccagcttcagcaccttcaagtgctacggcgtgtcccctaccaagctgaacgacctgtgcttcacaaacgtgtac gccgacagcttcgtgatccggggagatgaagtgcggcagattgcccctggacagacaggcaagatcgccgactacaactac aagctgcccgacgacttcaccggctgtgtgattgcctggaacagcaacaacctggactccaaagtcggcggcaactacaat tacctgtaccggctgttccggaagtccaatctgaagcccttcgagcgggacatctccaccgagatctatcaggccggcagc accccttgtaacggcgtggaaggcttcaactgctacttcccactgcagtcctacggctttcagcccacaaatggcgtgggc tatcagccctacagagtggtggtgctgagcttcgaactgctgcatgcccctgccacagtgtgcggccctaagaaaagcacc tgatagtagttaattaactcgcgatcgctgaatacagcagcaattggcaagctgcttacatagaactcgcggcgattggca tgccgccttaaaatttttattttattttttcttttcttttccgaatcggattttgtttttaatatttcaaaaaaaaaaaaa aaaaaaaaaacgcgtcgaggggaattaattcttgaagacgaaagggccaggtggcacttttcggggaaatgtgcgcggaac ccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataata ttgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttt tgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatct caacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtgg cgcggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagta ctcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataa cactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgt aactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggc ggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcg tgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtca ggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagt ttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatct catgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgaga tcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaaga gctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagtt aggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtgg cgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttc gtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgct tcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggggg aaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggg gcggagcctatggaaaaacgccagcaacgcgagctcgatttaggtgacactatagatgggcggcgcatgagagaagcccag accaattacctacccaaaatggagaaagttcacgttgacatcgaggaagacagcccattcctcagagctttgcagcggagc ttcccgcagtttgaggtagaagccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttca aaactgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgcagaatgtattctaag cacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacagattgtataagtatgcaactaagctgaagaaa aactgtaaggaaataactgataaggaattggacaagaaaatgaaggagctggccgccgtcatgagcgaccctgacctggaa actgagactatgtgcctccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggtt gacggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggctttgacaccacccctttt atgtttaagaacttggctggagcatatccatcatactctaccaactgggccgacgaaaccgtgttaacggctcgtaacata ggcctatgcagctctgacgttatggagcggtcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaac aatgttctattctctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcacctgccgtctgtattt cacttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacgggtacgtcgttaaaagaatagct atcagtccaggcctgtatgggaagccttcaggctatgctgctacgatgcaccgcgagggattcttgtgctgcaaagtgaca gacacattgaacggggagagggtctcttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcata ctggcaacagatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacggtcgcacc cagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatttgctaggtgggcaaaggaatataag gaagatcaagaagatgaaaggccactaggactacgagatagacagttagtcatggggtgttgttgggcttttagaaggcac aagataacatctatttataagcgcccggatacccaaaccatcatcaaagtgaacagcgatttccactcattcgtgctgccc aggataggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcacaaggagccgtcacct ctcattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggctaaggaggtgcgtgaagccgaggagttgcgc gcagctctaccacctttggcagctgatgttgaggagcccactctggaggcagacgtcgacttgatgttacaagaggctggg gccggctcagtggagacacctcgtggcttgataaaggttaccagctacgctggcgaggacaagatcggctcttacgctgtg ctttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagtcatagtgataacacac tctggccgaaaagggcgttatgccgtggaaccataccatggtaaagtagtggtgccagagggacatgcaatacccgtccag gactttcaagctctgagtgaaagtgccaccattgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgcc acacatggaggagcgctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgacggcgaatacctgtac gacatcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggcgagctggtggatcctcccttc catgaattcgcctacgagagtctgagaacacgaccagccgctccttaccaagtaccaaccataggggtgtatggcgtgcca ggatcaggcaagtctggcatcattaaaagcgcagtcaccaaaaaagatctagtggtgagcgccaagaaagaaaactgtgca gaaattataagggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctcttgaatggatgc aaacaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtactctcagagcgctcatagccattata agacctaaaaaggcagtgctctgcggggatcccaaacagtgcggtttttttaacatgatgtgcctgaaagtgcattttaac cacgagatttgcacacaagtcttccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaaccttg ttttacgacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggcagtaccaaacctaag caggacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaatagattacaaaggcaacgaaataatgacg gcagctgcctctcaagggctgacccgtaaaggtgtgtatgccgttcggtacaaggtgaatgaaaatcctctgtacgcaccc acctcagaacatgtgaacgtcctactgacccgcacggaggaccgcatcgtgtggaaaacactagccggcgacccatggata aaaacactgactgccaagtaccctgggaatttcactgccacgatagaggagtggcaagcagagcatgatgccatcatgagg cacatcttggagagaccggaccctaccgacgtcttccagaataaggcaaacgtgtgttgggccaaggctttagtgccggtg ctgaagaccgctggcatagacatgaccactgaacaatggaacactgtggattattttgaaacggacaaagctcactcagca gagatagtattgaaccaactatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgttccg ttatccattaggaataatcactgggataactccccgtcgcctaacatgtacgggctgaataaagaagtggtccgtcagctc tctcgcaggtacccacaactgcctcgggcagttgccactggaagagtctatgacatgaacactggtacactgcgcaattat gatccgcgcataaacctagtacctgtaaacagaagactgcctcatgctttagtcctccaccataatgaacacccacagagt gacttttcttcattcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccgtcccaggcaaaatggttgactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggcatcccaggtgatgtgcccaaatat gacataatatttgttaatgtgaggaccccatataaataccatcactatcagcagtgtgaagaccatgccattaagcttagc atgttgaccaagaaagcttgtctgcatctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggcc agcgaaagcatcattggtgctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcctcacttgaagagacg gaagttctgtttgtattcattgggtacgatcgcaaggcccgtacgcacaatccttacaagctttcatcaaccttgaccaac atttatacaggttccagactccacgaagccggatgtgcaccctcatatcatgtggtgcgaggggatattgccacggc [ SEQ ID NO: 14 ]

[0210] The plasmid 43_CHIKV-ERR-B2-P2A-GFP_610921701_30BG2. annotations may have the nucleic acid sequence represented by SEQ ID NO: 15: gtgcggtacgtatccagtaatccacgctgttggaccaaacttctctaattattcggagtctgaaggggaccgggaattggc agctgcctatcgagaagtcgcaaaggaagtaactaggctgggagtaaatagtgtagctatacctctcctctccacaggtgt atactcaggagggaaagacaggctgacccagtcactgaaccacctctttacagccatggactcgacggatgcagacgtggt catctactgccgcgacaaagaatgggagaagaaaatatctgaggccatacagatgcggacccaagtagagctgctggatga gcacatctccatagactgcgatattgttcgcgtgcaccctgacagcagcttggcaggcagaaaaggatacagcaccacgga aggcgcactgtactcatatctagaagggacccgttttcatcagacggctgtggatatggcggagatacatactatgtggcc aaagcaaacagaggccaatgagcaagtctgcctatatgccctgggggaaagtattgaatcgatcaggcagaaatgcccggt ggatgatgcagacgcatcatctccccccaaaactgtcccgtgcctttgccgttacgctatgactccagaacgcgtcacccg gcttcgcatgaaccacgtcacaagcataattgtgtgttcttcgtttcccctcccaaagtacaaaatagaaggagtgcaaaa agtcaaatgctctaaggtaatgctatttgaccacaacgtgccatcgcgcgtaagtccaagggaatatagatcttcccagga gtctgcacaggaggcgagtacaatcacgtcactgacgcatagtcaattcgacctaagcgttgatggcgagatactgcccgt cccgtcagacctggatgctgacgccccagccctagaaccagcactagacgacggggcgacacacacgctgccatccacaac cggaaaccttgcggccgtgtctgattgggtaatgagcaccgtacctgtcgcgccgcccagaagaaggcgagggagaaacct gactgtgacatgtgacgagagagaagggaatataacacccatggctagcgtccgattctttagggcagagctgtgtccggt cgtacaagaaacagcggagacgcgtgacacagcaatgtctcttcaggcaccaccgagtaccgccacggaaccgaatcatcc gccgatctccttcggagcatcaagcgagacgttccccattacatttggggacttcaacgaaggagaaatcgaaagcttgtc ttctgagctactaactttcggagacttcttaccaggagaagtggatgacttgacagacagcgactggtccacgtgctcaga cacggacgacgagttaagactagacagggcaggtgggtatatattctcgtcggacaccggtccaggtcatttacaacagaa gtcagtacgccagtcagtgctgccggtgaacaccctggaggaagtccacgaggagaagtgttacccacctaagctggatga agcaaaggagcaactattacttaagaaactccaggagagtgcatccatggccaacagaagcaggtatcagtcgcgcaaagt agaaaacatgaaagcagcaatcatccagagactaaagagaggctgtagactatacttaatgtcagagaccccaaaagtccc tacttaccggactacatatccggcgcctgtgtactcgcctccgatcaacgtccgattgtccaatcccgagtccgcagtggc agcatgcaatgagttcttagctagaaactatccaactgtctcatcataccaaattaccgacgagtatgatgcatatctaga catggtggacgggtcggagagttgcctggaccgagcgacattcaatccgtcaaaactcaggagctacccgaaacagcacgc ttaccacgcgccctccatcagaagcgctgtaccgtccccattccagaacacactacagaatgtactggcagcagccacgaa aagaaactgcaacgtcacacagatgagggaattacccactttggactcagcagtattcaacgtggagtgtttcaaaaaatt cgcatgcaaccaagaatactgggaagaatttgctgccagccctattaggataacaactgagaatttagcaacctatgttac taaactaaaagggccaaaagcagcagcgctattcgcaaaaacccataatctactgccactacaggaagtaccaatggatag gttcacagtagatatgaaaagggacgtaaaggtgactcctggtacaaagcatacagaggaaagacctaaggtgcaggttat acaggcggctgaacccttggcgacagcatacctatgtgggattcacagagagctggttaggaggctgaacgccgtcctcct acccaatgtacatacactatttgacatgtctgccgaggatttcgatgccatcatagccgcacactttaagccaggagacac tgttttggaaacggacatagcctcctttgataagagccaagatgattcacttgcgcttactgctttgatgctgttagagga tttaggggtggatcactccctgctggacttgatagaggctgctttcggagagatttccagctgtcacctaccgacaggtac gcgcttcaagttcggcgccatgatgaaatcaggtatgttcctaactctgttcgtcaacacattgttaaacatcaccatcgc cagccgagtgctggaagatcgtctgacaaaatccgcgtgcgcggccttcatcggcgacgacaacataatacatggagtcgt ctccgatgaattgatggcagccagatgtgccacttggatgaacatggaagtgaagatcatagatgcagttgtatccttgaa agccccttacttttgtggagggtttatactgcacgatactgtgacaggaacagcttgcagagtggcagacccgctaaaaag gctttttaaactgggcaaaccgctagcggcaggtgacgaacaagatgaagatagaagacgagcgctggctgacgaagtgat cagatggcaacgaacagggctaattgatgagctggagaaagcggtatactctaggtacgaagtgcagggtatatcagttgt ggtaatgtccatggccacctttgcaagctccagatccaacttcgagaagctcagaggacccgtcataactttgtacggcgg tcctaaataggtacgcactacagctacctattttgcagaagccgacagcaagtatctaaacactaatcagctacctaggaa gttcgaatataggcgcgccgccaccatgacaaacatgtcatgcgcttacgagctaatcaagtcacttccagccaagctgga gcagctggctcaggagacgcaagcaacgatccaaacgctcatgatcgcggatcccaacgtcaacaaggatctgcgagcgtt ctgcgagttcctgaccgtgcagcaccagcgggcgtatcgagcgacgaacagcctgctcatcaaaccgcgagtcgcagcagc gcttcgcggggaggagctggacctgggcgaggcggacgtcgccgcccgggtccgccagctaaaacaacagctggcggagct cgagatggaaatcaagccagggcaccaacaagtggcccaagtaagcggcaggcggaaggccgcagccgcagctcccgtggcccagctgggtcgcgtgggcgtggtaaatgaggctagcggcagcggcgccaccaacttcagcctgctgaagcaggccggcga cgtggaggagaaccccgggcccgaattcatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcga gctggacggcgacgtaaacggccacaagttcagcgtccgcggcgagggcgagggcgatgccaccaacggcaagctgaccct gaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttaggctacggcgtggcctgctt cgcccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccat ctctttcaaggacgacggtacctacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgtgct gaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaacttcaacagccacaaggtctatat cacggccgacaagcagaagaacggcatcaaggctaacttcaagacccgccacaacgttgaggacggcggcgtgcagctcgc cgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagccatcagtccaa actgagcaaagaccccaacgagaagcgcgatcacatggtcctgaaggagagggtgaccgccgccgggattacacatgacat ggacgagctgtacaagtgatagtagttaattaactcgcgatcgctaacttgacaattaagtatgaaggtatatgtgtcccc taagagacacactgtacatagcaaataatctatagatcaaagggctacgcaacccctgaatagtaacaaaatacaaaatca ctaaaaattataaaaacagaaaaatacataaataggtatacgtgtcccctaagagacacattgtatgtaggtgataagtat agatcaaagggccgaataacccctgaatagtaacaaaatatgaaaatcaataaaaatcataaaatagaaaaaccataaaca gaagtagttcaaagggctataaaacccctgaatagtaacaaaacataaaattaataaaaatcaaatgaataccataattgg caaacggaagagatgtaggtacttaagcttcctaaaagcagccgaactcactttgagaagtaggcatagcataccgaactc ttccacgattctccgaacccacagggacgtaggagatgttattttgtttttaatatttcaaaaaaaaaaaaaaaaaaaaaa gcggccgcttccgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaacatggtcatagctgtttcc ttgcgtattgggcgctctccgcttcctcgctcactgactcgctgcgctcggtcgttcgggtaaagcctggggtgcctaatg agcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacga gcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaag ctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgct ttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgt tcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagc agccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggcta cactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccgg caaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaaga tcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaa aaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctga cagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgt cgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagaaccacgctcaccggc tccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatcca gtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacagg catcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccc catgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcat ggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaa gtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcag aactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttc gatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaag gcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaag catttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcac atttccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctca ttttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtggccgctac agggcgctcccattcgccattcaggctgcgcaactgttgggaagggcgtttcggtgcgggcctcttcgctattacgccagc tggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacgg ccagtgatttaaatacgatttaggtgacactatagatggctgcgtgagacacacgtagcctaccagtttcttactgctcta ctctgcaaagcaagagattaataacccatcatggatcctgtgtacgtggacatagacgctgacagcgcctttttgaaggcc ctgcaacgtgcgtaccccatgtttgaggtggaaccaaggcaggtcacaccgaatgaccatgctaatgctagagcgttctcg catctagctataaaactaatagagcaggaaattgaccccgactcaaccatcctggatatcggcagtgcgccagcaaggagg atgatgtcggacaggaagtaccactgcgtctgcccgatgcgcagtgcggaagatcccgagagactcgccaattatgcgaga aagctagcatctgccgcaggaaaagtcctggacagaaacatctctggaaagatcggggacttacaagcagtaatggccgtg ccagacacggagacgccaacattctgcttacacacagacgtctcatgtagacagagagcagacgtcgctatataccaagac gtctatgctgtacacgcacccacgtcgctataccaccaggcgattaaaggggtccgagtggcgtactgggttgggttcgac acaaccccgttcatgtacaatgccatggcgggtgcctacccctcatactcgacaaactgggcagatgagcaggtactgaag gctaagaacataggattatgttcaacagacctgacggaaggtagacgaggcaagttgtctattatgagagggaaaaagcta aaaccgtgcgaccgtgtgctgttctcagtagggtcaacgctctacccggaaagccgcaagctacttaagagctggcacctg ccatcggtgttccatttaaagggcaaactcagcttcacatgccgctgtgatacagtggtttcgtgtgagggctacgtcgtt aagagaataacgatgagcccaggcctttatggaaaaaccacagggtatgcggtaacccaccacgcagacggattcctgatgtgcaagactaccgacacggttgacggcgaaagaatgtcattctcggtgtgcacatacgtgccggcgaccatttgtgatcaa atgaccggcatccttgctacagaagtcacgccggaggatgcacagaagctgttggtggggctgaaccagagaatagtggtt aacggcagaacgcaacggaatacgaacaccatgaaaaattatctgcttcccgtggtcgcccaagccttcagtaagtgggca aaggagtgccggaaagacatggaagatgaaaaactcctgggggtcagagaaagaacactgacctgctgctgtctatgggca ttcaagaagcagaaaacacacacggtctacaagaggcctgatacccagtcaattcagaaggttcaggccgagtttgacagc tttgtggtaccgagtctgtggtcgtccgggttgtcaatccctttgaggactagaatcaaatggttgttaagcaaggtgcca aaaaccgacctgatcccatacagcggagacgcccgagaagcccgggacgcagaaaaagaagcagaggaagaacgagaagca gaactgactcgcgaagccctaccacctctacaggcagcacaggaagatgttcaggtcgaaatcgacgtggaacagcttgag gacagagcgggcgcaggaataatagagactccgagaggagctatcaaagttactgcccaaccaacagaccacgtcgtggga gagtacctggtactctccccgcagaccgtactacgtagccagaagctcagtctgattcacgctttggcggagcaagtgaag acgtgcacgcacaacggacgagcagggaggtatgcggtcgaagcgtacgacggccgagtcctagtgccctcaggctatgca atctcgcctgaagacttccagagtctaagcgaaagcgcaacgatggtgtataacgaaagagagttcgtaaacagaaagcta caccatattgcgatgcacggaccagccctgaacaccgacgaagagtcgtatgagctggtgagggcagagaggacagaacac gagtacgtctacgacgtggatcagagaagatgctgtaagaaggaagaagccgcaggactggtactggtgggcgacttgact aatccgccctaccacgaattcgcatatgaagggctaaaaatccgccctgcctgcccatacaaaattgcagtcataggagtc ttcggagtaccgggatctggcaagtcagctattatcaagaacctagttaccaggcaggacctggtgactagcggaaagaaa gaaaactgccaagaaatcaccaccgacgtgatgagacagagaggtctagagatatctgcacgtacggttgactcgctgctc ttgaatggatgcaacagaccagtcgacgtgttgtacgtagacgaggcgtttgcgtgccactctggaacgctacttgctttg atcgccttggtgagaccaaggcagaaagttgtactttgtggtgacccgaagcagtgcggcttcttcaatatgatgcagatg aaagtcaactataatcacaacatctgcacccaagtgtaccacaaaagtatctccaggcggtgtacactgcctgtgaccgcc attgtgtcatcgttgcattacgaaggcaaaatgcgcactacgaatgagtacaacaagccgattgtagtggacactacaggc tcaacaaaacctgaccctggagacctcgtgttaacgtgcttcagagggtgggttaaacaactgcaaattgactatcgtgga tacgaggtcatgacagcagccgcatcccaagggttaaccagaaaaggagtttacgcagttagacaaaaagttaatgaaaac ccgctctatgcatcaacgtcagagcacgtcaacgtactcctaacgcgtacggaaggtaaactggtatggaagacactttcc ggcgacccgtggataaagacgctgcagaacccaccgaaaggaaacttcaaagcaactattaaggagtgggaggtggagcat gcatcaataatggcgggcatctgcagtcaccaaatgaccttcgatacattccaaaataaagccaacgtttgttgggctaag agcttggtccctatcctcgaaacagcggggataaaactaaatgataggcagtggtctcagataattcaagccttcaaagaa gacaaagcatactcacctgaagtagccctgaatgaaatatgtacgcgcatgtatggggtggatctagacagcgggctattt tctaaaccgttggtgtctgtgtattacgcggataaccactgggataataggcctggagggaaaatgttcggatttaacccc gaggcagcatccattctagaaagaaagtatccattcacaaaagggaagtggaacatcaacaagcagatctgcgtgactacc aggaggatagaagactttaaccctaccaccaacatcataccggccaacaggagactaccacactcattagtggccgaacac cgcccagtaaaaggggaaagaatggaatggctggttaacaagataaacggccaccacgtgctcctggtcagtggctataac cttgcactgcctactaagagagtcacttgggtagcgccgttaggtgtccgcggagcggactacacatacaacctagagttg ggtctgccagagagaaggggtaggtatgacctagtggtcataaacatccacacaccttttcgcatacaccattaccaacag tgcgtcgaccacgcaatgaaactgcaaatgctcgggggtgactcattgagactgctcaaaccgggcggctctctattgatc agagcatatggttacgcagatagaaccagtgaacgagtcatctgcgtattgggacgcaagtttagatcgtctagagcgttg aaaccaccatgtgtcaccagcaacactgagatgtttttcctattcagcaactttgacaatggcagaaggaatttcacaact catgtcatgaacaatcaactgaatgcagccttcgtaggacaggtcacccgagcaggatgtgcaccgtcgtaccgggtaaaa cgcatggacatcgcgaagaacgatgaagagtgcgtagtcaacgccgctaaccctcgcgggttaccgggtggcggtgtttgc aaggcagtatacaaaaaatggccggagtcctttaagaacagtgcaacaccagtgggaaccgcaaaaacagttat [ SEQ ID NO: 15]

[0211] The plasmid 45_VEEVatt-B2-P2A-GFP_611089101_84. annotations may have the nucleic acid sequence represented by SEQ ID NO: 16: accctaccgacgtcttccagaataaggcaaacgtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatag acatgaccactgaacaatggaacactgtggattattttgaaacggacaaagctcactcagcagagatagtattgaaccaac tatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgttccgttatccattaggaataatc actgggataactccccgtcgcctaacatgtacgggctgaataaagaagtggtccgtcagctctctcgcaggtacccacaac tgcctcgggcagttgccactggaagagtctatgacatgaacactggtacactgcgcaattatgatccgcgcataaacctag tacctgtaaacagaagactgcctcatgctttagtcctccaccataatgaacacccacagagtgacttttcttcattcgtca gcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccgtcccaggcaaaatggttgactggttgtcagacc ggcctgaggctaccttcagagctcggctggatttaggcatcccaggtgatgtgcccaaatatgacataatatttgttaatg tgaggaccccatataaataccatcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagctt gtctgcatctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggccagcgaaagcatcattggtg ctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcctcacttgaagagacggaagttctgtttgtattca ttgggtacgatcgcaaggcccgtacgcacaatccttacaagctttcatcaaccttgaccaacatttatacaggttccagactccacgaagccggatgtgcaccctcatatcatgtggtgcgaggggatattgccacggccaccgaaggagtgattataaatg ctgctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaattcccggaaagcttcgatttacagc cgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacatatcattcatgccgtaggaccaaacttcaacaaag tttcggaggttgaaggtgacaaacagttggcagaggcttatgagtccatcgctaagattgtcaacgataacaattacaagt cagtagcgattccactgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaaccatttgctga cagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatgggaaatgactctcaaggaagcagtgg ctaggagagaagcagtggaggagatatgcatatccgacgactcttcagtgacagaacctgatgcagagctggtgagggtgc atccgaagagttctttggctggaaggaagggctacagcacaagcgatggcaaaactttctcatatttggaagggaccaagt ttcaccaggcggccaaggatatagcagaaattaatgccatgtggcccgttgcaacggaggccaatgagcaggtatgcatgt atatcctcggagaaagcatgagcagtattaggtcgaaatgccccgtcgaagagtcggaagcctccacaccacctagcacgc tgccttgcttgtgcatccatgccatgactccagaaagagtacagcgcctaaaagcctcacgtccagaacaaattactgtgt gctcatcctttccattgccgaagtatagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccga aagtgcctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgagactccggagccatcggcagaga ac ca atccacagagggga ca cctgaacaac caeca ct tataaccgaggatgagaccaggactagaacgc ct gage cgatca tcatcgaagaggaagaagaggatagcataagtttgctgtcagatggcccgacccaccaggtgctgcaagtcgaggcagaca ttcacgggccgccctctgtatctagctcatcctggtccattcctcatgcatccgactttgatgtggacagtttatccatac ttgacaccctggagggagctagcgtgaccagcggggcaacgtcagccgagactaactcttacttcgcaaagagtatggagt ttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctccacatcccgctccgcgcacaagaacaccgtcac ttgcacccagcagggcctgctcgagaaccagcctagtttccaccccgccaggcgtgaatagggtgatcactagagaggagc tcgaggcgcttaccccgtcacgcactcctagcaggtcggtctcgagaaccagcctggtctccaacccgccaggcgtaaata gggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatgacggtttgatgcgggtgcatacatcttttcct ccgacaccggtcaagggcatttacaacaaaaatcagtaaggcaaacggtgctatccgaagtggtgttggagaggaccgaat tggagatttcgtatgccccgcgcctcgaccaagaaaaagaagaattactacgcaagaaattacagttaaatcccacacctg ctaacagaagcagataccagtccaggaaggtggagaacatgaaagccataacagctagacgtattctgcaaggcctagggc attatttgaaggcagaaggaaaagtggagtgctaccgaaccctgcatcctgttcctttgtattcatctagtgtgaaccgtg ccttttcaagccccaaggtcgcagtggaagcctgtaacgccatgttgaaagagaactttccgactgtggcttcttactgta ttattccagagtacgatgcctatttggacatggttgacggagcttcatgctgcttagacactgccagtttttgccctgcaa agctgcgcagctttccaaagaaacactcctatttggaacccacaatacgatcggcagtgccttcagcgatccagaacacgc tc ca ga aegt cctggcag ctgccacaaaaagaaattgca at gtea eg caaatgagagaattgcccgtattggatt eggegg cctttaatgtggaatgcttcaagaaatatgcgtgtaataatgaatattgggaaacgtttaaagaaaaccccatcaggctta ctgaagaaaacgtggtaaattacattaccaaattaaaaggaccaaaagctgctgctctttttgcgaagacacataatttga atatgttgcaggacataccaatggacaggtttgtaatggacttaaagagagacgtgaaagtgactccaggaacaaaacata ctgaagaacggcccaaggtacaggtgatccaggctgccgatccgctagcaacagcgtatctgtgcggaatccaccgagagc tggttaggagattaaatgcggtcctgcttccgaacattcatacactgtttgatatgtcggctgaagactttgacgctatta tagccgagcacttccagcctggggattgtgttctggaaactgacatcgcgtcgtttgataaaagtgaggacgacgccatgg ctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttgacgctgattgaggcggctttcggcgaaa tttcatcaatacatttgcccactaaaactaaatttaaattcggagccatgatgaaatctggaatgttcctcacactgtttg tgaacacagtcattaacattgtaatcgcaagcagagtgttgagagaacggctaaccggatcaccatgtgcagcattcattg gagatgacaatatcgtgaaaggagtcaaatcggacaaattaatggcagacaggtgcgccacctggttgaatatggaagtca agattatagatgctgtggtgggcgagaaagcgccttatttctgtggagggtttattttgtgtgactccgtgaccggcacag cgtgccgtgtggcagaccccctaaaaaggctgtttaagcttggcaaacctctggcagcagacgatgaacatgatgatgaca ggagaagggcattgcatgaagagtcaacacgctggaaccgagtgggtattctttcagagctgtgcaaggcagtagaatcaa ggtatgaaaccgtaggaacttccatcatagttatggccatgactactctagctagcagtgttaaatcattcagctacctga gaggggcccctataactctctacggctaacctgaatggactacgacatagtctagtccgccaagttcgaatataggcgcgc cgccaccatgacaaacatgtcatgcgcttacgagctaatcaagtcacttccagccaagctggagcagctggctcaggagac gcaagcaacgatccaaacgctcatgatcgcggatcccaacgtcaacaaggatctgcgagcgttctgcgagttcctgaccgt gcagcaccagcgggcgtatcgagcgacgaacagcctgctcatcaaaccgcgagtcgcagcagcgcttcgcggggaggagct ggacctgggcgaggcggacgtcgccgcccgggtccgccagctaaaacaacagctggcggagctcgagatggaaatcaagcc agggcaccaacaagtggcccaagtaagcggcaggcggaaggccgcagccgcagctcccgtggcccagctgggtcgcgtggg cgtggtaaatgaggctagcggcagcggcgccaccaacttcagcctgctgaagcaggccggcgacgtggaggagaaccccgg gcccgaattcatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaa cggccacaagttcagcgtccgcggcgagggcgagggcgatgccaccaacggcaagctgaccctgaagttcatctgcaccac cggcaagctgcccgtgccctggcccaccctcgtgaccaccttaggctacggcgtggcctgcttcgcccgctaccccgacca catgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatctctttcaaggacgacgg tacctacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgtgctgaagggcatcgacttcaa ggaggacggcaacatcctggggcacaagctggagtacaacttcaacagccacaaggtctatatcacggccgacaagcagaa ga aegg ca tcaa gg ct aacttcaagacc eg ccacaacgttgagga eggegg eg tgcagctcgccgaccactaccagcagaa cacccc categg cgacggccccgtgctgctgcccgacaaccactacctgagccatcagtccaaactgagcaaagaccccaacgagaagcgcgatcacatggtcctgaaggagagggtgaccgccgccgggattacacatgacatggacgagctgtacaagtg atagtagttaattaactcgcgatcgctgaatacagcagcaattggcaagctgcttacatagaactcgcggcgattggcatg ccgccttaaaatttttattttattttttcttttcttttccgaatcggattttgtttttaatatttcaaaaaaaaaaaaaaa aaaaaaaacgcgtcgaggggaattaattcttgaagacgaaagggccaggtggcacttttcggggaaatgtgcgcggaaccc ctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatatt gaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttg ctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctca acagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcg cggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtact caccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataaca ctgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaa ctcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatgg caacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcgg ataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtg ggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcagg caactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagttt actcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctca tgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatc ctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagc taccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttag gccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcg ataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgt gcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttc ccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaa acgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggc ggagcctatggaaaaacgccagcaacgcgagctcgatttaggtgacactatagatgggcggcgcatgagagaagcccagac caattacctacccaaaatggagaaagttcacgttgacatcgaggaagacagcccattcctcagagctttgcagcggagctt cccgcagtttgaggtagaagccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttcaaa actgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgcagaatgtattctaagca caagtatcattgtatctgtccgatgagatgtgcggaagatccggacagattgtataagtatgcaactaagctgaagaaaaa ctgtaaggaaataactgataaggaattggacaagaaaatgaaggagctggccgccgtcatgagcgaccctgacctggaaac tgagactatgtgcctccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggttga cggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggctttgacaccaccccttttat gtttaagaacttggctggagcatatccatcatactctaccaactgggccgacgaaaccgtgttaacggctcgtaacatagg cctatgcagctctgacgttatggagcggtcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaacaa tgttctattctctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcacctgccgtctgtatttca cttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacgggtacgtcgttaaaagaatagctat cagtccaggcctgtatgggaagccttcaggctatgctgctacgatgcaccgcgagggattcttgtgctgcaaagtgacaga cacattgaacggggagagggtctcttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcatact ggcaacagatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacggtcgcaccca gagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatttgctaggtgggcaaaggaatataagga agatcaagaagatgaaaggccactaggactacgagatagacagttagtcatggggtgttgttgggcttttagaaggcacaa gataacatctatttataagcgcccggatacccaaaccatcatcaaagtgaacagcgatttccactcattcgtgctgcccag gataggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcacaaggagccgtcacctct cattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggctaaggaggtgcgtgaagccgaggagttgcgcgc agctctaccacctttggcagctgatgttgaggagcccactctggaggcagacgtcgacttgatgttacaagaggctggggc cggctcagtggagacacctcgtggcttgataaaggttaccagctacgctggcgaggacaagatcggctcttacgctgtgct ttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagtcatagtgataacacactc tggccgaaaagggcgttatgccgtggaaccataccatggtaaagtagtggtgccagagggacatgcaatacccgtccagga ctttcaagctctgagtgaaagtgccaccattgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccac acatggaggagcgctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgacggcgaatacctgtacga catcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggcgagctggtggatcctcccttcca tgaattcgcctacgagagtctgagaacacgaccagccgctccttaccaagtaccaaccataggggtgtatggcgtgccagg atcaggcaagtctggcatcattaaaagcgcagtcaccaaaaaagatctagtggtgagcgccaagaaagaaaactgtgcaga aattataagggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctcttgaatggatgcaa acaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtactctcagagcgctcatagccattataag acctaaaaaggcagtgctctgcggggatcccaaacagtgcggtttttttaacatgatgtgcctgaaagtgcattttaacca cgagatttgcacacaagtcttccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaaccttgttttacgacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggcagtaccaaacctaagca ggacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaatagattacaaaggcaacgaaataatgacggc agctgcctctcaagggctgacccgtaaaggtgtgtatgccgttcggtacaaggtgaatgaaaatcctctgtacgcacccac ctcagaacatgtgaacgtcctactgacccgcacggaggaccgcatcgtgtggaaaacactagccggcgacccatggataaa aacactgactgccaagtaccctgggaatttcactgccacgatagaggagtggcaagcagagcatgatgccatcatgaggca catcttggagagaccgg

[0212] [ SEQ ID NO: 16]

[0213] The plasmid 49_CHIKV-ERR-B2-P2A-HA-influenza_611815201_1.annotations may have the nucleic acid sequence represented by SEQ ID NO: 17: ttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggc gacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcat gagegg atacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctaaattgta agcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaa atcccttataaatcaaaagaatagaccgagatagggttgagtggccgctacagggcgctcccattcgccattcaggctgcg caactgttgggaagggcgtttcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgat taagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgatttaaatacgatttaggtgacac tatagatggctgcgtgagacacacgtagcctaccagtttcttactgctctactctgcaaagcaagagattaataacccatc atggatcctgtgtacgtggacatagacgctgacagcgcctttttgaaggccctgcaacgtgcgtaccccatgtttgaggtg gaaccaaggcaggtcacaccgaatgaccatgctaatgctagagcgttctcgcatctagctataaaactaatagagcaggaa attgaccccgactcaaccatcctggatatcggcagtgcgccagcaaggaggatgatgtcggacaggaagtaccactgcgtc tgcccgatgcgcagtgcggaagatcccgagagactcgccaattatgcgagaaagctagcatctgccgcaggaaaagtcctg gacagaaacatctctggaaagatcggggacttacaagcagtaatggccgtgccagacacggagacgccaacattctgctta cacacagacgtctcatgtagacagagagcagacgtcgctatataccaagacgtctatgctgtacacgcacccacgtcgcta taccaccaggcgattaaaggggtccgagtggcgtactgggttgggttcgacacaaccccgttcatgtacaatgccatggcg ggtgcctacccctcatactcgacaaactgggcagatgagcaggtactgaaggctaagaacataggattatgttcaacagac ctgacggaaggtagacgaggcaagttgtctattatgagagggaaaaagctaaaaccgtgcgaccgtgtgctgttctcagta gggtcaacgctctacccggaaagccgcaagctacttaagagctggcacctgccatcggtgttccatttaaagggcaaactc agcttcacatgccgctgtgatacagtggtttcgtgtgagggctacgtcgttaagagaataacgatgagcccaggcctttat ggaaaaaccacagggtatgcggtaacccaccacgcagacggattcctgatgtgcaagactaccgacacggttgacggcgaa agaatgtcattctcggtgtgcacatacgtgccggcgaccatttgtgatcaaatgaccggcatccttgctacagaagtcacg ccggaggatgcacagaagctgttggtggggctgaaccagagaatagtggttaacggcagaacgcaacggaatacgaacacc atgaaaaattatctgcttcccgtggtcgcccaagccttcagtaagtgggcaaaggagtgccggaaagacatggaagatgaa aaactcctgggggtcagagaaagaacactgacctgctgctgtctatgggcattcaagaagcagaaaacacacacggtctac aagaggcctgatacccagtcaattcagaaggttcaggccgagtttgacagctttgtggtaccgagtctgtggtcgtccggg ttgtcaatccctttgaggactagaatcaaatggttgttaagcaaggtgccaaaaaccgacctgatcccatacagcggagac gcccgagaagcccgggacgcagaaaaagaagcagaggaagaacgagaagcagaactgactcgcgaagccctaccacctcta caggcagcacaggaagatgttcaggtcgaaatcgacgtggaacagcttgaggacagagcgggcgcaggaataatagagact ccgagaggagctatcaaagttactgcccaaccaacagaccacgtcgtgggagagtacctggtactctccccgcagaccgta ctacgtagccagaagctcagtctgattcacgctttggcggagcaagtgaagacgtgcacgcacaacggacgagcagggagg tatgcggtcgaagcgtacgacggccgagtcctagtgccctcaggctatgcaatctcgcctgaagacttccagagtctaagc gaaagcgcaacgatggtgtataacgaaagagagttcgtaaacagaaagctacaccatattgcgatgcacggaccagccctg aacaccgacgaagagtcgtatgagctggtgagggcagagaggacagaacacgagtacgtctacgacgtggatcagagaaga tgctgtaagaaggaagaagccgcaggactggtactggtgggcgacttgactaatccgccctaccacgaattcgcatatgaa gggctaaaaatccgccctgcctgcccatacaaaattgcagtcataggagtcttcggagtaccgggatctggcaagtcagct attatcaagaacctagttaccaggcaggacctggtgactagcggaaagaaagaaaactgccaagaaatcaccaccgacgtg atgagacagagaggtctagagatatctgcacgtacggttgactcgctgctcttgaatggatgcaacagaccagtcgacgtg ttgtacgtagacgaggcgtttgcgtgccactctggaacgctacttgctttgatcgccttggtgagaccaaggcagaaagtt gtactttgtggtgacccgaagcagtgcggcttcttcaatatgatgcagatgaaagtcaactataatcacaacatctgcacc caagtgtaccacaaaagtatctccaggcggtgtacactgcctgtgaccgccattgtgtcatcgttgcattacgaaggcaaa atgcgcactacgaatgagtacaacaagccgattgtagtggacactacaggctcaacaaaacctgaccctggagacctcgtg ttaacgtgcttcagagggtgggttaaacaactgcaaattgactatcgtggatacgaggtcatgacagcagccgcatcccaa gggttaaccagaaaaggagtttacgcagttagacaaaaagttaatgaaaacccgctctatgcatcaacgtcagagcacgtc aacgtactcctaacgcgtacggaaggtaaactggtatggaagacactttccggcgacccgtggataaagacgctgcagaac ccaccgaaaggaaacttcaaagcaactattaaggagtgggaggtggagcatgcatcaataatggcgggcatctgcagtcac caaatgaccttcgatacattccaaaataaagccaacgtttgttgggctaagagcttggtccctatcctcgaaacagcggggataaaactaaatgataggcagtggtctcagataattcaagccttcaaagaagacaaagcatactcacctgaagtagccctg aatgaaatatgtacgcgcatgtatggggtggatctagacagcgggctattttctaaaccgttggtgtctgtgtattacgcg gataaccactgggataataggcctggagggaaaatgttcggatttaaccccgaggcagcatccattctagaaagaaagtat ccattcacaaaagggaagtggaacatcaacaagcagatctgcgtgactaccaggaggatagaagactttaaccctaccacc aacatcataccggccaacaggagactaccacactcattagtggccgaacaccgcccagtaaaaggggaaagaatggaatgg ctggttaacaagataaacggccaccacgtgctcctggtcagtggctataaccttgcactgcctactaagagagtcacttgg gtagcgccgttaggtgtccgcggagcggactacacatacaacctagagttgggtctgccagagagaaggggtaggtatgac ctagtggtcataaacatccacacaccttttcgcatacaccattaccaacagtgcgtcgaccacgcaatgaaactgcaaatg ctcgggggtgactcattgagactgctcaaaccgggcggctctctattgatcagagcatatggttacgcagatagaaccagt gaacgagtcatctgcgtattgggacgcaagtttagatcgtctagagcgttgaaaccaccatgtgtcaccagcaacactgag atgtttttcctattcagcaactttgacaatggcagaaggaatttcacaactcatgtcatgaacaatcaactgaatgcagcc ttcgtaggacaggtcacccgagcaggatgtgcaccgtcgtaccgggtaaaacgcatggacatcgcgaagaacgatgaagag tgcgtagtcaacgccgctaaccctcgcgggttaccgggtggcggtgtttgcaaggcagtatacaaaaaatggccggagtcc tttaagaacagtgcaacaccagtgggaaccgcaaaaacagttatgtgcggtacgtatccagtaatccacgctgttggacca aacttctctaattattcggagtctgaaggggaccgggaattggcagctgcctatcgagaagtcgcaaaggaagtaactagg ctgggagtaaatagtgtagctatacctctcctctccacaggtgtatactcaggagggaaagacaggctgacccagtcactg aaccacctctttacagccatggactcgacggatgcagacgtggtcatctactgccgcgacaaagaatgggagaagaaaata tctgaggccatacagatgcggacccaagtagagctgctggatgagcacatctccatagactgcgatattgttcgcgtgcac cctgacagcagcttggcaggcagaaaaggatacagcaccacggaaggcgcactgtactcatatctagaagggacccgtttt catcagacggctgtggatatggcggagatacatactatgtggccaaagcaaacagaggccaatgagcaagtctgcctatat gccctgggggaaagtattgaatcgatcaggcagaaatgcccggtggatgatgcagacgcatcatctccccccaaaactgtc ccgtgcctttgccgttacgctatgactccagaacgcgtcacccggcttcgcatgaaccacgtcacaagcataattgtgtgt tcttcgtttcccctcccaaagtacaaaatagaaggagtgcaaaaagtcaaatgctctaaggtaatgctatttgaccacaac gtgccatcgcgcgtaagtccaagggaatatagatcttcccaggagtctgcacaggaggcgagtacaatcacgtcactgacg catagtcaattcgacctaagcgttgatggcgagatactgcccgtcccgtcagacctggatgctgacgccccagccctagaa ccagcactagacgacggggcgacacacacgctgccatccacaaccggaaaccttgcggccgtgtctgattgggtaatgagc accgtacctgtcgcgccgcccagaagaaggcgagggagaaacctgactgtgacatgtgacgagagagaagggaatataaca cccatggctagcgtccgattctttagggcagagctgtgtccggtcgtacaagaaacagcggagacgcgtgacacagcaatg tctcttcaggcaccaccgagtaccgccacggaaccgaatcatccgccgatctccttcggagcatcaagcgagacgttcccc attacatttggggacttcaacgaaggagaaatcgaaagcttgtcttctgagctactaactttcggagacttcttaccagga gaagtggatgacttgacagacagcgactggtccacgtgctcagacacggacgacgagttaagactagacagggcaggtggg tatatattctcgtcggacaccggtccaggtcatttacaacagaagtcagtacgccagtcagtgctgccggtgaacaccctg gaggaagtccacgaggagaagtgttacccacctaagctggatgaagcaaaggagcaactattacttaagaaactccaggag agtgcatccatggccaacagaagcaggtatcagtcgcgcaaagtagaaaacatgaaagcagcaatcatccagagactaaag agaggctgtagactatacttaatgtcagagaccccaaaagtccctacttaccggactacatatccggcgcctgtgtactcg cctccgatcaacgtccgattgtccaatcccgagtccgcagtggcagcatgcaatgagttcttagctagaaactatccaact gtctcatcataccaaattaccgacgagtatgatgcatatctagacatggtggacgggtcggagagttgcctggaccgagcg acattcaatccgtcaaaactcaggagctacccgaaacagcacgcttaccacgcgccctccatcagaagcgctgtaccgtcc ccattccagaacacactacagaatgtactggcagcagccacgaaaagaaactgcaacgtcacacagatgagggaattaccc actttggactcagcagtattcaacgtggagtgtttcaaaaaattcgcatgcaaccaagaatactgggaagaatttgctgcc agccctattaggataacaactgagaatttagcaacctatgttactaaactaaaagggccaaaagcagcagcgctattcgca aaaacccataatctactgccactacaggaagtaccaatggataggttcacagtagatatgaaaagggacgtaaaggtgact cctggtacaaagcatacagaggaaagacctaaggtgcaggttatacaggcggctgaacccttggcgacagcatacctatgt gggattcacagagagctggttaggaggctgaacgccgtcctcctacccaatgtacatacactatttgacatgtctgccgag gatttcgatgccatcatagccgcacactttaagccaggagacactgttttggaaacggacatagcctcctttgataagagc caagatgattcacttgcgcttactgctttgatgctgttagaggatttaggggtggatcactccctgctggacttgatagag gctgctttcggagagatttccagctgtcacctaccgacaggtacgcgcttcaagttcggcgccatgatgaaatcaggtatg ttcctaactctgttcgtcaacacattgttaaacatcaccatcgccagccgagtgctggaagatcgtctgacaaaatccgcg tgcgcggccttcatcggcgacgacaacataatacatggagtcgtctccgatgaattgatggcagccagatgtgccacttgg atgaacatggaagtgaagatcatagatgcagttgtatccttgaaagccccttacttttgtggagggtttatactgcacgat actgtgacaggaacagcttgcagagtggcagacccgctaaaaaggctttttaaactgggcaaaccgctagcggcaggtgac gaacaagatgaagatagaagacgagcgctggctgacgaagtgatcagatggcaacgaacagggctaattgatgagctggag aaagcggtatactctaggtacgaagtgcagggtatatcagttgtggtaatgtccatggccacctttgcaagctccagatcc aacttcgagaagctcagaggacccgtcataactttgtacggcggtcctaaataggtacgcactacagctacctattttgca gaagccgacagcaagtatctaaacactaatcagctacctaggaagttcgaatataggcgcgccgccaccatgacaaacatg tcatgcgcttacgagctaatcaagtcacttccagccaagctggagcagctggctcaggagacgcaagcaacgatccaaacg ctcatgatcgcggatcccaacgtcaacaaggatctgcgagcgttctgcgagttcctgaccgtgcagcaccagcgggcgtat cgagcgacgaacagcctgctcatcaaaccgcgagtcgcagcagcgcttcgcggggaggagctggacctgggcgaggcggacgtcgccgcccgggtccgccagctaaaacaacagctggcggagctcgagatggaaatcaagccagggcaccaacaagtggcc caagtaagcggcaggcggaaggccgcagccgcagctcccgtggcccagctgggtcgcgtgggcgtggtaaatgaggctagc ggcagcggcgccaccaacttcagcctgctgaagcaggccggcgacgtggaggagaaccccgggcccgaattcatgaaggca aacctactggtcctgttatgtgcacttgcagctgcagatgcagacacaatatgtataggctaccatgcgaacaattcaacc gacactgttgacacagtactcgagaagaatgtgacagtgacacactctgttaacctgctcgaagacagccacaacggaaaa ctatgtagattaaaaggaatagccccactacaattggggaaatgtaacatcgccggatggctcttgggaaatccagaatgc gacccactgcttccagtgagatcatggtcctacattgtagaaacaccaaactctgagaatggaatatgttatccaggagat ttcatcgactatgaggagctgagggagcaattgagctcagtgtcatcattcgaaagattcgaaatatttcccgaagaaagc tcatggcccaaccacaacacaaacggagtaacggcagcatgctcccatgaggggaaaagcagtttttacagaaatttgcta tggctgacggagaaggagggctcatacccaaagctgaaaaattcttatgtgaacaaaaaagggaaagaagtccttgtactg tggggtattcatcacccgtctaacagtaaggaacaacagaatctctatcagaatgaaaatgcttatgtctctgtagtgact tcaaattataacaggagatttaccccggaaatagcagaaagacccaaagtaagagatcaagctgggaggatgaactattac tggaccttgctaaaacccggagacacaataatatttgaggcaaatggaaatctaatagcaccaatgtatgctttcgcactg agtagaggctttgggtccggcatcatcacctcaaacgcatcaatgcatgagtgtaacacgaagtgtcaaacacccctggga gctataaacagcagtctcccttaccagaatatacacccagtcacaataggagagtgcccaaaatacgtcaggagtgccaaa ttgaggatggttacaggactaaggaacattccgtccattcaatccagaggtctatttggagccattgccggttttattgaa gggggatggactggaatgatagatggatggtatggttatcatcatcagaatgaacagggatcaggctatgcagcggatcaa aaaagcacacaaaatgccattaacgggattacaaacaaggtgaacactgttatcgagaaaatgaacattcaattcacagct gtgggtaaagaattcaacaaattagaaaaaaggatggaaaatttaaataaaaaagttgatgatggatttctggacatttgg acatataatgcagaattgttagttctactggaaaatgaaaggactctggatttccatgactcaaatgtgaagaatctgtat gagaaagtaaaaagccaattaaagaataatgccaaagaaatcggaaatagatgttttgagttctaccacaagtgtgacaat gaatgcatggaaagtgtaagaaatgggacttatgattatcccaaatattcagaagagtcaaagttgaacagggaaaaggta gatggagtgaaattggaatcaatggggatctatcagattctggcgatctactcaactgtcgccagttcactggtgcttttg gtctccctgggggcaatcagtttctggatgtgttctaatggatctttgcagtgcagaatatgcatctagtagttaattaac tcgcgatcgctaacttgacaattaagtatgaaggtatatgtgtcccctaagagacacactgtacatagcaaataatctata gatcaaagggctacgcaacccctgaatagtaacaaaatacaaaatcactaaaaattataaaaacagaaaaatacataaata ggtatacgtgtcccctaagagacacattgtatgtaggtgataagtatagatcaaagggccgaataacccctgaatagtaac aaaatatgaaaatcaataaaaatcataaaatagaaaaaccataaacagaagtagttcaaagggctataaaacccctgaata gtaacaaaacataaaattaataaaaatcaaatgaataccataattggcaaacggaagagatgtaggtacttaagcttccta aaagcagccgaactcactttgagaagtaggcatagcataccgaactcttccacgattctccgaacccacagggacgtagga gatgttattttgtttttaatatttcaaaaaaaaaaaaaaaaaaaaaaaagcggccgcttccgctcactgcccgctttccag tcgggaaacctgtcgtgccagctgcattaacatggtcatagctgtttccttgcgtattgggcgctctccgcttcctcgctc actgactcgctgcgctcggtcgttcgggtaaagcctggggtgcctaatgagcaaaaggccagcaaaaggccaggaaccgta aaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggt ggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgc cgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagtt cggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaact atcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggt atgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctc tgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggttttt ttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctc agtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaa aatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcaccta tctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttac catctggccccagtgctgcaatgataccgcgagaaccacgctcaccggctccagatttatcagcaataaaccagccagccg gaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaa gtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatgg cttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcg gtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactg tcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccga gttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgtt cttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatc [ SEQ ID NO: 17 ]

[0214] The plasmid

[0215] 51_CHIKV-ERR-B2-P2A-FireFlyLuciferase_611815201_7. annotations

[0216] may have the nucleic acid sequence represented by SEQ ID NO: 18:tgataggcagtggtctcagataattcaagccttcaaagaagacaaagcatactcacctgaagtagccctgaatgaaatatg tacgcgcatgtatggggtggatctagacagcgggctattttctaaaccgttggtgtctgtgtattacgcggataaccactg ggataataggcctggagggaaaatgttcggatttaaccccgaggcagcatccattctagaaagaaagtatccattcacaaa agggaagtggaacatcaacaagcagatctgcgtgactaccaggaggatagaagactttaaccctaccaccaacatcatacc ggccaacaggagactaccacactcattagtggccgaacaccgcccagtaaaaggggaaagaatggaatggctggttaacaa gataaacggccaccacgtgctcctggtcagtggctataaccttgcactgcctactaagagagtcacttgggtagcgccgtt aggtgtccgcggagcggactacacatacaacctagagttgggtctgccagagagaaggggtaggtatgacctagtggtcat aaacatccacacaccttttcgcatacaccattaccaacagtgcgtcgaccacgcaatgaaactgcaaatgctcgggggtga ctcattgagactgctcaaaccgggcggctctctattgatcagagcatatggttacgcagatagaaccagtgaacgagtcat ctgcgtattgggacgcaagtttagatcgtctagagcgttgaaaccaccatgtgtcaccagcaacactgagatgtttttcct attcagcaactttgacaatggcagaaggaatttcacaactcatgtcatgaacaatcaactgaatgcagccttcgtaggaca ggtcacccgagcaggatgtgcaccgtcgtaccgggtaaaacgcatggacatcgcgaagaacgatgaagagtgcgtagtcaa cgccgctaaccctcgcgggttaccgggtggcggtgtttgcaaggcagtatacaaaaaatggccggagtcctttaagaacag tgcaacaccagtgggaaccgcaaaaacagttatgtgcggtacgtatccagtaatccacgctgttggaccaaacttctctaa ttattcggagtctgaaggggaccgggaattggcagctgcctatcgagaagtcgcaaaggaagtaactaggctgggagtaaa tagtgtagctatacctctcctctccacaggtgtatactcaggagggaaagacaggctgacccagtcactgaaccacctctt tacagccatggactcgacggatgcagacgtggtcatctactgccgcgacaaagaatgggagaagaaaatatctgaggccat acagatgcggacccaagtagagctgctggatgagcacatctccatagactgcgatattgttcgcgtgcaccctgacagcag cttggcaggcagaaaaggatacagcaccacggaaggcgcactgtactcatatctagaagggacccgttttcatcagacggc tgtggatatggcggagatacatactatgtggccaaagcaaacagaggccaatgagcaagtctgcctatatgccctggggga aagtattgaatcgatcaggcagaaatgcccggtggatgatgcagacgcatcatctccccccaaaactgtcccgtgcctttg ccgttacgctatgactccagaacgcgtcacccggcttcgcatgaaccacgtcacaagcataattgtgtgttcttcgtttcc cctcccaaagtacaaaatagaaggagtgcaaaaagtcaaatgctctaaggtaatgctatttgaccacaacgtgccatcgcg cgtaagtccaagggaatatagatcttcccaggagtctgcacaggaggcgagtacaatcacgtcactgacgcatagtcaatt cgacctaagcgttgatggcgagatactgcccgtcccgtcagacctggatgctgacgccccagccctagaaccagcactaga cgacggggcgacacacacgctgccatccacaaccggaaaccttgcggccgtgtctgattgggtaatgagcaccgtacctgt cgcgccgcccagaagaaggcgagggagaaacctgactgtgacatgtgacgagagagaagggaatataacacccatggctag cgtccgattctttagggcagagctgtgtccggtcgtacaagaaacagcggagacgcgtgacacagcaatgtctcttcaggc accaccgagtaccgccacggaaccgaatcatccgccgatctccttcggagcatcaagcgagacgttccccattacatttgg ggacttcaacgaaggagaaatcgaaagcttgtcttctgagctactaactttcggagacttcttaccaggagaagtggatga cttgacagacagcgactggtccacgtgctcagacacggacgacgagttaagactagacagggcaggtgggtatatattctc gtcggacaccggtccaggtcatttacaacagaagtcagtacgccagtcagtgctgccggtgaacaccctggaggaagtcca cgaggagaagtgttacccacctaagctggatgaagcaaaggagcaactattacttaagaaactccaggagagtgcatccat ggccaacagaagcaggtatcagtcgcgcaaagtagaaaacatgaaagcagcaatcatccagagactaaagagaggctgtag actatacttaatgtcagagaccccaaaagtccctacttaccggactacatatccggcgcctgtgtactcgcctccgatcaa cgtccgattgtccaatcccgagtccgcagtggcagcatgcaatgagttcttagctagaaactatccaactgtctcatcata ccaaattaccgacgagtatgatgcatatctagacatggtggacgggtcggagagttgcctggaccgagcgacattcaatcc gtcaaaactcaggagctacccgaaacagcacgcttaccacgcgccctccatcagaagcgctgtaccgtccccattccagaa cacactacagaatgtactggcagcagccacgaaaagaaactgcaacgtcacacagatgagggaattacccactttggactc agcagtattcaacgtggagtgtttcaaaaaattcgcatgcaaccaagaatactgggaagaatttgctgccagccctattag gataacaactgagaatttagcaacctatgttactaaactaaaagggccaaaagcagcagcgctattcgcaaaaacccataa tctactgccactacaggaagtaccaatggataggttcacagtagatatgaaaagggacgtaaaggtgactcctggtacaaa gcatacagaggaaagacctaaggtgcaggttatacaggcggctgaacccttggcgacagcatacctatgtgggattcacag agagctggttaggaggctgaacgccgtcctcctacccaatgtacatacactatttgacatgtctgccgaggatttcgatgc catcatagccgcacactttaagccaggagacactgttttggaaacggacatagcctcctttgataagagccaagatgattc acttgcgcttactgctttgatgctgttagaggatttaggggtggatcactccctgctggacttgatagaggctgctttcgg agagatttccagctgtcacctaccgacaggtacgcgcttcaagttcggcgccatgatgaaatcaggtatgttcctaactct gttcgtcaacacattgttaaacatcaccatcgccagccgagtgctggaagatcgtctgacaaaatccgcgtgcgcggcctt catcggcgacgacaacataatacatggagtcgtctccgatgaattgatggcagccagatgtgccacttggatgaacatgga agtgaagatcatagatgcagttgtatccttgaaagccccttacttttgtggagggtttatactgcacgatactgtgacagg aacagcttgcagagtggcagacccgctaaaaaggctttttaaactgggcaaaccgctagcggcaggtgacgaacaagatga agatagaagacgagcgctggctgacgaagtgatcagatggcaacgaacagggctaattgatgagctggagaaagcggtata ctctaggtacgaagtgcagggtatatcagttgtggtaatgtccatggccacctttgcaagctccagatccaacttcgagaa gctcagaggacccgtcataactttgtacggcggtcctaaataggtacgcactacagctacctattttgcagaagccgacag caagtatctaaacactaatcagctacctaggaagttcgaatataggcgcgccgccaccatgacaaacatgtcatgcgctta cgagctaatcaagtcacttccagccaagctggagcagctggctcaggagacgcaagcaacgatccaaacgctcatgatcgc ggatcccaacgtcaacaaggatctgcgagcgttctgcgagttcctgaccgtgcagcaccagcgggcgtatcgagcgacgaacagcctgctcatcaaaccgcgagtcgcagcagcgcttcgcggggaggagctggacctgggcgaggcggacgtcgccgcccg ggtccgccagctaaaacaacagctggcggagctcgagatggaaatcaagccagggcaccaacaagtggcccaagtaagcgg caggcggaaggccgcagccgcagctcccgtggcccagctgggtcgcgtgggcgtggtaaatgaggctagcggcagcggcgc caccaacttcagcctgctgaagcaggccggcgacgtggaggagaaccccgggcccgaattcatggaagacgccaaaaacat aaagaaaggcccggcgccattctatccgctggaagatggaaccgctggagagcaactgcataaggctatgaagagatacgc cctggttcctggaacaattgcttttacagatgcacatatcgaggtggacatcacttacgctgagtacttcgaaatgtccgt tcggttggcagaagctatgaaacgatatgggctgaatacaaatcacagaatcgtcgtatgcagtgaaaactctcttcaatt ctttatgccggtgttgggcgcgttatttatcggagttgcagttgcgcccgcgaacgacatttataatgaacgtgaattgct caacagtatgggcatttcgcagcctaccgtggtgttcgtttccaaaaaggggttgcaaaaaattttgaacgtgcaaaaaaa gctcccaatcatccaaaaaattattatcatggattctaaaacggattaccagggatttcagtcgatgtacacgttcgtcac atctcatctacctcccggttttaatgaatacgattttgtgccagagtccttcgatagggacaagacaattgcactgatcat gaactcctctggatctactggtctgcctaaaggtgtcgctctgcctcatagaactgcctgcgtgagattctcgcatgccag agatcctatttttggcaatcaaatcattccggatactgcgattttaagtgttgttccattccatcacggttttggaatgtt tactacactcggatatttgatatgtggatttcgagtcgtcttaatgtatagatttgaagaagagctgtttctgaggagcct tcaggattacaagattcaaagtgcgctgctggtgccaaccctattctccttcttcgccaaaagcactctgattgacaaata cgatttatctaatttacacgaaattgcttctggtggcgctcccctctctaaggaagtcggggaagcggttgccaagaggtt ccatctgccaggtatcaggcaaggatatgggctcactgagactacatcagctattctgattacacccgagggggatgataa accgggcgcggtcggtaaagttgttccattttttgaagcgaaggttgtggatctggataccgggaaaacgctgggcgttaa tcaaagaggcgaactgtgtgtgagaggtcctatgattatgtccggttatgtaaacaatccggaagcgaccaacgccttgat tgacaaggatggatggctacattctggagacatagcttactgggacgaagacgaacacttcttcatcgttgaccgcctgaa gtctctgattaagtacaaaggctatcaggtggctcccgctgaattggaatccatcttgctccaacaccccaacatcttcga cgcaggtgtcgcaggtcttcccgacgatgacgccggtgaacttcccgccgccgttgttgttttggagcacggaaagacgat gacggaaaaagagatcgtggattacgtcgccagtcaagtaacaaccgcgaaaaagttgcgcggaggagttgtgtttgtgga cgaagtaccgaaaggtcttaccggaaaactcgacgcaagaaaaatcagagagatcctcataaaggccaagaagggcggaaa gtccaaattgtgatagtagttaattaactcgcgatcgctaacttgacaattaagtatgaaggtatatgtgtcccctaagag acacactgtacatagcaaataatctatagatcaaagggctacgcaacccctgaatagtaacaaaatacaaaatcactaaaa attataaaaacagaaaaatacataaataggtatacgtgtcccctaagagacacattgtatgtaggtgataagtatagatca aagggccgaataacccctgaatagtaacaaaatatgaaaatcaataaaaatcataaaatagaaaaaccataaacagaagta gttcaaagggctataaaacccctgaatagtaacaaaacataaaattaataaaaatcaaatgaataccataattggcaaacg gaagagatgtaggtacttaagcttcctaaaagcagccgaactcactttgagaagtaggcatagcataccgaactcttccac gattctccgaacccacagggacgtaggagatgttattttgtttttaatatttcaaaaaaaaaaaaaaaaaaaaaaagcggc cgcttccgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaacatggtcatagctgtttccttgcg tattgggcgctctccgcttcctcgctcactgactcgctgcgctcggtcgttcgggtaaagcctggggtgcctaatgagcaa aaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatc acaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccc tcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctc atagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagc ccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagcca ctggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacacta gaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaac aaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctt tgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaagga tcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagtt accaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgt agataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagaaccacgctcaccggctccag atttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtcta ttaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcg tggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgt tgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggtta tggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcat tctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactt taaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgt aacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaa atgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcattt atcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttc cccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttt taaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtggccgctacagggc gctcccattcgccattcaggctgcgcaactgttgggaagggcgtttcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagt gatttaaatacgatttaggtgacactatagatggctgcgtgagacacacgtagcctaccagtttcttactgctctactctg caaagcaagagattaataacccatcatggatcctgtgtacgtggacatagacgctgacagcgcctttttgaaggccctgca acgtgcgtaccccatgtttgaggtggaaccaaggcaggtcacaccgaatgaccatgctaatgctagagcgttctcgcatct agctataaaactaatagagcaggaaattgaccccgactcaaccatcctggatatcggcagtgcgccagcaaggaggatgat gtcggacaggaagtaccactgcgtctgcccgatgcgcagtgcggaagatcccgagagactcgccaattatgcgagaaagct agcatctgccgcaggaaaagtcctggacagaaacatctctggaaagatcggggacttacaagcagtaatggccgtgccaga cacggagacgccaacattctgcttacacacagacgtctcatgtagacagagagcagacgtcgctatataccaagacgtcta tgctgtacacgcacccacgtcgctataccaccaggcgattaaaggggtccgagtggcgtactgggttgggttcgacacaac cccgttcatgtacaatgccatggcgggtgcctacccctcatactcgacaaactgggcagatgagcaggtactgaaggctaa gaacataggattatgttcaacagacctgacggaaggtagacgaggcaagttgtctattatgagagggaaaaagctaaaacc gtgcgaccgtgtgctgttctcagtagggtcaacgctctacccggaaagccgcaagctacttaagagctggcacctgccatc ggtgttccatttaaagggcaaactcagcttcacatgccgctgtgatacagtggtttcgtgtgagggctacgtcgttaagag aataacgatgagcccaggcctttatggaaaaaccacagggtatgcggtaacccaccacgcagacggattcctgatgtgcaa gactaccgacacggttgacggcgaaagaatgtcattctcggtgtgcacatacgtgccggcgaccatttgtgatcaaatgac cggcatccttgctacagaagtcacgccggaggatgcacagaagctgttggtggggctgaaccagagaatagtggttaacgg cagaacgcaacggaatacgaacaccatgaaaaattatctgcttcccgtggtcgcccaagccttcagtaagtgggcaaagga gtgccggaaagacatggaagatgaaaaactcctgggggtcagagaaagaacactgacctgctgctgtctatgggcattcaa gaagcagaaaacacacacggtctacaagaggcctgatacccagtcaattcagaaggttcaggccgagtttgacagctttgt ggtaccgagtctgtggtcgtccgggttgtcaatccctttgaggactagaatcaaatggttgttaagcaaggtgccaaaaac cgacctgatcccatacagcggagacgcccgagaagcccgggacgcagaaaaagaagcagaggaagaacgagaagcagaact gactcgcgaagccctaccacctctacaggcagcacaggaagatgttcaggtcgaaatcgacgtggaacagcttgaggacag agcgggcgcaggaataatagagactccgagaggagctatcaaagttactgcccaaccaacagaccacgtcgtgggagagta cctggtactctccccgcagaccgtactacgtagccagaagctcagtctgattcacgctttggcggagcaagtgaagacgtg cacgcacaacggacgagcagggaggtatgcggtcgaagcgtacgacggccgagtcctagtgccctcaggctatgcaatctc gcctgaagacttccagagtctaagcgaaagcgcaacgatggtgtataacgaaagagagttcgtaaacagaaagctacacca tattgcgatgcacggaccagccctgaacaccgacgaagagtcgtatgagctggtgagggcagagaggacagaacacgagta cgtctacgacgtggatcagagaagatgctgtaagaaggaagaagccgcaggactggtactggtgggcgacttgactaatcc gccctaccacgaattcgcatatgaagggctaaaaatccgccctgcctgcccatacaaaattgcagtcataggagtcttcgg agtaccgggatctggcaagtcagctattatcaagaacctagttaccaggcaggacctggtgac...

Claims

1. CLAIMS1. An isolated self-amplifying RNA (saRNA) comprising:(a) a nucleic acid sequence encoding an RNA-dependent-RNA-polymerase (RdRP); (b) a nucleic acid sequence encoding a heterologous protein; and(c) a nucleic acid sequence encoding a viral suppressor of RNAi (VSR) protein.

2. The saRNA of claim 1, wherein the VSR protein is a viral B2 protein.

3. The saRNA of claim 2, wherein the viral B2 protein is from the family Nodaviridae.

4. The saRNA of claim 3, wherein the viral B2 protein is derived from a Nodamuravirus B2 (NoV B2) protein or a Flock House virus B2 (FHV B2) protein.

5. The saRNA of claim 4, wherein the NoV B2 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of protein B2 [Nodamura virus], wherein the protein has an arginine residue at position 59 (R59) relative to the amino sequence of protein B2 [Nodamura virus],6. The saRNA of claim 5, wherein the NoV B2 protein comprises an amino acid sequence consisting of the amino acid sequence of protein B2 [Nodamura virus],7. The saRNA of claim 4, wherein the FHV B2 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of protein B2 [Flock House virus], wherein the protein has an arginine residue at position 54 (R54) relative to the amino sequence of protein B2 [Flock House virus],8. The saRNA of claim 7, wherein the FHV B2 protein comprises an amino acid sequence consisting of the amino acid sequence of protein B2 [Flock House virus],9. The saRNA according to any preceding claim, wherein the saRNA is an Alphavirus saRNA.

10. The saRNA according to claim 9, wherein the saRNA is a Chikungunya virus (CHIKV) saRNA or a Venezuelan equine encephalitis virus (VEEV) saRNA.

11. The saRNA according to any one of claims 1-10, wherein the nucleic acid encoding the RdRP is upstream of the nucleic acids encoding the VSR protein and heterologous protein.

12. The saRNA according to any one of claims 1-11, wherein the nucleic acid sequence encoding the VSR protein is upstream of the nucleic acid sequence encoding the heterologous protein.

13. A composition comprising a saRNA according to any one of claims 1-12.

14. A pharmaceutical composition comprising a saRNA according to any one of claims 1-12.

15. A vector encoding the saRNA according to any one of claims 1-12.

16. A cell comprising a saRNA according to any one of claims 1-12, or a vector according to claim 15.

17. A vaccine comprising a saRNA according to any one of claims 1-12, or a vector according to claim 15.

18. A method of modulating expression of a heterologous protein in a cell, comprising introducing to the cell a saRNA according to any one of claims 1-12 or a vector according to claim 15.

19. The method of claim 18, wherein the cell is a somatic cell.

20. The method of claim 18 or 19, wherein the cell is a stem cell.

21. A method of producing induced pluripotent stem (iPS) cells, comprising the steps(a) introducing to a somatic cell a saRNA according to any one of claims 1-12 or a vector according to claim 15, encoding one or more reprogramming factors; and(b) expressing the one or more reprogramming factors in the cell to initiate dedifferentiation of the somatic cell into an iPS cell.

22. The method of claim 19, wherein the somatic cell is a fibroblast cell.

23. The method according to claim 21 or 22, wherein the one or more reprogramming factors comprise Oct4, Klf4 and SOX2, expressed together with c-myc or GLIS1.

24. A method of inducing an immune response in a subject, comprising administering a saRNA according to any one of claims 1-12, a composition according to claim 13, a pharmaceutical composition according to claim 14, a vector according to claim 15 or a vaccine according to claim 17 to a subject.

25. A saRNA according to any one of claims 1-12, a composition according to claim 13, a pharmaceutical composition according to claim 14, a vector according to claim 15, or a vaccine according to claim 17 for use in a method of inducing an immune response in a subject.

26. A saRNA according to any one of claims 1-12, composition according to claim 13, pharmaceutical composition according to claim 14, vector according to claim 15, or cell according to claim 16 for use in medicine.

27. A method of treating a disease or a condition in a subject, comprising administering a saRNA according to any one of claims 1-12, composition according to claim 13, pharmaceutical composition according to claim 14, vector according to claim 15, or cell according to claim 16.

28. The use of a saRNA according to any one of claims 1-12, composition according to claim 13, pharmaceutical composition according to claim 14, vector according to claim 15, or cell according to claim 16 for the manufacture of a medicament for the treatment of a disease or condition in a subject.

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