Chimeric adenovirus vector

Abstract

This provides methods for treating coronavirus disease 2019 (COVID-19). [Solution] This disclosure provides a chimeric adenovirus vector comprising nucleic acids encoding coronavirus disease 2019 (COVID-19) protein and an adjuvant, and a method of using the vector to induce an immune response to the SARS-CoV-2 protein for the purpose of treating COVID-19.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 144,339 filed on 1 February 2021, U.S. Provisional Application No. 63 / 074,954 filed on 4 September 2020, U.S. Provisional Application No. 63 / 045,710 filed on 29 June 2020, and U.S. Provisional Application No. 63 / 035,490 filed on 5 June 2020, each of which is incorporated herein by reference for all purposes. [Background technology]

[0002] Background of this disclosure Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Some symptoms of this disease include fever, cough, shortness of breath, muscle pain, sputum production, diarrhea, sore throat, loss of smell, and abdominal pain. The majority of cases present with mild symptoms, but some progress to viral pneumonia and multiple organ failure. There is currently no cure for this disease, and it is spreading rapidly across several continents, with regional outbreaks occurring worldwide. [Overview of the project]

[0003] Summary of this disclosure In one aspect, the present disclosure provides a chimeric adenovirus expression vector comprising an expression cassette including: (a) a first promoter functionally ligated to a nucleic acid encoding a first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein; and (b) a second promoter functionally ligated to a nucleic acid encoding a Toll-like receptor-3 (TLR-3) agonist.

[0004] In some embodiments, the nucleic acid encoding the TLR-3 agonist includes a nucleic acid encoding dsRNA. In some embodiments, the nucleic acid encoding the TLR-3 agonist includes a sequence selected from the group consisting of SEQ ID NO: 13 to 20. In a particular embodiment, the nucleic acid encoding the TLR-3 agonist includes the sequence of SEQ ID NO: 13.

[0005] Furthermore, the chimeric adenovirus expression vector may include an additional element (c): a third promoter functionally linked to the nucleic acid encoding a second SARS-CoV-2 protein. In some embodiments, element (c) is positioned between elements (a) and (b) in the expression cassette. In certain embodiments, the first SARS-CoV-2 protein in (a) is different from the second SARS-CoV-2 protein in (c). In other embodiments, the SARS-CoV-2 protein in (a) is the same as the SARS-CoV-2 protein in (c).

[0006] In some aspects of this aspect, the nucleic acid encoding the first SARS-CoV-2 protein in element (a) and / or the nucleic acid encoding the second SARS-CoV-2 protein in element (c) includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% identity with the sequence of SEQ ID NO:3. In some aspects, the first and / or second SARS-CoV-2 protein includes a SARS-CoV-2 S protein having a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% identity with the sequence of SEQ ID NO:1 or SEQ ID NO:21 or SEQ ID NO:22. In some embodiments, the nucleic acid encoding the first SARS-CoV-2 protein in element (a) and / or the nucleic acid encoding the second SARS-CoV-2 protein in element (c) comprises a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% identity with the sequence of SEQ ID NO:23.

[0007] In some embodiments, the nucleic acid encoding the first SARS-CoV-2 protein in element (a) and / or the nucleic acid encoding the second SARS-CoV-2 protein in element (c) includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% identity with respect to the sequence of SEQ ID NO:4. In some embodiments, the first and / or second SARS-CoV-2 protein includes a SARS-CoV-2 N protein having a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% identity with respect to the sequence of SEQ ID NO:2.

[0008] In some aspects of this aspect, the nucleic acid encoding the first SARS-CoV-2 protein in element (a) and / or the nucleic acid encoding the second SARS-CoV-2 protein in element (c) includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% identity with respect to the sequence of SEQ ID NO: 5. In some aspects, the first and / or second SARS-CoV-2 protein includes a fusion protein comprising the S1 region of the SARS-CoV-2 S protein, the furin region, and the SARS-CoV-2 N protein, the fusion protein including a sequence having at least 85% identity with respect to the sequence of SEQ ID NO: 12.

[0009] Furthermore, the first and second promoters of the chimeric adenovirus vector may be the same or different. For example, the first and second promoters may each be CMV promoters.

[0010] In some aspects of this situation, when all three elements (a) to (c) are present, the first promoter may be a CMV promoter, the second promoter may be a CMV promoter, and the third promoter may be a β-actin promoter (e.g., a human β-actin promoter).

[0011] In another aspect, the present disclosure features a chimeric adenovirus expression vector comprising an expression cassette including the following elements: (a) a first promoter functionally ligated to a nucleic acid encoding the SARS-CoV-2 S protein; and (b) a second promoter functionally ligated to a nucleic acid encoding a Toll-like receptor-3 (TLR-3) agonist.

[0012] In some aspects of this situation, the nucleic acid encoding the SARS-CoV-2 S protein contains the sequence SEQ ID NO:3. In some aspects, the SARS-CoV-2 S protein contains the sequence SEQ ID NO:1, SEQ ID NO:21, or SEQ ID NO:22.

[0013] In some embodiments, both the first promoter and the second promoter are CMV promoters.

[0014] In some aspects of this phase, elements (a) and (b) are collectively encoded in the sequence SEQ ID NO:6. Furthermore, the chimeric adenovirus expression vector in this phase is encoded in the sequence SEQ ID NO:9.

[0015] In another aspect, the present disclosure features a chimeric adenovirus expression vector comprising an expression cassette containing the following elements: (a) a first promoter functionally ligated to a nucleic acid encoding the SARS-CoV-2 S protein; (b) a second promoter functionally ligated to a nucleic acid encoding a Toll-like receptor-3 (TLR-3) agonist; and (c) a third promoter functionally ligated to a nucleic acid encoding the SARS-CoV-2 N protein. Optionally, the N-terminus-C-terminus order of the elements in the expression cassette is element (a), element (c), and element (b).

[0016] In some aspects of this aspect, the nucleic acid encoding the SARS-CoV-2 S protein comprises the sequence of SEQ ID NO:3. In some aspects of this aspect, the nucleic acid encoding the SARS-CoV-2 S protein comprises the sequence of SEQ ID NO:23. In some aspects, the SARS-CoV-2 S protein comprises the sequence of SEQ ID NO:1 or SEQ ID NO:21 or SEQ ID NO:22.

[0017] In some aspects of this aspect, the nucleic acid encoding the SARS-CoV-2 N protein comprises the sequence of SEQ ID NO:4. In some aspects, the SARS-CoV-2 N protein comprises the sequence of SEQ ID NO:2.

[0018] Furthermore, in some aspects of this aspect, the first promoter of element (a) is a CMV promoter, the second promoter of element (b) is a CMV promoter, and the third promoter of element (c) is a β-actin promoter (e.g., human β-actin promoter).

[0019] In some aspects, elements (a), (b), and (c) are collectively encoded by the sequence of SEQ ID NO:7. Furthermore, the chimeric adenovirus expression vector of this aspect is encoded by the sequence of SEQ ID NO:10.

[0020] In another aspect, the present disclosure features a chimeric adenovirus expression vector comprising an expression cassette comprising the following elements: (a) a first promoter functionally linked to a nucleic acid encoding a SARS-CoV-2 fusion protein, wherein the SARS-CoV-2 fusion protein comprises the S1 region of the SARS-CoV-2 S protein, a furin site, and the SARS-CoV-2 N protein; and (b) a second promoter functionally linked to a nucleic acid encoding a Toll-like receptor-3 (TLR-3) agonist.

[0021] In some aspects of this aspect, the nucleic acid encoding the SARS-CoV-2 fusion protein comprises the sequence of SEQ ID NO:5. In some aspects, the SARS-CoV-2 fusion protein comprises the sequence of SEQ ID NO:12.

[0022] In some aspects of this aspect, both the first promoter and the second promoter are CMV promoters.

[0023] In some aspects of this aspect, elements (a) and (b) together are encoded by the sequence of SEQ ID NO:8. Furthermore, the chimeric adenovirus expression vector of this aspect is encoded by the sequence of SEQ ID NO:11.

[0024] In another aspect, the present disclosure features an immunogenic composition comprising the chimeric adenovirus expression vector described herein and a pharmaceutically acceptable carrier.

[0025] In a further aspect, the disclosure further features a chimeric adenovirus expression vector comprising an expression cassette including the following elements: (a) a first promoter functionally ligated to a nucleic acid encoding a first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein; (b) a second promoter functionally ligated to a nucleic acid encoding a Toll-like receptor-3 (TLR-3) agonist; and (c) a third promoter functionally ligated to a nucleic acid encoding a SARS-CoV-2 N protein. In some embodiments, the SARS-CoV-2 N protein comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:2. In some embodiments, element (c) is located between elements (a) and (b) in the expression cassette. In some embodiments, the first SARS-CoV-2 protein comprises a SARS-CoV-2 S protein having a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:1, SEQ ID NO:21, or SEQ ID NO:22. In some embodiments, the nucleic acid encoding the TLR-3 agonist comprises a nucleic acid encoding dsRNA. In some embodiments, the nucleic acid encoding the TLR-3 agonist comprises a sequence selected from the group consisting of SEQ ID NO:13 to 20. In some embodiments, the nucleic acid encoding the first SARS-CoV-2 protein in element (a) comprises a sequence that is at least 85%, 90%, 95%, 97%, 99%, or 100% identical to the sequence of SEQ ID NO:3. In some embodiments, the nucleic acid encoding the SARS-CoV-2 N protein contains a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% identity with the sequence of SEQ ID NO:4. In some embodiments, the first and second promoters are identical. In some embodiments, both the first and second promoters are CMV promoters.In some embodiments, the first promoter is a CMV promoter, the second promoter is a CMV promoter, and the third promoter is a β-actin promoter. In some embodiments, element (c) is located between elements (a) and (b), and elements (a), (c), and (b) together encode a sequence having at least 95% identity to SEQ ID NO:7, or encodes the sequence of SEQ ID NO:7. In some embodiments, the chimeric adenovirus expression vector contains a sequence having at least 95% identity to SEQ ID NO:10, or contains the sequence of SEQ ID NO:10.

[0026] In another aspect, the Disclosure provides a method for inducing an immune response in a subject to a SARS-CoV-2 protein (e.g., a SARS-CoV-2 protein having the sequence SEQ ID NO: 1, 2, or 12) or a variant thereof described herein (e.g., having at least 90% or at least 95% identity to SEQ ID NO: 1, 2, or 12), comprising administering an immunogenically effective amount of a chimeric adenovirus expression vector or an immunogenic composition described herein to the subject. In some embodiments, the route of administration is oral, intranasal, or mucosal (e.g., oral). In certain embodiments, the route of administration is oral delivery by swallowing a tablet.

[0027] In some embodiments of the method, an immune response is induced in the alveolar cells, absorptive intestinal cells, ciliated cells, goblet cells, club cells, and / or airway basal cells of the subject. In certain embodiments, the subject is human.

[0028] Also provided is a chimeric polynucleotide expression cassette comprising the following elements: (a) a first promoter functionally ligated to a nucleic acid encoding a first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein; (b) a second promoter functionally ligated to a nucleic acid encoding a Toll-like receptor-3 (TLR-3) agonist; and (c) a third promoter functionally ligated to a nucleic acid encoding a SARS-CoV-2 protein or a non-SARS-CoV-2 antigen protein (which can be used to induce an immune response in a subject, including, but not limited to, a CD8 T cell response).

[0029] In some embodiments, the chimeric polynucleotide is a chimeric adenovirus expression vector. In some embodiments, the nucleic acid encoding the TLR-3 agonist includes a nucleic acid encoding dsRNA. In some embodiments, the nucleic acid encoding the TLR-3 agonist includes a sequence selected from the group consisting of SEQ ID NO: 13-20. In some embodiments, element (c) is positioned between elements (a) and (b) in the expression cassette.

[0030] In a further aspect, the present disclosure provides a chimeric polynucleotide comprising an expression cassette comprising: (a) a first promoter functionally ligated to a nucleic acid encoding an antigen protein; (b) a second promoter functionally ligated to a nucleic acid encoding a Toll-like receptor-3 (TLR-3) agonist; and (c) a third promoter functionally ligated to a nucleic acid encoding a SARS-CoV-2 N protein. In some embodiments, the SARS-CoV-2 N protein has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to SEQ ID NO:2. In some embodiments, the chimeric polynucleotide is a chimeric adenovirus expression vector. In some embodiments, the nucleic acid encoding the TLR-3 agonist comprises a nucleic acid encoding dsRNA. In some embodiments, the nucleic acid encoding the TLR-3 agonist comprises a sequence selected from the group consisting of SEQ ID NO:13-20. In some embodiments, element (c) is positioned between elements (a) and (b) in the expression cassette. In some embodiments, the antigen protein is derived from bacteria, fungi, viruses, or parasites. In some embodiments, the antigen protein is a cancer antigen.

[0031] In a further context, the present disclosure provides a method for inducing an immune response in a subject, comprising administering the chimeric polynucleotide described in the preceding paragraph to the subject. [Brief explanation of the drawing]

[0032] [Figure 1] Figure 1 shows antigen expression in human cells after infection. [Figure 2] Figure 2 shows the IgG antibody titers against S1 after immunizing mice on day 0 and day 14. Titers were measured by standard ELISA. [Figure 3]Figures 3A and 3B show IgG antibody titers against S1 and S2 after immunization of mice on day 0 and day 14. Binding signals for both antigens were measured at multiple time points using MSD. No significant difference in signal was observed at the early time points, but a greater antibody response was detected in the higher dose group at later time points. [Figure 4] Figures 4A-4D. Transgene inserts developed to test vaccine-specific responses. Recombinant adenoviruses were generated using these inserts. a. rAd-S, b. rAd-SN, c. rAd-S1-N, d. rAd-S(immobilized)-N. [Figure 5A] Figures 5A-5D. Immunization with candidate rAd vaccines induces serum IgG and lung IgA responses. Antibody titers against S after immunization of Balb / c mice on day 0 and day 14 with 1 × 10⁸ IU of rAd, expressing full-length S (rAd-S), co-expressing full-length S and N (rAd-SN), or co-expressing a fusion protein containing the S1 domain and N (rAd-S1-N). (Figure 5A) Serum IgG endpoint titer against S1 was measured by standard ELISA (n=6 per vaccinated group; n=3 for the PBS-administered group). (Symbols represent mean titers, bars represent standard error). (Figure 5B) Neutralizing antibody responses comparing rAd-SN and rAd-S1-N using two different methods, namely surrogate VNT (sVNT) and cell-based VNT (cVNT). (Figure 5C) IgA lung antibody titers against S1 and S2 in immunized mice. Endpoint titers were measured by standard ELISA (n=10 per group). Lines represent the median and interquartile range. **p<0.01, ***p<0.001, as defined by Mann-Whitney t-test. Figure 5D. Neutralizing antibodies measured in immunized lungs. [Figure 5B] Refer to the explanation in Figure 5A. [Figure 5C] Refer to the explanation in Figure 5A. [Figure 5D] Refer to the explanation in Figure 5A. [Figure 6]Figures 6A-6B. Immunization with rAd co-expressing full-length S and N vaccines induces a dose-dependent IgG response. Figures 6A and 6B. Balb / c mice were immunized with IN on day 0 and day 14 with rAd (rAd-SN) co-expressing 1 × 10⁷ IU, 1 × 10⁸ IU, or 7.2 × 10⁸ IU of full-length S and N. The amount of specific IgG against S1 (Figure 6A) and S2 in serum diluted to 1 / 4000 was evaluated using a mesoscale binding assay. Dots represent the mean, and lines represent the standard deviation. [Figure 7A] Figures 7A-7C. Immunization with rAd co-expressing full-length S and N vaccines induces a dose-dependent pluripotent T cell response. (Figure 7A) Balb / c mice were immunized with IN on day 0 and day 14 with 1 × 10⁸ IU (high Ad-SN) and 1 × 10⁷ IU (low Ad-SN) rAd-SN. Frequency of CD4+ (upper panel) or CD8+ T cells (lower panel) producing only IFN-γ, TNF-α, IL-2, or IL-4 after spleen cells were stimulated with a 1 μg / ml (CD4+) or 5 μg / ml (CD8+) S peptide pool, as determined by ICS-FACS. (B) Frequency of pluripotent CD4+ (upper panel) or CD8+ T cells (lower panel) producing more than one cytokine after spleen cells were stimulated with a 1 μg / ml (CD4+) or 5 μg / ml (CD8+) S peptide pool. Bars represent the mean, and lines represent the standard error of the mean. (C) IFN-γ T cell response to S protein at week 4 after immunization at week 0 and week 4 with rAd-SN doses of 1 × 10⁶ IU, 1 × 10⁷ IU, and 1 × 10⁸ IU was measured by ELISPOT. Bars represent the mean, and lines represent the standard deviation. *p<0.05; one-way nonparametric ANOVA with multiple comparisons. [Figure 7B] Refer to the explanation in Figure 7A. [Figure 7C] Refer to the explanation in Figure 7A. [Figure 8]Figures 8A-8B: Antibodies against S were superior when the S protein was expressed in a wild-type configuration compared to the immobilized version. Balb / c mice were immunized with 1e8 IU per mouse (n=6) at weeks 0 and 4, and antibody titers were measured. (Figure 8A) IgG antibody titers over time. (Figure 8B) Neutralizing antibody response was measured at week 6. Note that 1:1000 was the maximum dilution performed. [Figure 9] Figures 9A-9F: (Figure 9A) (Left) Frequency of CD27++CD38++ plasmablasts in peripheral blood before (Day 1) and after (Day 8) vaccination, as measured by flow cytometry. Bars represent medians, while error bars correspond to 95% confidence intervals. The Wilcoxon test was used to compare frequencies before and after vaccination; (Right) Representative flow cytometry plot showing CD27++CD38++ plasmablasts before and 8 days post-vaccination for one vaccinated individual; (Figure 9B) Change ratio of plasmablast frequency (Day 8 / Day 1). In total, 24 / 35 (69%) of subjects showed a 2-fold or greater increase (median increase of 3.3-fold overall); (Figure 9C) Change ratio of IgA and B7-expressing plasmablasts (Day 8 / Day 1) in low and high-dose vaccine cohorts. The frequency between two different dose groups was compared using the Mann-Whitney test; (Figure 9D) Multiplier change in the number of IgA-positive antibody-secreting cells (ASCs) reactive to the S1 domain of the SArs-CoV-2 spike antigen (Day 8 / Day 1); (Figure 9E) Multiplier change in serum S, N, or RBD-specific IgA antibodies as measured by the MSD platform (Day 29 / Day 1). The red dotted line represents the median. The frequency between two different dose groups was compared using the Mann-Whitney test; (Figure 9F) Multiplier change in nasal and salivary S, N, or RBD-specific IgA antibodies as measured by the MSD platform (Day 29 / Day 1). [Figure 10]Figures 10A-10F: (Figure 10A) PBMCs were restimulated with SARS-CoV-2 peptide before and after immunization, surface-stained for CD4, CD8, and the degranulation marker CD107a, and intracellularly stained for cytokines. Percentages of IFNγ, TNFα, and CD107a in CD8 T cells increased after immunization compared to background in response to the SARS-CoV-2 spike protein; (Figure 10B) Percentages of IFNγ, TNFα, and CD107a in CD4 T cells increased after immunization compared to background in response to the SARS-CoV-2 spike protein; (Figure 10C) Increase in IFNγ-producing CD8 T cells after immunization on day 8 compared to day 1; (Figure 10D) Polarization to Th1 response compared to Th2 response in subjects immunized with VXA-CoV2-1. The percentage change compared to baseline is shown; (Figure 10E) Percentages of IFNγ, TNFα, and CD107a in CD8 T cells increase after immunization compared to background in response to SARS-CoV-2 nuclear proteins; (Figure 10F) Percentages of IFNγ, TNFα, and CD107a in CD4 T cells increase after immunization compared to background in response to SARS-CoV-2 nuclear proteins. [Figure 11] Figure 11: Percentages of IFNγ, TNFα, and CD107a in CD8 T cells increase after immunization compared to background in response to four distinct coronavirus-derived S&N peptides. [Figure 12A]Figures 12A-12D. Oral VXA-CoV-2 induces a larger number of antiviral CD8 T cells than intramuscular mRNA vaccines. PBMCs were restimulated with SARS-CoV-2 peptide before and after immunization, surface-stained for CD4, CD8, and the degranulation marker CD107a, and intracellularly stained for cytokines. PBMCs from all three vaccines were evaluated simultaneously (Figure 12A). The graph shows that the percentages of IFNγ, TNFα, and CD107a in CD8 T cells increased after immunization compared to background in response to the SARS-CoV-2 spike protein. (Figure 12B) IFNγ data from (Figure 12A) are plotted together with those from the vaxart cohort and the convalescent period. Day 1 is not subtracted for convalescent subjects as pre-infection samples are unavailable. (Figure 12C) Representative FACS plots comparing the three vaccines. Figure 12(D) Time course of the Pfizer and Moderna vaccines. [Figure 12B] Refer to the explanation in Figure 12A. [Figure 12C] Refer to the explanation in Figure 12A. [Figure 12D] Refer to the explanation in Figure 12A. [Modes for carrying out the invention]

[0033] Detailed explanation of this disclosure I. Introduction Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a mucosal viral pathogen that infects the epithelial cells of the lungs and possibly further into the intestines (9). Some symptoms of this disease include, for example, fever, cough, shortness of breath, muscle pain, sputum production, diarrhea, sore throat, loss of smell, and abdominal pain. The majority of cases present with mild symptoms, but some progress to viral pneumonia and multiple organ failure.

[0034] This virus is transmitted primarily through close contact and through respiratory droplets produced when coughing or sneezing. It is also possible to contract COVID-19 by touching a contaminated surface and then touching your face. While the infection can be transmitted before symptoms appear, it is most contagious when symptoms are present. Currently, there is no vaccine or specific antiviral treatment for COVID-19. Management of the disease includes symptomatic treatment, supportive care, isolation, and some experimental measures.

[0035] The SARS-CoV-2 virus genome encodes four main structural proteins, including the spike (S), nucleocapsid (N), membrane (M), and coat (E), which are necessary for the construction of a complete viral particle. After viral entry, 16 non-structural proteins are formed from two large precursor proteins. These viruses have relatively large positive-sense RNA strands (26–32 kb), and this RNA can mutate, evolve, and undergo homologous recombination with other family members without erroneous editing to create new viral species (6). The S protein is thought to be a primary antibody target for coronavirus vaccines because it is involved in receptor binding, membrane fusion, and tissue tropism. When comparing SARS-CoV-2 Wu-1 (GenBank accession number QHD43416.1) and SARS-CoV (GenBank accession number AY525636.1), the S protein was found to have 76.2% identity, 87.2% similarity, and 2% gaps (1273 locations) (7). Both SARS-CoV and SARS-CoV-2 are thought to use the same receptor for cell entry: angiotensin-converting enzyme 2 receptor (ACE2), which is expressed in several human cell types (8). As discussed in the paper by Xu et al., high levels of ACE2 expression are present in type II alveolar cells of the lung, absorptive enterocytes of the ileum and colon, and possibly even in oral tissues such as the tongue (9).

[0036] Vaccines, immunogenic compositions, and methods for treating COVID-19 are provided herein, including the use of a chimeric adenovirus vector comprising one or more nucleic acids encoding one or more SARS-CoV-2 proteins and a nucleic acid encoding a TLR-3 agonist.

[0037] II. Definition For example, as used herein with respect to nucleic acids, proteins, or vectors, the terms “chimeric” or “recombinant” indicate that a nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or by alteration of the native nucleic acid or protein. Therefore, for example, chimeric and recombinant vectors contain nucleic acid sequences not found in the native (non-chimeric or non-recombinant) form of the vector. A chimeric adenovirus expression vector refers to an adenovirus expression vector containing a nucleic acid sequence encoding a heterologous polypeptide, such as the SARS-CoV-2 protein.

[0038] The term "expression vector" refers to a nucleic acid construct generated recombinantly or synthetically using a set of specified nucleic acid elements that enable the transcription of a particular nucleic acid in a host cell. An expression vector may be a plasmid, a virus, or part of a nucleic acid fragment. Typically, an expression vector contains the nucleic acid to be transcribed, functionally linked to a promoter.

[0039] The term “promoter” refers to a set of nucleic acid regulatory sequences that direct the transcription of a nucleic acid. As used herein, a promoter includes the necessary nucleic acid sequences near the transcription start site, for example, the TATA element in the case of a polymerase type II promoter. Promoters may also optionally include distal enhancer or repressor elements, which may be located several thousand base pairs from the transcription start site. Promoters include constitutive and inducible promoters. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental control. The term “functionally linked” refers to a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter or a set of transcription factor binding sites) and a second nucleic acid sequence, where the expression regulatory sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0040] The term "SARS-CoV-2" or "Severe Acute Respiratory Syndrome Coronavirus 2" refers to a coronavirus within the large genus of betacoronaviruses, derived from the family Coronaviridae. Genbank accession number MN908947.3 is the publicly available DNA sequence of SARS-CoV-2. This virus is transmitted primarily through close contact and through respiratory droplets produced when coughing or sneezing.

[0041] The term "SARS-CoV-2 protein" refers to the protein or fragment of said protein encoded by the nucleic acid of SARS-CoV-2 (e.g., Genbank accession number MN908947.3). In some embodiments, the SARS-CoV-2 protein fragment contains at least 10, 20, or more consecutive amino acids from the full-length protein encoded by the sequence of Genbank accession number MN908947.3. For example, a SARS-CoV-2 protein may be a structural protein of the full-length protein encoded by the nucleic acid of the SARS-CoV-2 virus, such as the SARS-CoV-2 S protein (surface glycoprotein; e.g., SEQ ID NO:1 or SEQ ID NO:21 or SEQ ID NO:22, or their variants that are at least 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:1 or SEQ ID NO:21 or SEQ ID NO:22) or the SARS-CoV-2 N protein (nucleocapsidrin protein; SEQ ID NO:2). A SARS-CoV-2 protein may also be a fusion protein containing different parts of the full-length protein encoded by the nucleic acid of the SARS-CoV-2 virus. For example, a SARS-CoV-2 fusion protein may contain the S1 region of the SARS-CoV-2 S protein, the furin region, and the SARS-CoV-2 N protein (e.g., SEQ ID NO:12).

[0042] The term "COVID-19" or "coronavirus disease 2019" refers to an infectious disease caused by the SARS-CoV-2 virus.

[0043] As used herein, the terms “TLR agonist” or “Toll-like receptor agonist” refer to compounds that bind to and stimulate Toll-like receptors, including, for example, TLR-2, TLR-3, TLR-6, ​​TLR-7, or TLR-8. TLR agonists are outlined in MacKichan, IAVI Report. 9:1-5 (2005) and Abreu et al., J Immunol, 174(8), 4453-4460 (2005). Agonists induce signal transduction after binding to their receptors.

[0044] As used herein, the terms “TLR-3 agonist” or “Toll-like receptor 3 agonist” refer to compounds that bind to and stimulate TLR-3. TLR-3 agonists have been identified and include several chemosynthetic analogs to double-stranded RNA, including double-stranded RNA, viral dsRNA, polyinosine-polycytidylic acid (poly-I:C)-polyadenylic acid-polyuridylic acid (poly-A:U) and poly-I:poly-C, as well as antibodies against TLR-3 (or cross-linking of antibodies) that result in IFN-β production (Matsumoto, M, et al, Biochem Biophys Res Commun 24:1364 (2002), de Bouteiller, et al, J Biol Chem 18:38133-45 (2005)). In some embodiments, TLR-3 agonists contain one of the sequences among SEQ ID NO:13-20. In some embodiments, the TLR-3 agonist is a dsRNA (for example, a dsRNA encoded in a nucleic acid containing the sequence described in SEQ ID NO:13).

[0045] When the term "heterogeneous" is used in relation to a portion of a nucleic acid, it indicates that the nucleic acid contains two or more subsequences that are not found in nature in the same relation to each other. For example, a nucleic acid typically has two or more sequences from unrelated genes, such as a promoter from one source and a coding region from another, which are typically produced by recombination and prepared to create a new functional nucleic acid. Similarly, heterogeneous protein indicates that a protein contains two or more subsequences that are not found in nature in the same relation to each other (e.g., a fusion protein).

[0046] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein and refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-stranded or double-stranded forms. The terms encompass nucleic acids, including known nucleotide analogs or modified skeletal residues or linkages, which are synthetic, natural, and unnatural, possess similar binding properties to the reference nucleic acid, and are metabolized in a similar manner to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs).

[0047] Unless otherwise indicated, a given nucleic acid sequence includes its conservedly modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as sequences that are explicitly shown. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0048] The term "antigen" refers to a portion of a protein or polypeptide chain that can be recognized by T cell receptors and / or antibodies. Typically, antigens are derived from bacterial proteins, viral proteins, or fungal proteins.

[0049] The term “immunogenically effective dose or amount” in the compositions of this disclosure refers to the amount that induces or modulates a specific immune response to the SARS-CoV-2 protein. Immune responses include humoral and cell-mediated immune responses. Immunogenic compositions can be used therapeutically or prophylactically to treat or prevent a disease at any given time. Humoral immune responses are generally mediated by the cell-free components of blood, i.e., plasma or serum; immunity is transferred by transferring serum or plasma from one individual to another. Cell-mediated immune responses are generally mediated by antigen-specific lymphocytes; immunity is transferred by transferring antigen-specific lymphocytes from one individual to another.

[0050] The terms “therapeutic dose,” “therapeutic effective dose,” or “effective dose” of a chimeric adenovirus vector or a composition containing a chimeric adenovirus vector refer to the amount of the vector or composition containing said vector that prevents, mitigates, reduces, or diminishes the severity of symptoms of diseases and disorders associated with a source of SARS-CoV-2 protein (e.g., SARS-CoV-2 virus).

[0051] The term "adjuvant" refers to a nonspecific immune response enhancer. Suitable adjuvants include, for example, cholera toxin, monophosphoryl lipid A (MPL), Freund's complete adjuvant, Freund's incomplete adjuvant, Quil A, and Al(OH). Adjuvants can also be substances that trigger activation of antigen-presenting cells and enhancement of T cell presentation through a second signaling molecule, such as Toll-like receptors. Examples of Toll-like receptors include those that recognize double-stranded RNA, bacterial flagella, LPS, CpG DNA, and bacterial lipopeptides (recently outlined in Abreu et al., J Immunol, 174(8), 4453-4460 (2005)).

[0052] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to amino acid polymers, which are artificial chemical mimics of naturally occurring amino acids, as well as naturally occurring and non-naturally occurring amino acid polymers.

[0053] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded in the genetic code, as well as such amino acids that are later modified, such as hydroxyproline and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimes refer to chemical compounds that have a different structure from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[0054] Amino acids may be referred to herein by either their generally known three-letter code or by the one-letter code recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides may be referred to by their generally accepted one-letter codes.

[0055] As used herein, the terms “percent identity” or “identical percent” as used in the context of nucleic acids or polypeptides refer to a sequence having at least 50% sequence identity with a reference sequence. Alternatively, the percentage identity may be any integer from 50% to 100%. In some embodiments, a sequence is substantially identical to a reference sequence if, when determined using the methods described herein; preferably using BLAST with standard parameters such as those described below, it has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to a reference sequence. Percent identity can also be determined by manual alignment.

[0056] For sequence comparison, typically one sequence acts as the reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into the computer, sub-sequence coordinates are specified if necessary, and program parameters for the sequence algorithm are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence to the reference sequence based on the program parameters.

[0057] The comparison window includes a reference to any one segment of several consecutive positions, for example, a segment of at least 10 residues. In some embodiments, the comparison window has 10 to 600 residues, for example, about 10 to about 30 residues, about 10 to about 20 residues, about 50 to about 200 residues, or about 100 to about 150 residues, and after optimally aligning the two sequences, the sequences can be compared to reference sequences of the same number of consecutive positions.

[0058] Suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website. This algorithm involves initially identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in the database sequence, either match or satisfy a certain positive threshold score T. T is referred to as the adjacent word score threshold (Altschul et al., ibid.). These initial adjacent word hits serve as seeds to initiate a search to find longer HSPs that contain them. Word hits are then extended in both directions along each sequence as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a score matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is stopped if: the cumulative alignment score decreases by quantity X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of one or more negative score residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) uses a word size of 28 (W), an expected value of 10 (E), M=1, N=-2, and a comparison of both strands as defaults.For amino acid sequences, the BLASTP program uses a word size (W) of 3, an expected value (E) of 10, and a BLOSUM62 score matrix by default (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0059] The BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indicator of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, for amino acid sequences, the minimum sum probability of comparing a test amino acid sequence with a reference amino acid sequence should be less than about 0.01, more preferably about 10. -5 Less than, most preferably about 10 -20 If it is less than [a certain value], it is considered similar to the reference array.

[0060] III. Compositions and Methods of the Disclosure This disclosure provides compositions comprising a chimeric adenovirus vector. The chimeric adenovirus vector may contain one or more nucleic acids encoding one or more SARS-CoV-2 proteins. The chimeric adenovirus vector may also contain nucleic acids encoding a Toll-like receptor (TLR) agonist (e.g., a TLR-3 agonist), which can act as an effective adjuvant when administered in conjunction with the viral vector.

[0061] In some embodiments, the chimeric adenovirus vector of the present disclosure comprises an expression cassette comprising: (a) a first promoter functionally ligated to a nucleic acid encoding a first SARS-CoV-2 protein; and (b) a second promoter functionally ligated to a nucleic acid encoding a Toll-like receptor-3 (TLR-3) agonist. The first SARS-CoV-2 protein may be a full-length protein (or substantially identical protein) encoded by the nucleic acid of SARS-CoV-2 (e.g., Genbank accession number MN908947.3) or a fragment thereof. For example, the first SARS-CoV-2 protein could be the structural protein of the full-length protein encoded by the nucleic acid of the SARS-CoV-2 virus, such as the SARS-CoV-2 S protein (surface glycoprotein; e.g., SEQ ID NO:1, or substantially identical thereto, e.g., SEQ ID NO:21 or SEQ ID NO:22, or their variants that are at least 90%, or at least 95%, 96%, 97%, 98%, or 99% identical to, e.g., SEQ ID NO:1, SEQ ID NO:21, or SEQ ID NO:22); or the SARS-CoV-2 N protein (nucleocapsidrin protein; SEQ ID NO:2 or substantially identical thereto, e.g., its variants that are at least 90%, or at least 95%, 96%, 97%, 98%, or 99% identical to, e.g., SEQ ID NO:2). In other embodiments, the first SARS-CoV-2 protein may be a protein encoded by another part of the nucleic acid of the SARS-CoV-2 virus, for example, a protein encoded by the ORF1ab gene, a protein encoded by the ORF3a gene, a protein encoded by the E gene (encoding the coat protein), a protein encoded by the M gene (encoding the membrane glycoprotein), a protein encoded by the ORF6 gene, a protein encoded by the ORF7a gene, a protein encoded by the ORF8 gene, or a protein encoded by the ORF10 gene.

[0062] In a further embodiment, the first SARS-CoV-2 protein may be a fusion protein containing different portions of the full-length protein encoded by the nucleic acid of the SARS-CoV-2 virus. For example, the SARS-CoV-2 fusion protein may contain the S1 region of the SARS-CoV-2 S protein, the furin site, and the SARS-CoV-2 N protein (e.g., SEQ ID NO:12).

[0063] The nucleic acid encoding the first SARS-CoV-2 protein in element (a) may include a sequence that has at least 85%, 90%, 95%, 96%, 97%, 99%, or 100% identity (e.g., 85%, 87%, 89%, 91%, 93%, 95%, 97%, 99%, or 100%) with respect to the sequence of SEQ ID NO:3 encoding the amino acid sequence of the SARS-CoV-2 S protein (SEQ ID NO:1). In some embodiments, the first SARS-CoV-2 protein in element (a) may contain a sequence having at least 85%, 90%, 95%, 96%, 97%, 99%, or 100% identity (e.g., 85%, 87%, 89%, 91%, 93%, 95%, 97%, 99%, or 100%) with respect to the sequence of SEQ ID NO:3, and may encode the SARS-CoV-2 S protein of SEQ ID NO:21 or SEQ ID NO:22. In some embodiments, the first SARS-CoV-2 protein in element (a) may contain a sequence having at least 85%, 90%, 95%, 96%, 97%, 99%, or 100% identity (e.g., 85%, 87%, 89%, 91%, 93%, 95%, 97%, or 99%) with the sequence of SEQ ID NO:3, encoding a SARS-CoV-2 S protein variant that is at least 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:1 or SEQ ID NO:21 or SEQ ID NO:22. In other embodiments, the nucleic acid encoding the first SARS-CoV-2 protein in element (a) may include a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% identity (e.g., 85%, 87%, 89%, 91%, 93%, 95%, 97%, 99%, or 100%) with respect to the sequence of SEQ ID NO:4 encoding the amino acid sequence of SARS-CoV-2 N protein (SEQ ID NO:2).In some embodiments, the first SARS-CoV-2 protein in element (a) may contain a sequence having at least 85%, 90%, 95%, 96%, 97%, 99%, or 100% identity (e.g., 85%, 87%, 89%, 91%, 93%, 95%, 97%, or 99%) with respect to the sequence of SEQ ID NO:4, encoding a SARS-CoV-2 N protein variant that is at least 90% identical to, or at least 95%, 96%, 97%, 98%, or 99% identical to, SEQ ID NO:2. In a further embodiment, the nucleic acid encoding the first SARS-CoV-2 protein in element (a) may include a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% (e.g., 85%, 87%, 89%, 91%, 93%, 95%, 97%, 99%, or 100%) identity with the sequence of SEQ ID NO:5 encoding the amino acid sequence (SEQ ID NO:12) of the SARS-CoV-2 fusion protein, which includes the S1 region of the SARS-CoV-2 S protein, the furin region, and the SARS-CoV-2 N protein.

[0064] In addition to the first SARS-CoV-2 protein, the chimeric adenovirus vector of this disclosure may further include element (c), which is a third promoter functionally linked to the nucleic acid encoding the second SARS-CoV-2 protein. In certain embodiments, the N-terminus-C-terminus order of the elements in the expression cassette is element (a), element (c), and element (b). In some embodiments, the first and second SARS-CoV-2 proteins encoded by their respective nucleic acids in elements (a) and (c) in the expression cassette are the same. In some embodiments, the first and second SARS-CoV-2 proteins encoded by their respective nucleic acids in elements (a) and (c) in the expression cassette are different.

[0065] For example, the first SARS-CoV-2 protein could be a SARS-CoV-2 S protein (a variant of SEQ ID NO:1, SEQ ID NO:21, or SEQ ID NO:22 that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:1, SEQ ID NO:21, or SEQ ID NO:22, or a variant of SEQ ID NO:3 that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:3), and the second SARS-CoV-2 protein could be a SARS-CoV-2 N protein (for example, SEQ ID SEQ ID NO:2 may be encoded by a nucleic acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 99%, or 100% identity (e.g., 85%, 87%, 89%, 91%, 93%, 95%, 97%, 99%, or 100%) with the sequence of NO:4. In another example, the first SARS-CoV-2 protein could be the SARS-CoV-2 N protein (e.g., SEQ ID NO:2 encoded by a nucleic acid sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% identity (e.g., 85%, 87%, 89%, 91%, 93%, 95%, 97%, 99%, or 100%) to the sequence of SEQ ID NO:4), and the second SARS-CoV-2 protein could be the SARS-CoV-2 S protein (e.g., SEQ ID NO:1 or SEQ ID NO:21 or SEQ ID NO:22, or, for example, SEQ ID It may be a variant of SEQ ID NO:1 or SEQ ID NO:21 or SEQ ID NO:22 that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence of NO:3, and encodes a nucleic acid sequence that is at least 85%, 90%, 95%, 97%, 99%, or 100% identical to the sequence of NO:3 (for example, 85%, 87%, 89%, 91%, 93%, 95%, 97%, 99%, or 100%).

[0066] In another example, the first SARS-CoV-2 protein could be a SARS-CoV-2 N protein (e.g., SEQ ID NO:2; or a variant thereof that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2) or a SARS-CoV-2 S protein (e.g., SEQ ID NO:1, SEQ ID NO:21, or SEQ ID NO:22, or a variant thereof that is at least 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:1, SEQ ID NO:21, or SEQ ID NO:22), and the second SARS-CoV-2 protein could be a SARS-CoV-2 fusion protein (e.g., SEQ ID SEQ ID NO:12) may be encoded by a nucleic acid sequence that has at least 85%, 90%, 95%, 97%, 99%, or 100% identity (e.g., 85%, 87%, 89%, 91%, 93%, 95%, 97%, 99%, or 100%) with respect to the sequence of NO:5.

[0067] In yet another example, the first SARS-CoV-2 protein could be a SARS-CoV-2 fusion protein (e.g., SEQ ID NO:12 encoded by a nucleic acid sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% identity to the sequence of SEQ ID NO:5 (e.g., 85%, 87%, 89%, 91%, 93%, 95%, 97%, 99%, or 100%)), and the second SARS-CoV-2 protein could be a SARS-CoV-2 N protein (e.g., SEQ ID NO:2; or a variant that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2) or a SARS-CoV-2 S protein (e.g., SEQ ID NO:1 or SEQ ID NO:21 or SEQ ID NO:22, or, for example, SEQ ID NO:1 or SEQ ID NO:21 or SEQ ID These could be variants of NO:22 that are at least 90%, 95%, 97%, 98%, or 99% identical to them.

[0068] Those skilled in the art understand that variants of the SARS-CoV-2 protein, such as the SARS-CoV-2 S protein, emerge rapidly. Two examples of mutant S protein sequences, namely the UK B.1.1.1.7 variant and the South Africa B.1.351 501Y.V2 variant, are provided under SEQ ID NO: 21 and 22, respectively. Among the many, other S protein variants are known, including the Brazilian variant, P.1 (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I); and the Indian variant B.1.617 (L452R, E484Q, D614G). Thus, in some embodiments, the SARS-CoV-2 S protein sequence is a variant sequence identified in patient populations.

[0069] In addition to the vectors described above that induce an immune response to the SARS-CoV-2 protein, and taking into consideration the data shown in Example 6, embodiments are provided herein that can stimulate a CD8 T cell immune response to a second antibody by co-introducing the SARS-CoV-2 N protein with any second antigen which may originate from a non-SARS-CoV-2 antigen source.

[0070] Accordingly, the Disclosure also provides a polynucleotide encoding the SARS-CoV-2 N protein (e.g., SEQ ID NO:2, or a variant thereof having at least 90% identity or at least 95% identity to SEQ ID NO:2, or a fragment thereof) and a second antigenic protein of any source. For example, the second antigenic protein may be derived from a non-SARS-CoV-2 virus, bacteria, other pathogens, or cancer. For example, in some embodiments, the second antigen may be: Herpes simplex, type 1; Herpes simplex, type 2; encephalitis virus; papillomavirus; varicella-zoster virus; Epstein-Barr virus; human cytomegalovirus; human herpesvirus, type 8; human papillomavirus; BK virus; JC virus; smallpox; poliovirus; hepatitis B virus; human bocavirus; parvovirus B19; human astrovirus; Norwalk virus. virus; coxsackievirus; hepatitis A virus; poliovirus; rhinovirus; severe acute respiratory syndrome virus; hepatitis C virus; yellow fever virus; dengue virus; West Nile virus;Rubella virus; Hepatitis E virus; Human immunodeficiency virus (HIV); Influenza virus; Guanarito virus; Junin virus; Lassa virus; Machupo virus; Sabia virus; Crimean-Congo hemorrhagic fever virus; Ebola virus; Marburg virus; Measle virus; Mumps virus; Parainfluenza virus; Respiratory syncytial virus; Human metapneumovirus; Hendra virus; Nipah virus; Rabies virus; Hepatitis D D); Rotavirus; Orbivirus; Coltivirus; Bannavirus; Human Enterovirus; Hantavirus; West Nile virus; Middle East Respiratory Syndrome Coronavirus; Japanese encephalitis virus; Vesicular exanthernavirus; or proteins or fragments thereof derived from Eastern equine encephalitis. See also U.S. Patent No. 8,222,224 for a list of usable antigens.

[0071] Specific examples of second antigens that can be used in combination with the SARS-CoV-2 N protein as described herein include, but are not limited to, norovirus (e.g., VP1), respiratory syncytial virus (RSV), influenza virus (e.g., HA, NA, M1, NP), human immunodeficiency virus (HIV, e.g., gag, pol, env, etc.), human papillomavirus (HPV, e.g., capsid proteins such as L1), Venezuelan Equine Encephalomyelitis (VEE) virus, Epstein-Barr virus, herpes simplex virus (HSV), and human herpesvirus. Examples include those derived from hepatitis A, B, C, E, and G viruses (HAV, HBV, HCV, HEV, HGV, e.g., surface antigens), mumps virus, rubella virus, measles virus, poliovirus, smallpox virus, rabies virus, and varicella-zoster virus.

[0072] Suitable viral antigens useful as second antigens described herein include viral non-structural proteins, such as proteins encoded by viral nucleic acids that do not encode structural polypeptides, in contrast to those that produce the capsid or proteins surrounding the virus. Examples of non-structural proteins include those that promote viral nucleic acid replication, viral gene expression, or post-translational processing, such as non-structural proteins 1, 2, 3, and 4 (NS1, NS2, NS3, and NS4, respectively) from Venezuelan Equine encephalitis (VEE), Eastern Equine Encephalitis (EEE), or Semliki Forest.

[0073] Useful bacterial antigens as second antigens described herein include, for example, Staphylococcus aureus, Staphylococcus epidermis, Helicobacter pylori, Streptococcus bovis, Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium leprae, Corynebacterium diphtheriae, Borrelia burgdorferi, Bacillus anthracis, and Bacillus cereus. cereus), Clostridium botulinum, Clostridium difficile, Salmonella typhi, Vibrio chloerae, Haemophilus influenzae, Bordetella pertussis, Yersinia pestis, Neisseria gonorrhoeae, Treponema pallidum, Mycoplasma species, Legionella pneumophila, Rickettsia typhi, Chlamydia trachomatis, and Shigella dysenteriae, Vibrio cholerae (For example, cholera toxin subunit B, cholera toxin-coregulated pilus (TCP));It may originate from Helicobacter pylori (e.g., VacA, CagA, NAP, Hsp, catalase, urea) or Escherichia coli (e.g., heat-labile enterotoxin, ciliary antigen).

[0074] Parasitic antigens useful as second antigens described herein include, for example, Giardia lamblia, species of the genera Leishmania, Trypanosoma, Trichomonas, and Plasmodium (e.g., Plasmodium falciparum). Surface protein antigens of *Mycobacterium falciparum*, e.g., pfs25, pfs28, pfs45, pfs84, pfs48 / 45, pfs230, Pvs25, and Pvs28); species of the genus *Schistosoma*; may be derived from *Mycobacterium tuberculosis* (e.g., Ag85, MPT64, ESAT-6, CFP10, R8307, MTB-32, MTB-39, CSP, LSA-1, LSA-3, EXP1, SSP-2, SALSA, STARP, GLURP, MSP-1, MSP-2, MSP-3, MSP-4, MSP-5, MSP-8, MSP-9, AMA-1, type 1 intrinsic membrane protein, RESA, EBA-175, and DBA).

[0075] Useful fungal antigens as second antigens described herein may be derived, for example, from Tinea pedis, Tinea corporus, Tinea cruris, Tinea unguium, Cladosporium carionii, Coccidioides immitis, species of Candida, Aspergillus fumigatus, and Pneumocystis carinii.

[0076] Useful cancer antigens as second antigens described herein include, for example, antigens expressed or overexpressed in colorectal cancer, gastric cancer, pancreatic cancer, lung cancer, ovarian cancer, prostate cancer, breast cancer, skin cancer (e.g., melanoma), leukemia, or lymphoma. Exemplary cancer antigens include, for example, HPV L1, HPV L2, HPV E1, HPV E2, placental alkaline phosphatase, AFP, BRCA1, Her2 / neu, CA 15-3, CA 19-9, CA-125, CEA, HCG, urokinase-type plasminogen activator (Upa), plasminogen activator inhibitor, CD53, CD30, CD25, C5, CD11a, CD33, CD20, ErbB2, and CTLA-4. For further cancer targets, see Sliwkowski & Mellman (2013) Science 341:6151.

[0077] Attenuated adenoviruses can be used to express the SARS-CoV-2 N protein and a second antigen protein (e.g., to induce a CD8 T cell response), but other polynucleotides or vectors can also be used. Examples of expression vectors include virus-derived vectors, e.g., recombinant adeno-associated virus (AAV) vectors, retroviral vectors, adenovirus vectors, modified vaccinia ankara (MVA) vectors, and lentivirus (e.g., HSV-1-derived) vectors (see, e.g., Brouard et al. (2009) British J. Pharm. 157:153). In other embodiments, the SARS-CoV-2 N protein (e.g., SEQ ID NO:2) and the second antigen protein may be encoded by polynucleotides, e.g., naked or encapsulated DNA, or RNA, e.g., mRNA (see, e.g., U.S. Patent Application Publication 2020 / 0254086 for details on various aspects of RNA-based vaccines).

[0078] In some embodiments, a vector containing a region encoding the SAR-CoV-2 N protein and a region encoding a second antigen protein further contains a nucleic acid encoding a TLR agonist (e.g., a TLR-3 agonist), which can act as an effective adjuvant when administered in conjunction with a vector such as a viral vector.

[0079] In some embodiments, the vector, for example, a viral vector, encodes the SARS-Co-V2 N protein (e.g., the N protein sequence of SEQ ID NO:2, or a variant thereof that is at least 90% identical to or at least 95% identical to SEQ ID NO:2) and a second antigen protein, and the expression of the N protein and the second antigen protein is driven by different promoters. In further embodiments, the vector includes a ribosome skipping element located between the nucleic acid region encoding the N protein and the region encoding the second antigen protein. In some embodiments, the vector includes an IRES located between the N protein and the second antigen protein to produce a bicistronic transcript. In some embodiments, the ribosome skipping element is a sequence encoding viral 2A peptide (T2A), porcine rhinitis virus-1 2A peptide (P2A), foot-and-mouth disease virus 2A peptide (F2A), equine rhinitis A virus 2A peptide (E2A), cytoplasmic polyhedron disease virus 2A peptide (BmCPV 2A), or silkworm (B. mori) flacherie virus 2A peptide (BmIFV 2A); located between the N protein and the second antigen protein. In some embodiments, the construct further encodes a TLR agonist.

[0080] In some embodiments, the vector comprises a first promoter functionally linked to a polynucleotide sequence encoding the SARS-CoV-2 N protein, and a second promoter functionally linked to a second antigen protein. In some embodiments, the vector, for example, a viral vector, may further comprise a third promoter functionally linked to a TLR agonist, for example, a TLR-3 agonist.

[0081] In certain embodiments, the N-terminus-C-terminus order of the elements in the expression cassette is the sequence encoding the antigen protein, the sequence encoding the SARS-Co-V2 N protein, and the sequence encoding the TLR agonist (e.g., a TLR3 agonist).

[0082] In a further embodiment, the antigen protein may be fused with an N protein sequence. For example, the fusion protein may include the antigen protein, a furin site, and a variant thereof that is at least 90% identical to, or at least 95% identical to, the SARS-CoV-2 N protein, or, for example, SEQ ID NO:2.

[0083] In some embodiments, the SARS-CoV-2 N protein encoded by the vector has at least 90% identity with SEQ ID NO:2. In some embodiments, the N protein encoded by the vector has at least 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:2.

[0084] In some embodiments, the vector includes an expression cassette described herein in a construct provided herein that encodes both the N protein and the SARS-CoV-2 S protein, in which the second antigen protein replaces the SARS-CoV-2 S protein. Thus, for example, in some embodiments, the vector includes the following sequence (5'-3): CMV-2 antigen protein-BGH-β-actin-N protein-SPA-BGH-CMV-dsRNA-SPA Here, "CMV" is the CMV promoter; "second antigen protein" is a nucleic acid sequence encoding a second antigen protein derived from an infectious agent or cancer antigen, for example, as described herein; "BGH" is a bovine growth hormone polyadenylation signal sequence; "β-actin" is a β-actin promoter, for example, a human β-actin promoter; "N protein" is a nucleic acid sequence encoding a SARS-CoV2 N protein, for example, SEQ ID NO:2, or a protein having at least 90% identity or at least 95% identity to SEQ ID NO:2, for example; "SPA" is a synthetic poly(A) sequence; and "dsRNA" is a nucleic acid sequence encoding a TLR agonist, for example, a TLR-3 agonist.

[0085] In some embodiments, in a construct containing an N protein and an antigen protein, such as an infectious disease antigen or a cancer antigen, an N protein derived from another coronavirus is used instead of the SARS-CoV-2 N protein. Thus, for example, in some embodiments, such a construct may contain the SARS-CoV or MERS N protein.

[0086] In some embodiments, the vector is an adenovirus vector, such as the adenovirus 5 (Ad5) vector described below.

[0087] Appropriate adenovirus vector In some embodiments, the adenovirus vector described herein is adenovirus 5 (Ad5), which may include, for example, Ad5 with deletions in the E1 / E3 region and Ad5 with deletions in the E4 region. Other suitable adenovirus vectors include lineage 2, orally tested lineages 4 and 7, enteric adenoviruses 40 and 41, and other lineages (e.g., Ad34) that are sufficient to deliver an antigen and induce an adaptive immune response to the transgene antigen (Lubeck et al., Proc Natl Acad Sci USA, 86(17), 6763-6767 (1989); Shen et al., J Virol, 75(9), 4297-4307 (2001); Bailey et al., Virology, 202(2), 695-706 (1994)). In some embodiments, the adenovirus vector is a living, non-replicating adenovirus vector (e.g., rAd5 with E1 and E3 deleted), a living, attenuated adenovirus vector (e.g., E1B55K deleted virus), or a living adenovirus vector having wild-type replication.

[0088] Transcriptional and translational regulatory sequences for expression vectors used to transform vertebrate cells in vivo may be supplied by viral sources. For example, commonly used promoters and enhancers are derived from, for instance, β-actin, adenovirus, Simian virus (SV40), and human cytomegalovirus (CMV). Suitable promoters include, for example, vectors that enable protein expression under the direction of a CMV promoter, β-actin promoters, SV40 early promoters, SV40 late promoters, metallothionein promoters, mouse mammary cancer virus promoters, Rous sarcoma virus promoters, transducer promoters, or other promoters that have been shown to be effective for expression in mammalian cells. Furthermore, promoters, regulatory, and / or signal sequences from viral genomes may be used, provided that such regulatory sequences are compatible with the host cell of choice.

[0089] Various promoters can be used in the chimeric adenovirus vectors described herein. The promoters used for elements (a), (b), and / or (c) may be the same or different. For example, in some embodiments, the first promoter used for element (a) and the second promoter used for element (b) may both be CMV promoters. In other embodiments, if element (c) is included, the third promoter may be the same as and / or different from the first and / or second promoters. For example, the first and second promoters may both be CMV promoters, and the third promoter may be a β-actin promoter (e.g., a human β-actin promoter).

[0090] TLR Agonist The chimeric adenovirus vectors described herein may also contain nucleic acids encoding Toll-like receptor (TLR) agonists, which can act as effective adjuvants when administered in conjunction with the viral vector. TLR agonists can be used to enhance the immune response to the SARS-CoV-2 protein. In some embodiments, TLR-3 agonists are used. In some embodiments, the TLR agonists described herein can be delivered simultaneously with an expression vector encoding the antigen of interest (e.g., the SARS-CoV-2 protein). In other embodiments, the TLR agonist can be delivered separately (i.e., temporally or spatially) from the expression vector encoding the antigen of interest (e.g., the SARS-CoV-2 protein). For example, the expression vector can be administered via a non-extraintestinal route (e.g., orally, intranasally, or mucosally), while the TLR agonist can be delivered via an extraintestinal route (e.g., intramuscularly, intraperitoneally, or subcutaneously).

[0091] In certain embodiments, TLR-3 agonists can be used to stimulate immunorecognition of an antigen of interest. Examples of TLR-3 agonists include small hairpin RNAs, viral RNAs, short segments of RNA capable of forming double-stranded or small hairpin RNAs, and small interfering RNAs (siRNAs). In one embodiment of this disclosure, the TLR-3 agonist is a viral dsRNA, such as a dsRNA or dsRNA viral intermediate derived from Sindbis virus (Alexopoulou et al, Nature 413:732-8 (2001)). In some embodiments, the TLR-3 agonist is a small hairpin RNA. A small hairpin RNA sequence typically contains two complementary sequences linked by a linker sequence. A specific linker sequence is not a critical aspect of this disclosure. Any suitable linker sequence can be used as long as it does not interfere with the binding of the two complementary sequences to form the dsRNA.

[0092] In some embodiments, TLR-3 agonists may contain sequences that have at least 85%, 90%, 95%, 97%, 99%, or 100% identity (e.g., 85%, 87%, 89%, 91%, 93%, 95%, 97%, 99%, or 100%) to the sequences described in SEQ ID NO:13-20. In certain embodiments, the TLR-3 agonist contains the sequence of SEQ ID NO:13. In certain embodiments, the dsRNA that is a TLR-3 agonist does not encode a specific polypeptide, but when it comes into contact with responder cells (e.g., dendritic cells, peripheral blood mononuclear cells, or macrophages) in vitro or in vivo, it causes the production of pro-inflammatory cytokines (e.g., IL-6, IL-8, TNF-α, IFN-α, IFN-β).

[0093] In certain embodiments, the TLR agonists described herein (e.g., TLR-3 agonists) can be delivered simultaneously within the same expression vector encoding the SARS-CoV-2 protein. In other embodiments, the TLR agonists (e.g., TLR-3 agonists) can be delivered separately (i.e., separately in time or space) from the expression vector encoding the SARS-CoV-2 protein. In some cases where the TLR-3 agonist is delivered separately from the expression vector, the nucleic acid encoding the TLR-3 agonist (e.g., expression dsRNA) and the chimeric adenovirus vector containing the nucleic acid encoding the SARS-CoV-2 protein can be administered in the same formulation. In other cases, the nucleic acid encoding the TLR-3 agonist and the chimeric adenovirus vector containing the nucleic acid encoding the SARS-CoV-2 protein can be administered in different formulations. When nucleic acids encoding a TLR-3 agonist and adenovirus vectors containing nucleic acids encoding the SARS-CoV-2 protein are administered in different formulations, they may be administered simultaneously or sequentially. For example, nucleic acids encoding a TLR-3 agonist may be administered first, followed by the chimeric adenovirus vector (e.g., 1, 2, 4, 8, 12, 16, 20, or 24 hours, 2, 4, 6, 8, or 10 days later). Alternatively, the adenovirus vector may be administered first, followed by the nucleic acids encoding a TLR-3 agonist (e.g., 1, 2, 4, 8, 12, 16, 20, or 24 hours, 2, 4, 6, 8, or 10 days later). In some embodiments, the nucleic acids encoding a TLR-3 agonist and the nucleic acids encoding the SARS-CoV-2 protein are under the control of the same promoter. In other embodiments, the nucleic acid encoding the TLR-3 agonist and the nucleic acid encoding the SARS-CoV-2 protein are under the control of different promoters.

[0094] IV. Pharmaceutical composition and route of administration Immunogenic pharmaceutical compositions may include chimeric adenovirus vectors and pharmaceutically acceptable carriers as described herein. Suitable carriers include, for example, water, saline, alcohol, fat, wax, buffer, solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, tarcan, cellulose, glucose, sucrose, and magnesium carbonate, or biodegradable microspheres (e.g., polylactic acid, polyglycolic acid). Suitable biodegradable microspheres are disclosed, for example, in U.S. Patents 4,897,268; 5,075,109; 5,928,647; 5,811,128; and 5,820,883. Immunogenic polypeptides and / or carrier expression vectors may be encapsulated in biodegradable microspheres or conjugated to the surface of microspheres.

[0095] The components in an immunogenic pharmaceutical composition are closely related to factors such as the route of administration, the timeline and / or duration of drug release, and the targeted delivery site, but are not limited to these. In some embodiments, a delayed-release coating or further coating of the formulation may include other film-forming polymers that are insensitive to luminal conditions, for technical reasons or for chronographic control of drug release. Materials used for such purposes include, but are not limited to, sugars, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, hydroxypropyl cellulose, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, and sodium carboxymethylcellulose, used alone or in mixtures.

[0096] Additives such as dispersants, colorants, dyes, further polymers (e.g., poly(ethyl acrylic acrylate, methyl methacrylate)), anti-tacks, and defoamers may be included in the coating layer. Other compounds may be added to increase the film thickness and to reduce the diffusion of acidic gastric juice into the core material. The coating layer may also contain pharmaceutically acceptable plasticizers to obtain desired mechanical properties. Such plasticizers include, but are not limited to, triacetin, citrate esters, phthalate esters, dibutyl sebacate, cetyl alcohol, polyethylene glycol, glycerol monoesters, polysorbate, or other plasticizers and mixtures thereof. The amount of plasticizer can be optimized for each formulation, as well as in relation to the selected polymer, the selected plasticizer, and the amount of said polymer applied.

[0097] Such immunogenic pharmaceutical compositions may also include non-immunogenic buffers (e.g., neutral buffered saline or phosphate-buffered saline), carbohydrates (e.g., glucose, mannose, sucrose, or dextran), mannitol, proteins, polypeptides, or amino acids (e.g., glycine), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), adjuvants (e.g., aluminum hydroxide), suspending agents, thickeners, and / or preservatives. Alternatively, the compositions of this disclosure may be formulated as lyophilates. Compounds may also be encapsulated in liposomes using well-known techniques.

[0098] Furthermore, pharmaceutical compositions can be prepared to protect them from degradation in the stomach, thereby ensuring that the administered immunogenic biological agent reaches the desired location. Methods for microencapsulating DNA and drugs for oral delivery are described, for example, in US2004043952. For the oral environment, several of these are available, including the Eudragit and TimeClock release systems and other methods specifically designed for adenoviruses (Lubeck et al., Proc Natl Acad Sci USA, 86(17), 6763-6767 (1989); Chourasia and Jain, J Pharm Timeclock Sci, 6(1), 33-66 (2003)). In some embodiments, the Eudragit system can be used to deliver chimeric adenovirus vectors to the lower small intestine.

[0099] In certain embodiments, the immunogenic composition is in the form of a tablet or capsule, for example, a compressed tablet covered with an enteric coating. In some embodiments, the immunogenic composition is encapsulated in a polymer capsule containing gelatin, hydroxypropyl methylcellulose, starch, or pullulan. In some embodiments, the immunogenic composition is in the form of microparticles less than 2 mm in diameter, for example, each microparticle being covered with an enteric coating as described herein. In certain embodiments, the immunogenic composition in the form of a tablet, capsule, or microparticles can be administered orally. In some embodiments, site-specific delivery can be achieved via a tablet or capsule that releases based on an externally generated signal. As disclosed in Digenis et al. (1998) Pharm. Sci. Tech. Today 1:160, an initial model was published for radio frequency (HF) signals. The concept of the original HF capsule was subsequently updated, and the result was commercialized as InteliSite®. The updated capsule is a radio frequency activated non-disintegrating delivery system. Radiation labeling of the capsule allows its position within a specific region of the GI tube to be determined by gamma machinography. Once the capsule reaches the desired position in the GI tube, external activation opens a series of windows for the capsule to access the drug reservoir.

[0100] In some embodiments, the immunogenic composition can be encapsulated in a wirelessly controlled capsule, which is then tracked and, once it reaches the delivery site, a signal is sent. In some embodiments, the capsule is signaled at a given post-administration time corresponding to when it is expected to arrive at the delivery site, whether detected or not.

[0101] The compositions described herein can be administered as part of a sustained-release formulation (i.e., a formulation such as a capsule or sponge that provides sustained release of the compound after administration). Such formulations can generally be prepared using well-known techniques (see, for example, Coombes et al. (1996) Vaccine 14:1429-1438). The sustained-release formulation may include polypeptides, polynucleotides, or antibodies dispersed in a carrier matrix and / or contained within a reservoir surrounded by a rate-controlling membrane.

[0102] The carriers for use in such formulations may be biocompatible and biodegradable; preferably, the formulation provides a relatively constant level of active ingredient release. Examples of such carriers include poly(lactide-co-glycolide) microparticles, as well as polyacrylates, latex, starch, cellulose, and dextran. Other delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., cross-linked polysaccharides or oligosaccharides) and optionally an outer layer containing amphiphilic compounds (see, e.g., WO 94 / 20078; WO 94 / 23701; and WO 96 / 06638). The amount of active compound contained in a sustained-release formulation depends on the implantation site, the rate of release and the expected duration, as well as the nature of the condition being treated or prevented.

[0103] In some embodiments, the immunogenic composition is contained in unit-dose containers or multi-dose containers, such as sealed ampoules or vials. Such containers are preferably sealed to maintain the sterility of the formulation until use. Generally, the formulation can be stored as a suspension, liquid, or emulsion in an oily or aqueous vehicle. Alternatively, the immunogenic composition can be stored in a lyophilized state requiring only the addition of a sterile liquid carrier immediately before use.

[0104] Composition for Targeted Delivery In some embodiments of targeted delivery, enteric coatings are used to protect the substance from the low pH environment of the stomach and to delay its release until the encapsulated substance reaches the desired target in the gastrointestinal tract. Enteric coatings are well known and commercially available. Examples include pH-sensitive polymers, biodegradable polymers, hydrogels, sustained-release systems, and osmotic delivery systems (see, for example, Chourasia & Jain (2003) J. Pharm. Pharmaceutical Sci. 6:33).

[0105] In some embodiments, the targeted delivery site is the ileum. The pH of the gastrointestinal tract (GIT) progresses from strongly acidic in the stomach (pH approximately 2) to more neutral in the ileum (pH approximately 5.8–7.0). pH-sensitive coatings that dissolve in or just before the ileum can be used. Examples include Eudragit® L and S polymers (pH thresholds ranging from 5.5 to 7.0); polyvinyl phthalate phthalate acetate (pH 5.0) hydroxypropyl methylcellulose phthalate 50 and 55 (pH 5.2 and 5.4, respectively); and cellulose phthalate acetate (pH 5.0). Thakral et al. (2013) Expert Opin. Drug Deliv. 10:131 outlines Euragit® formulations for ileal delivery, particularly the L and S combination to ensure delivery at pH ≤ 7.0. Crotts et al. (2001) Eur. J Pharm. Biol. 51:71 describes an Eudragit® formulation with appropriate disintegration properties. Vijay et al. (2010) J. Mater. Sci. Mater. Med. 21:2583 outlines an acrylic acid (AA)-methyl methacrylate (MMA)-based copolymer for ileal delivery at pH 6.8.

[0106] For ileal delivery, polymer coatings typically dissolve at approximately pH 6.8, allowing for complete release within approximately 40 minutes (see, e.g., Huyghebaert et al. (2005) Int. J. Pharm. 298:26). To achieve this, the therapeutic substance can be covered with layers of different coatings, such that the outermost layer protects the substance from low pH conditions and dissolves as the tablet leaves the stomach, and at least one inner layer that dissolves as the tablet passes through rising pH conditions. Examples of layered coatings for distal ileum delivery are described, e.g., WO 2015 / 127278, WO 2016 / 200951, and WO 2013 / 148258.

[0107] Biodegradable polymers (e.g., pectin, azopolymers) typically rely on the enzymatic activity of the microflora residing in the GIT. The ileum contains a greater number of bacteria, including lactobacilli and enterobacteria, than earlier stages.

[0108] Osmotically controlled release oral delivery systems (OROS®; Alza) are examples of osmotic systems that degrade over time under aqueous conditions. Such materials can be manipulated with other coatings or at various thicknesses for specific delivery to the ileum (see, for example, Conley et al. (2006) Curr. Med. Res. Opin. 22:1879).

[0109] Combination polymers for ileal delivery are reported in WO2000062820. Examples include Eudragit® L100-55 (25 mg / capsule) containing triethyl citrate (2.4 mg / capsule) and povidone K-25 (20 mg / tablet), followed by Eudragit® FS30D (30 mg / tablet). As described above, pH-sensitive polymers can be applied to result in ileal delivery, such as methacrylic acid copolymers (e.g., poly(methacrylate-co-methyl methacrylate) 1:1), cellulose phthalate acetate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose succinate acetate, polyvinyl phthalate acetate, cellulose trimellitate acetate, carboxymethyl ethyl cellulose, shellac, or other suitable polymers. The coating layer may also consist of film-forming polymers that are more sensitive to other luminal components than pH, e.g., bacterial degradation, or components that have such sensitivity when mixed with another film-forming polymer. Examples of such components that result in delayed release in the ileum include polymers containing azo bonds, polysaccharides such as pectin and its salts, galactomannan, amylose and chondroitin, disulfide polymers and glycosides.

[0110] To target the therapeutic composition to the ileum, a combination of components with varying pH, water, and enzyme sensitivity can be used. Coating thickness can also be used to control release. The components can also be used to form a matrix in which the therapeutic composition is embedded. Generally, Frontiers in Drug Design & Discovery See (Bentham Science Pub. 2009) vol. 4.

[0111] Adjuvant In some aspects of this disclosure, in addition to the TLR agonist encoded in the chimeric adenovirus vector (e.g., a TLR-3 agonist), the composition may further include additional adjuvants. Suitable adjuvants include, for example, lipid and non-lipid compounds, cholera toxin (CT), CT subunit B, CT derivative CTK63, heat-labile enterotoxin (LT) of Escherichia coli, LT derivative LTK63, Al(OH)3, and polyionic organic acids as described in, for example, WO 04 / 020592, Anderson and Crowle, Infect. Immun. 31(1):413-418 (1981), Roterman et al., J. Physiol. Pharmacol., 44(3):213-32 (1993), Arora and Crowle, J. Reticuloendothel. 24(3):271-86 (1978), and Crowle and May, Infect. Immun. 38(3):932-7 (1982). Suitable polyionic organic acids include, for example, 6,6'-[3,3'-dimethyl[1,1'-biphenyl]-4,4'-diyl]bis(azo)bis[4-amino-5-hydroxy-1,3-naphthalene disulfonic acid] (Evans Blue) and 3,3'-[1,1'-biphenyl]-4,4'-diylbis(azo)bis[4-amino-1-naphthalene sulfonic acid] (Congo Red). It will be recognized by those skilled in the art that polyionic organic acids can be used in any genetic vaccination method in combination with any dosage type.

[0112] Other suitable adjuvants include topical immunomodulators, such as members of the imidazoquinoline family, e.g., imiquimod and reximod (see, for example, Hengge et al., Lancet Infect. Dis. 1(3):189-98 (2001)).

[0113] Further suitable adjuvants include, for example, further alum-based adjuvants (e.g., Alhydrogel, Rehydragel, aluminum phosphate, algammulin); oily adjuvants (Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, Mich.), Specol, RIBI, TiterMax, Montanide ISA50, or Seppic MONTANIDE ISA 720); nonionic block copolymer-based adjuvants, cytokines (e.g., GM-CSF or Flat3-ligand); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, Pa.); salts of calcium, iron, or zinc; insoluble suspensions of acylated tyrosine; acylated sugars; cationic or anionic derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A, and commercially available as Quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, are also suitable adjuvants. Hemocyanins (e.g., keyhole limpet hemocyanin) and hemoerythrin can also be used in this disclosure. Polysaccharide adjuvants, such as chitin, chitosan, and deacetylated chitin, are also suitable adjuvants. Other suitable adjuvants include muramyl dipeptides (MDP, N-acetylmuramyl L-alanyl D-isoglutamine), bacterial peptidoglycans and their derivatives (e.g., threonyl-MDP and MTPPE). BCG and BCG cell wall skeleton (CWS) can also be used in this disclosure as adjuvants, either together with trehalose dimycolic acid or on their own. Trehalose dimycolic acid can be used on its own (see, for example, U.S. Patent No. 4,579,945).Detoxified endotoxins are also useful as adjuvants, either alone or in combination with other adjuvants (see, for example, U.S. Patent Nos. 4,866,034; 4,435,386; 4,505,899; 4,436,727; 4,436,728; 4,505,900; and 4,520,019). Saponins QS21, QS17, and QS7 are also useful as adjuvants (see, for example, U.S. Patent No. 5,057,540; EP 0362279; WO 96 / 33739; and WO 96 / 11711). Other suitable adjuvants include Montanide ISA 720 (Seppic, France) and SAF (Chiron, Calif., United). Examples include (United States), ISCOMS (CSL), MF-59 (Chiron), adjuvants of the SBAS series (e.g., SBAS-2, SBAS-4, or SBAS-6 or their variants, available from SmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton, Mont.), and RC-529 (Corixa, Hamilton, Mont.).

[0114] Within the pharmaceutical compositions provided herein, adjuvant compositions can be designed, for example, to primarily induce a Th1 or Th2 type immune response. High levels of Th1 cytokines (e.g., IFN-gamma, TNF-α, IL-2, and IL-12) tend to favor the induction of a cell-mediated immune response to an administered antigen. In contrast, high levels of Th2 cytokines (e.g., IL-4, IL-5, IL-6, and IL-10) tend to favor the induction of a humoral immune response. Following oral delivery of compositions comprising immunogenic polypeptides provided herein, immune responses, including Th1 and Th2 type responses, are typically expected to be induced.

[0115] Route of administration Compositions containing a chimeric adenovirus vector can be administered by any non-intestinal route (e.g., orally, intranasally, or via mucosa through the vagina, lungs, salivary glands, nasal cavity, small intestine, colon, rectum, tonsils, or Peyer's patches). The composition can be administered alone or with the adjuvant described above. In certain embodiments, the immunogenic composition is administered orally in the form of a tablet or capsule. In further embodiments, the immunogenic composition is administered orally in the form of a tablet or capsule for targeted delivery in the ileum.

[0116] V. Therapeutic uses One aspect of this disclosure involves using the immunogenic compositions described herein to induce an antigen-specific immune response to the SARS-CoV-2 protein (e.g., the SARS-CoV-2 protein having sequence SEQ ID NO: 1, 2, or 12) in a subject. In some embodiments, the immune response is induced in the alveolar cells, absorptive intestinal cells, ciliated cells, goblet cells, club cells, and / or airway basal cells of the subject. As used herein, “subject” refers to any warm-blooded animal, e.g., rodents, felines, canines, or primates, preferably humans. To prevent the disease, the immunogenic compositions may be used before the subject develops COVID-19. The disease can be diagnosed using diagnostic criteria generally accepted in the art. For example, viral infection can be diagnosed by measuring the viral titer in a biological sample from the subject (e.g., a nasal swab or mucosal sample).

[0117] As shown in the examples, the vaccines described herein are CD8 +This may be particularly effective in eliciting a T cell immune response. In some embodiments, this crucial CD8 response may be triggered by the presence of the SARS-CoV-2 N protein (e.g., SEQ ID NO:2 or a substantially identical variant thereof), which reacts to a second antigen protein (in the examples, this was the SARS-CoV-2 S protein, but may be a different SARS-CoV-2 protein, or a non-SARS-CoV-2 protein, as will be described in more detail below) with CD8 + It acts to stimulate the T cell response. Thus, in some embodiments, the vaccines described herein result in the expression of the SARS-CoV-2 N protein and a second antigen protein in a subject, e.g., a human subject, CD8 + It can be used to induce an immune response, including a T cell response. In some embodiments, the human subject has a low ability to produce an antibody-based immune response, or otherwise, CD8 + These are subjects who would benefit from a T-cell immune response. Examples of subjects include, but are not limited to, elderly individuals aged at least 50, at least 60, or at least 70 years, or elderly individuals with antibody deficiency disorders (see, for example, Angel A. Justiz Vaillant; Kamleshun Ramphul, ANTIBODY DEFICIENCY DISORER (Treasure Island (FL): StatPearls Publishing; 2020)) which may include, but are not limited to, subjects with X-linked agammaglobulinemia (Bruton's disease), transient neonatal hypogammaglobulinemia, selective Ig immunodeficiency, e.g., selective IgA deficiency, hyper-IgM syndrome, and common variable immunodeficiency disorder.

[0118] Immunotherapy is typically active immunotherapy, and the treatment relies on in vivo stimulation of the endogenous host immune system to respond to virus-infected cells, for example, by administering an immunogenic composition comprising a chimeric adenovirus vector as described herein.

[0119] The frequency and dosage of immunogenic compositions described herein are expected to vary from individual to individual and can be easily established using standard techniques. In some embodiments, 1 to 10 doses (e.g., 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) may be administered over a period of 52 weeks. In some embodiments, 2 or 3 doses may be administered at one-month intervals; or, for example, 2 to 3 doses every 2 to 3 months. For certain treatments, the interval may be once a year. Thereafter, booster vaccinations can be administered regularly.

[0120] The appropriate dose is, for example, an amount of the compound at least 10–50% higher than the basal (i.e., untreated) level, which can promote an antiviral immune response when administered as described above. Such a response can be monitored by measuring the patient's antiviral antibodies or by vaccine-dependent production of cytolytic T cells that can kill the patient's virus-infected cells, for example, in vitro. The immunogenic response can also be measured by detecting immune complexes formed between immunogenic polypeptides and antibodies in body fluids that are specific to immunogenic polypeptides. Body fluid samples taken from an individual before and after the start of therapy can be analyzed for immune complexes. Briefly, the number of immune complexes detected in both samples can be compared. A substantial change in the number of immune complexes in the second sample (after the start of therapy) compared to the first sample (before therapy) reflects the success of the therapy. Such vaccines should also be able to induce an immune response in vaccinated patients that leads to the prevention of COVID-19 disease compared to unvaccinated patients.

[0121] Exemplary dosages can be measured in infectious units (I.U.). Replication-deficient recombinant Ad5 vectors can be titrated and quantified using I.U. units. This is accomplished by performing an IU assay in an adherent human embryonic kidney (HEK) 293 cell line that permits the growth of replication-deficient Ad5. HEK293 cells are plated and allowed to adhere in a 24-well sterile tissue culture plate. The viral material is diluted in serial 10-fold dilutions and infected into individual wells of the plated HEK293 cells in an appropriate number of replicates, usually in duplicate or triplicate. Infection is allowed to proceed by incubation at 37 °C, 5% CO2 for about 40 - 42 hours. The cells are then fixed with methanol to permeabilize, washed, and blocked with a buffer solution containing bovine serum albumin (BSA). The cells are then incubated with a rabbit-derived primary antibody against the hexon surface protein of Ad5, washed, and probed again with an anti-rabbit secondary antibody conjugated with HRP. The infected cells are then stained by incubation with 3,3'-diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide. Infected cells are visualized using a phase contrast microscope and a dilution showing modest individual infection events is selected - these appear as darkly stained cells that are very well visible against the translucent monolayer of non-infected cells. The total number of infected cells per field is counted in at least 10 fields of an appropriate dilution. The virus titer can be calculated by multiplying the average number of these counts by the dilution factor used in the counting, in conjunction with the total number of fields of the magnification of the objective / eyepiece lenses used.

[0122] In some embodiments, the vaccine administered is 10 7 ~10 11 , for example, 10 8 ~10 11 , 10 9 ~10 11 , 5×10 9 ~5×10 10Dosages may be in the form of IUs. Appropriate dose sizes are considered to vary depending on the patient's size, but are typically in the range of approximately 0.01 ml to 10 ml, more typically 0.025 ml to 7.5 ml, and most typically 0.05 ml for the vaccine being injected. For the final product of tablets or capsules, the size will be between 10 mg and 1000 mg, most typically between 100 and 400 mg. Those skilled in the art will recognize that dose sizes can be adjusted based on the specific patient or the specific disease or disorder being treated. [Examples]

[0123] The following examples are intended to illustrate the Disclosure and not to limit it.

[0124] Example 1. Generation of recombinant adenovirus construct We developed several different recombinant adenovirus (rAd) constructs for preventing SARS-CoV-2 infection using the same vector platform (14, 15) previously clinically evaluated, except for the use of different antigens. Several rAd SARS-CoV-2 vaccines were generated by standard methods (e.g., as described by He, et al (17)).

[0125] Based on the publicly available DNA sequence of SARS-CoV-2, publicly available as Genbank accession number MN908947.3, three vaccine constructs were created. Specifically, codon-optimized nucleic acid sequences for expression in Homo sapiens cells were synthesized using the publicly available amino acid sequences of the SARS-CoV-2 S protein (or surface glycoprotein; hereafter SEQ 1) and the SARS-CoV-2 N protein (or nucleocapsidrin protein; hereafter SEQ 2). The codon-optimized nucleic acid sequences for the SARS-CoV-2 S gene and the SARS-CoV-2 N gene are shown in SEQ ID NO:3 and 4, respectively. Using these sequences, recombinant plasmids (pAds) containing a transgene cloned into the E1 region of adenovirus type 5 were created.

[0126] Two recombinant pAd plasmids were constructed using sequences derived from SARS-CoV-2: 1. ED81.4.1: pAd-CMV-SARS-CoV-2-S-BGH-CMV-dsRNA-SPA. Recombinant Ad5 vector containing SEQ ID NO:3 under the control of the CMV promoter. The sequence of the entire transgene cassette from the initial CMV promoter to the SPA following the dsRNA adjuvant is included as SEQ ID NO:6. The sequence of the entire recombinant adenovirus genome containing this transgene construct is included as SEQ ID NO:9. 2. ED84A6.4.1: pAd-CMV-SARS-CoV-2-S-BGH-βactin-SARS-CoV-2-N-SPA-BGH-CMV-dsRNA-SPA. A recombinant Ad5 vector containing SEQ ID NO:3 under the control of the CMV promoter and SEQ ID NO:4 under the control of the β-actin promoter. The sequence of the entire transgene cassette from the initial CMV promoter to the SPA following the dsRNA adjuvant is included as SEQ ID NO:7. The sequence of the entire recombinant adenovirus genome containing this transgene construct is included as SEQ ID NO:10.

[0127] Furthermore, a third pAd plasmid was constructed using a fusion sequence (SEQ ID NO: 5) that linked the S1 region of the SARS-CoV-2 S gene (including the native furin site between S1 and S2) to the full-length SARS-CoV-2 N gene: 3. ST05.1.3.3: pAd-CMV-SARS-CoV-2-S1-furin-N-BGH-CMV-dsRNA-SPA. Recombinant Ad5 vector containing SEQ ID NO:5 under the control of the CMV promoter. The sequence of the entire transgene cassette from the initial CMV promoter to the SPA following the dsRNA adjuvant is included as SEQ ID NO:8. The sequence of the entire recombinant adenovirus genome containing this transgene construct is included as SEQ ID NO:11.

[0128] The sequence was cloned into a shuttle plasmid using restriction sites (e.g., Sthl and Sgfl). Using the shuttle plasmid, the transgene was locked into a plasmid (pAd) containing the complete sequence of adenovirus type 5 lacking the E1 gene (pAd). The pAd plasmid, which provides the E1 gene product in trans, was transfected into human cells, enabling the replication and purification of recombinant adenovirus for use as a vaccine API.

[0129] Example 2. Expression of antigen protein The expression of three different candidate proteins was evaluated by intracellular staining / flow cytometry. HEK293 cells were tissue cultured in 24-well plates at a rate of 3e5 cells / well. After 4 hours, various constructs were used to infect the cells with an MOI of 1. After 40 hours, the cells were harvested, and separate wells were stained using a human monoclonal antibody (Genscript) that recognizes either the S1 or N protein. Expression in fixed cells was visualized using an anti-human IgG PE secondary antibody. The candidate protein that expressed the full-length SARS-CoV-2 S protein but not the N protein (rAd-S; plasmid pAd-CMV-SARS-CoV-2-S-BGH-CMV-dsRNA-SPA) clearly showed such an expression pattern. The candidate expressing the S1-N fusion protein (rAd-S1-N; plasmid pAd-CMV-SARS-CoV-2-S1-furin-N-BGH-CMV-dsRNA-SPA) expressed both the S and N proteins, similar to the candidate expressing S and N from separate promoters (rAd-SN; plasmid pAd-CMV-SARS-CoV-2-S-BGH-bactin-SARS-CoV-2-N-SPA-BGH-CMV-dsRNA-SPA) (Figure 1).

[0130] Example 3. Immunogenicity in mice The primary objective of the initial mouse immunogenicity study was to determine which rAd vectors would induce a significant antibody response. These results were used to determine which candidate vaccines would be selected for GMP production. Animals were immunized with (N=6) and antibody titers were measured over time. rAd vectors expressing both S and N from separate promoters (the plasmid pAd-CMV-SARS-CoV-2-S-BGH-β-actin-SARS-CoV-2-N-SPA-BGH-CMV-dsRNA-SPA) yielded titers equivalent to the S1 component of the SARS-CoV-2-derived S protein. The rAd-SN vector showed a slightly higher S1 antibody response than the fusion protein expressing rAd-S1-N (Figure 2).

[0131] Next, the dose-response of the selected vaccine rAd-SN was performed to test its immunogenicity. Three different dose levels were tested, and antibody responses to both S1 and S2 were measured using a mesoscale instrument. At the initial time point, similar responses were observed at all three dose levels, but at the later time point, the higher dose group showed an improved antibody response (Figures 3A and 3B).

[0132] Example 4. Immunogenicity in humans The rAd-SN plasmid (the above-mentioned pAd-CMV-SARS-CoV-2-S-BGH-βactin-SARS-CoV-2-N-SPA-BGH-CMV-dsRNA-SPA) is manufactured in a GMP facility, dried, and encapsulated in tablets. Human clinical trials will evaluate the ability of rAd-SN to induce an immune response in humans at different dose levels.

[0133] Example 5. Preclinical study of recombinant adenovirus mucosal vaccine for preventing SARS-CoV-2 infection. Introduction The emergence of a novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 disease in 2019, triggered a global pandemic and unprecedentedly high morbidity, mortality, and socioeconomic disruption. Coronavirus disease 2019 (COVID-19) is a respiratory illness of varying severity, ranging from asymptomatic infection to mild infection with fever and cough, to severe pneumonia and acute respiratory distress (1). Current reports suggest that asymptomatic transmission is considerable (2), and that SARS-CoV-2 infection induces a transient antibody response in most individuals (3). Therefore, the development of effective therapeutic interventions is urgently needed to protect the global population from infection and transmission of this virus and its associated clinical and social consequences. Mass vaccination with effective vaccines has been highly successful in preventing the transmission of many other infectious diseases and can also prevent disease in the vulnerable through the induction of herd immunity. Considerable effort and resources have been invested in urgently identifying an effective SARS-CoV-2 vaccine. Several different vaccine platforms have demonstrated preclinical immunogenicity and efficacy against pneumonia (4, 5). Some vaccines have demonstrated safety and immunogenicity in Phase I or Phase II trials (6-8). However, at present, no vaccine has demonstrated efficacy in this area.

[0134] All cutting-edge SARS-CoV-2 vaccine candidates are administered via the intramuscular (IM) route, some requiring storage at -80°C. This poses a significant obstacle to vaccine distribution and deployment during a pandemic when people are required to practice social distancing and avoid gatherings. The ultimate goal of any vaccine campaign is not to administer a certain number of vaccine doses, but to protect against disease by providing sufficient herd immunity to inhibit viral transmission. Injectable solutions require long-term and costly logistics for management and distribution, meaning that the effectiveness of administration does not immediately translate to immunity. Furthermore, systemic immunization can induce immunity in the peripheral and lower respiratory tracts. However, these vaccines cannot induce mucosal immunity in the upper respiratory tract, as evidenced by insufficient mucosal IgA reported by van Doremalen, et al., 4. Mucosal IgA (which has a polymeric structure and is associated with secretory components) can induce more potent viral neutralization (9), block viral transmission (10, 11), and is likely to induce sterilizing immunity, given that it is generally the first line of defense against respiratory pathogens.

[0135] Mucosal vaccines can induce mucosal immune responses, antibodies, and T cells on moist surfaces. The inventors have developed oral vaccines for multiple indications, including influenza and norovirus, delivered in tablet form for human use. The inventors' vaccine platform is a replication-deficient adenovirus type 5 vectorized vaccine expressing an antigen together with a novel Toll-like receptor 3 agonist as an adjuvant. These vaccines have shown good tolerability and can generate robust humoral and cellular immune responses to the expressed antigen (12-14). Protective efficacy in humans has been demonstrated against respiratory viruses for more than 90 days after vaccination, as shown in well-characterized experimental influenza infection models (15). Furthermore, the vaccine offers advantages such as room-temperature stability and ease of needle-free administration, providing several advantages in vaccine deployment and availability compared to injectable vaccine approaches.

[0136] In this specification, the inventors describe the preclinical development of a SARS-CoV-2 vaccine based on Vaxart's oral adenovirus platform. A key approach was to develop several vaccine candidates in parallel to create pre-production seeds while initial immunogenicity experiments were underway. Given that vaccines are to be produced during a pandemic, rapid decisions are needed to prevent manufacturing and regulatory timelines from running out. The inventors evaluated the relative immunogenicity of four candidate vaccines expressing antigens based on the spike (S) and nucleocapsid (N) SARS-CoV-2 proteins. These proteins are well characterized as antigens for related coronaviruses, e.g., SARS-CoV and MERS (outlined in Yong, et al., (16)), and increasingly as antigens for the SARS-CoV-2 spike. The objectives of our vaccine are to induce immunogenicity through three levels: firstly, by inducing potent serum neutralizing antibodies against S; secondly, by inducing a mucosal immune response; and thirdly, by inducing a T-cell response to both vaccine antigens. This three-part approach aims to induce robust and widespread immunity that can protect individuals from viral infection and disease, to facilitate rapid vaccine distribution during a pandemic, and to protect populations from viral transmission through herd immunity.

[0137] In this specification, we report the induction of neutralizing antibody (Nab), IgG and IgA antibody responses, as well as T cell responses, in mice immunized with rAd vectors expressing one or more SARS-CoV-2 antigens.

[0138] result Vector construction First, three different rAd vectors were constructed to express different SARS-CoV-2 antigens. These were a vector expressing the full-length S protein (rAd-S), a vector expressing both the S and N proteins (rAd-SN), and a vector expressing a fusion protein of the S1 domain and the N protein (rAd-S1-N). The N protein in rAd-SN was expressed under the control of the human β-actin promoter, which is far more potent in human cells than in mouse cells. As a control to explore the neutralizing antibody response, a further construct (rAd-S(immobilized)-N) in which the expressed S protein was fixed to its pre-fusion conformation was later constructed. These are shown in Figure 4. Flow cytometry and monoclonal antibodies against the S or N proteins were used to confirm the expression of various transgenes after infection of 293 cells.

[0139] Immunogenicity of rAd vectors expressing S and N antigens The primary goal of the initial mouse immunogenicity study was to determine which rAd vectors would induce a significant antibody response against S, and to obtain such results quickly enough to provide GMP seeds in time for production. Since we (17) observed that transgene expression by orally administered vaccine vectors could be suppressed in the gut environment of mice, we evaluated immunogenicity after intranasal (in) immunization. Animals were immunized in, and antibody titers were measured over time by IgG ELISA. All three rAd vectors induced nearly equivalent anti-S1 IgG titers at weeks 2 and 4, and IgG titers in all animals were significantly boosted by the second immunization (p<0.05, Mann-Whitney t-test) (Figure 5A). However, the vector expressing full-length S (rAd-SN) induced a higher neutralizing titer compared to the vector expressing S1 alone (Figure 5B). This was measured by two different neutralization assays: one based on SARS-CoV-2 infection of Vero cells (cVNT) and another based on a surrogate neutralization assay (sVNT). Furthermore, rAd-SN induced a higher pulmonary IgA response to S1 and, consequently, to S2 compared to rAd-S1-N two weeks after final immunization (Figure 5C). Notably, pulmonary neutralization titers were also significantly higher when using rAd-SN compared to the S1-containing vaccine (rAd-S1-N) (Figure 5D). This indicated that the rAd-SN candidate induced a greater functional response (NAb and IgA) compared to a vaccine simply containing the S1 domain. Since the N protein is far more conserved than the S protein and is a target of the long-term T cell response induced by infection (18), the vector rAd-SN was selected for GMP production.

[0140] Next, to understand the dose-responsiveness of this vaccine, three dose levels of rAd-SN were tested. Antibody responses to both S1 (Figure 6A) and S2 (Figure 6B) were measured. Similar responses were observed at all three dose levels at all time points. Responses to S1 and S2 significantly increased at week 6 compared to earlier times in all groups.

[0141] Next, the induction of S-specific T cells by rAd-SN at different doses was evaluated. Induction of antigen-specific CD4+ and CD8+ T cells producing effector cytokines such as IFN-γ, TNF-α, and IL-2 was observed 2 weeks after two immunizations (Figure 7A). Notably, IL-4 was hardly induced by this vaccine and was induced only in CD4+ T cells, which provides a level that ensures a very low risk of vaccine-dependent enhancement of the disease. Furthermore, immunization with rAd-SN induced bipolar and tripolar, multifunctional IFN-γ, TNF-α, and IL-2 CD4+ T cells (Figure 7B). Four weeks after the final immunization (week 8 of this study), a second dose-response experiment was performed to focus on the T cell response to the S protein. Splenocytes were stimulated overnight with peptide libraries against the S protein, divided into two separate peptide pools. The T cell responses in the two pools were summed and plotted (Figure 7C). Animals administered at dose levels of 1e7 IU and 1e8 IU showed a significantly higher T-cell response compared to untreated animals, but produced similar numbers of IFN-γ secreting cells, indicating a dose plateau at the 1e7 IU dose. Notably, this T-cell analysis was performed 4 weeks after a second immunization, possibly after the peak of the T-cell response.

[0142] Wild-type S, which expresses rAd, induces a superior neutralization response compared to pre-stabilization / pre-fusion S. Further studies were conducted to compare rAd-SN with a vaccine candidate in which the S protein was stabilized and the transmembrane region was removed (rAd-S(immobilized)-N). The stabilized version of the S protein has been proposed as a way to improve the neutralizing antibody response and produce less non-neutralizing antibody. The S protein was stabilized by the modification described in Amanat et al., (19). rAd-SN induced higher serum IgG titers against S1 at both time points tested (Figure 8A), although these were not statistically significant at 6 weeks according to Mann-Whitney (p=0.067). However, rAd-SN induced a significantly higher neutralizing antibody response than the stabilized version (Figure 8B) (p=0.0152). These results suggest that the wild-type version of the S protein is superior for rAd-based vaccines in mice.

[0143] Consideration The final stage of the COVID-19 pandemic requires the identification and manufacture of a safe and effective vaccine, followed by a global immunization campaign. Several vaccine candidates have been advanced to Phase III global efficacy trials, and if these trials are successful, they could form the first generation of the immunization campaign. However, all of these advanced candidates are injectable S-based vaccines. While such approaches are considered unlikely to prevent viral transmission, they should prevent pneumonia, as well as viral replication and damage to the lower respiratory tract and periphery, as demonstrated in offensive infection studies in macaques (4, 5).

[0144] One major constraint in the global COVID-19 immunization campaign is considered to be the bottleneck of cold chain distribution logistics and the requirement for properly trained healthcare workers (HCWs) to inject vaccines. Current logistical costs, including cold chain and training, could double the cost of adequately immunizing individuals in low- and middle-income countries (LMICs) (20). Implementing mass immunization campaigns that require HCWs trained on injection-based vaccines is expected to have a significant impact on healthcare resources in all countries. The need for cold chain, disposal of biohazardous sharp waste, and training is expected to lead to increased costs, vaccine inequality, uptake delays, and a prolongation of this pandemic. If vaccines cannot provide long-term protection (natural immunity to other beta-coronaviruses is temporary (21)) and annual injection-based campaigns are required, these costs are expected to be amplified. Vaxart's oral tablet vaccine platform offers solutions to these immunological, as well as logistical, economic, availability, and acceptability issues. In this study, the inventors demonstrated the immunogenicity of a SARS-CoV-2 vaccine using the Vaxart vaccine platform in a preclinical animal model; that is, the induction of serum and mucosal neutralizing antibodies as well as pluripotent T cells.

[0145] Mouse studies were designed to rapidly test the immunogenicity of the candidate vaccine in the spring of 2020 before moving on to critical manufacturing and clinical studies to address the pandemic. Vaxart's oral tablet vaccine platform has been previously demonstrated in humans to be able to evoke reliable mucosal responses (respiratory and intestinal), T-cell responses, and antibody responses against several different pathogens (12, 14, 22, 23). From our previous human influenza virus attack infection studies, we know that oral immunization was able to induce protective effects 90 days after immunization; this was comparable to commercially available quadrivalent inactivated vaccines (15). These features give us confidence that the adoption of this platform for COVID-19 could lead to efficacy against this pathogenic coronavirus and provide lasting protection from viral infection. Finally, a tablet vaccine campaign is far easier as it does not require qualified medical assistance to administer it. This ease of administration is expected to lead to increased vaccine availability and potentially greater acceptance, as demonstrated by the success of easily administered oral polio vaccines in eliminating poliovirus (24). Given the global level of COVID-19 denialism, distrust, and vaccine hesitation (25, 26), these features may become even more important during a SARS-CoV-2 immunization campaign compared to other vaccines, as substantially more resources may be needed to ensure vaccination. The tablet vaccine requires neither refrigeration nor freezing, neither needles nor vials, and can potentially be transported via standard mail or by delivery drones. These features significantly enhance deployment and distribution logistics and also enable access to isolated areas with limited technological resources.Finally, from an immunological perspective, oral administration of this adenovirus is neither compromised by existing immunity to adenoviruses nor causes substantial anti-vector immunity, which has been shown to significantly reduce vaccine efficacy in rAd5-based SARS-CoV-2 vaccines (27) (12, 13), and can prevent the development of persistent immunity when the same adenovirus platform is re-administered via the IM pathway (28).

[0146] Antigen selection can be challenging during a novel pandemic, a period in which critical decisions need to be made quickly. The S protein is considered a primary neutralizing antibody target for coronavirus vaccines because it is involved in receptor binding, membrane fusion, and tissue tropism. When comparing SARS-CoV-2 Wu-1 and SARS-CoV, the S protein was found to have 76.2% identity (29). Both SARS-CoV and SARS-CoV-2 are thought to use the same receptor for cell entry: the angiotensin-converting enzyme 2 receptor (ACE2), which is expressed in several human cell types (30). Therefore, the SARS-CoV-2 S protein has been used as a primary target antigen in vaccine development and is an ideal target, given that it functions as a crucial mechanism for viral binding to target cells. However, overall reliance on the S protein and IgG serum response in vaccines could ultimately lead to viral escape. In the case of influenza, small changes in hemagglutinin-binding proteins, including a single glycosylation site, can significantly affect the protective ability of the injected vaccine (31). Although SARS-CoV-2 appears to be more stable than most RNA viruses, mutations in the S protein have already been observed without the selective pressure of widely distributed vaccines. Once vaccine pressure begins, escape mutations may emerge. To address this problem, we have taken two approaches: firstly, to include a more conserved N protein in the vaccine, and secondly, to induce a broader immune response, namely through mucosal IgA.

[0147] High levels of ACE2 expression are present in type II alveolar cells of the lungs, absorptive enterocytes of the ileum and colon, and possibly even in oral tissues such as the tongue (32). Although viral transmission is thought to occur primarily through respiratory droplets and vectors between unprotected individuals in close contact (33), there is some evidence of transmission via the oral-fecal route, as seen in both SARS-CoV and MERS-CoV viruses, in which case coronavirus may be secreted in fecal samples of infected individuals (34). There is also evidence of a subpopulation of individuals with no respiratory symptoms but gastrointestinal symptoms, and that this subpopulation is likely to shed the virus for a longer period (35). Driving an immune mucosal immune response against S in both the respiratory tract and the intestinal tract may provide a greater ability to block broader immunity and transmission than simply targeting a single mucosal site. Blocking transmission, not just disease, is considered essential to reducing infection rates and ultimately eradicating SARS-CoV-2. The inventors have previously shown that a tablet-formed rAd-based oral vaccine can induce not only intestinal immunity against norovirus antigens in humans (12) but also protection from respiratory infection and shedding after an attack infection with the influenza virus (15). Furthermore, mucosal IgA is more likely to cope with any heterogeneity of the S protein of circulating viruses than monomeric IgG responses. mIgA has also been shown to be more cross-reactive than IgG for other respiratory pathogens (36). IgA may be a more neutralizing isotype than IgG in COVID-19 infection, and indeed, neutralizing IgA is dominant in the initial immune response (37). Notably, the inventors also found in their mouse studies that the ratio of neutralizing to non-neutralizing antibodies in the lungs was higher compared to serum antibody results, which supports the idea that IgA may have greater potency than IgG. Polymer IgA can transform weak single interactions into higher overall affinity binding and activation signals through multiple binding interactions to antigens and Fc receptors, resulting in greater cross-protection against heterologous viruses (38).

[0148] Our second strategy to mitigate this potential vaccine-driven escape problem was to include the N protein in the vaccine construct. The N protein is highly conserved among β-coronaviruses (over 90% identity), contains several immunodominant T cell epitopes, and long-term memory for N can be found in subjects who have recovered from SARS-CoV and in individuals whose exposure to either SARS-CoV or SARS-CoV-2 is not known (18, 39). In infection situations, the T cell response to the N protein appears to correlate with an increased neutralizing antibody response (40). Based on all of this evidence, we added N to our vaccine approach. We expressed this protein in 293A cells. However, since the human β-actin promoter is more active in human cells than in mice, we did not explore the immune response in Balb / c mice, but we intend to investigate this more carefully in future NHP and human studies.

[0149] The optimal sequence and structure of the S protein in SARS-CoV-2 vaccines is a subject of debate. Several laboratories have suggested that reducing the S protein to a key neutralizing domain within the receptor-binding domain (RBD) would promote a higher neutralizing antibody response and fewer non-neutralizing antibodies (41, 42). In an attempt to advance this approach, we constructed a vaccine candidate consisting of an S1 domain including the RBD. While the S1-based vaccine yielded a similar IgG binding titer to S1, the neutralizing antibody response was significantly lower compared to the full-length S antigen. Other gene-based vaccines also showed that reductionist approaches to S did not work very well, indicating that DNA vaccines expressing the full-length S protein produced higher neutralizing antibodies than shorter S segments (5). Consistent with these studies in macaques, we observed that the sequence of the antigen encoded by Ad has a significant impact on antibody function, with respect to neutralization as herein. Reducing the likelihood of exposure to non-neutralizing antibody epitopes seems theoretically reasonable, but this may reduce the assistance of T cells that enable the generation of more neutralizing antibodies. In fact, of the spike protein T cell responses that constitute 54% of the response to SARS-CoV-2, only 11% are located in the receptor-binding domain (43). Stabilizing the S protein may be important for protein vaccines, but not necessarily for gene-based vaccines. The former is generated in vitro and produced to maintain a uniform, defined structure that is injectable. The latter, on the other hand, is expressed on the cell surface in vivo, as in natural infection, in substantially pre-fusion form, and further stabilization may be unnecessary for B cells to produce antibodies against important neutralizing epitopes. We directly compared stabilized versions of S with wild-type versions in constructs encoding S and N proteins, as described in this embodiment. The wild-type versions induced a significantly better neutralizing antibody response.Interestingly, this has also been observed in DNA vaccine studies in NHP, where the stabilized version appears to induce lower neutralizing antibody (NAb) titers compared to wild-type S5. In NHP, a slightly different result was observed in a study of the rAd26 vector by Mercado et al., where expressing a stabilized version of the S protein appeared to improve NAb but reduce the T cell response (44). In summary, stabilization does not invariably improve the immune response in gene-based or vector-based vaccines.

[0150] Several vaccine candidates are in clinical trials or are about to begin them. Due to known safety and immunogenicity for epidemic pathogens such as the Ebola virus, two major candidate vaccines are based on recombinant adenovirus vectors: ChAdOx1-nCov from the University of Oxford and the AdVac platform from Janssen Pharmaceutical (45-48). We observed more potent serum IgG and NAb titers in our study compared to ChAdOx1-nCov in Balb / c mice (4). However, this may reflect differences in assay components. A study of the rAd36 vaccine was conducted by Hassan et al., who administered a dose of 1e10 VP by intranasal delivery (49). The results were significant in terms of blocking lung infection in a mouse SARS-CoV-2 attack infection model. They reported a serum antibody titer of 1e4 above the background titer, which was similar to our results despite using a dose 2-3 log-fold higher than our study. Indeed, in our studies, equally potent T cell and antibody responses were observed using 1e7 IU and 1e8 IU via the intranasal pathway. Using these doses, we observed a high percentage of CD8+ T cell responses (up to 14%) secreting IFN-γ and TNF-α, as well as potent CD4+ T cells, after peptide restimulation. Although we did not evaluate the trafficking characteristics of these antigen-specific T cells, we know that oral administration of this Ad-based vaccine induces high levels of mucosal homing lymphocytes in humans (12, 15). In this mouse study, the proportion of antigen-specific CD4+ and CD8+ T cells was multifunctional. Vaccine-induced T cells with multiple functions may provide more effective elimination of the virus after infection and thus may be involved in disease prevention; however, it is currently uncertain what the optimal T cell phenotype required for protection from disease is.

[0151] In summary, these studies in mice, as presented, were the first steps for the inventors in generating vaccine candidates, demonstrating the immunogenicity of constructs even at low vaccine doses, and elucidating the full-length spike protein as a key candidate antigen for inducing T cell responses and superior systemic and mucosal neutralizing antibodies. Future research is expected to focus on the immune response in humans.

[0152] method Vaccine construct For this study, four recombinant adenovirus vaccine constructs were created based on the publicly available DNA sequence of SARS-CoV-2, publicly available as Genbank accession number MN908947.3. Specifically, codon-optimized nucleic acid sequences for expression in Homo sapiens cells were synthesized using the publicly available amino acid sequences of the SARS-CoV-2 spike protein (S protein) and the SARS-CoV-2 nucleocapsid protein (N protein) (Blue Heron Biotechnology, Bothell, WA). Using these sequences, recombinant plasmids (rAd5) containing a transgene cloned into the E1 region of adenovirus type 5 were created using the same vector backbone as used in previous clinical trials of oral rAd tablets (12, 15), as described by He, et al. (50). The following four constructs were created, as shown in Figure 4: a. rAd-S: An rAd5 vector containing the full-length SARS-CoV-2 S gene under the control of the CMV promoter. b. rAd-SN: An rAd5 vector containing the full-length SARS-CoV-2 S gene under the control of the CMV promoter and the full-length SARS-CoV-2 N gene under the control of the human β-actin promoter. c. rAd-S1-N: An rAd5 vector that uses a fusion sequence to bind the S1 region of the SARS-CoV-2 S gene (including the native furin site between S1 and S2) to the full-length SARS-CoV-2 N gene. d. rAd-S(immobilized)-N: An rAd5 vector containing a stabilized S gene with its transmembrane region removed under the control of the CMV promoter, and a full-length SARS-CoV-2 N gene under the control of the human β-actin promoter. The S gene is stabilized by the following modifications: a) The native furin cleavage sites were removed by deleting arginine residues at amino acid positions 682, 683, and 685. b) Two stabilizing mutations were introduced: K986P and V987P c) The transmembrane region is removed following P1213, resulting in the bacteriophage T4 fibrin trimerized foldon domain sequence (51). Replaced with TIFF2026086500000002.tif4128.

[0153] All vaccines were grown in the Expi293F suspension cell line (Thermo Fisher Scientific), purified by CsCl density centrifugation, and provided in liquid form for animal testing.

[0154] Animal experiments The study was ethically approved by the Animal Husbandry and Use Committee (IACUC). All procedures followed local, state, and federal guidelines and regulations. Six-to-eight-week-old female Balb / c mice were purchased from Jackson Labs (Bar Harbor, ME). Since the mice do not swallow pills, liquid formulations were administered intranasally at a rate of 10 μl per nostril and 20 μl per mouse to test the immunogenicity of various constructs. Serum was obtained by puncturing the cheek at various time points.

[0155] Antibody evaluation ELISA Specific antibody titers against the protein were measured in the same manner as previously described (52). Briefly, microtiter plates (MaxiSorp: Nunc) were coated in 1 carbonate buffer (0.1 M at pH 9.6) containing 1.0 ug / ml of S1 protein (GenScript). The plates were incubated overnight at 4°C in a humidified chamber, then blocked for 1 hour in PBS + 0.05% Tween 20 (PBST) solution with 1% BSA, and then washed. Plasma samples were serially diluted in PBST. After incubation for 2 hours, the plates were washed at least 5 times with PBST. Antibodies were then added as a mixture of anti-mouse IgG1-horseradish peroxidase (HRP) and anti-mouse IgG2a-HRP (Bethyl Laboratories, Montgomery, TX). Each secondary antibody was used at a 1:5,000 dilution. After incubation for 1 hour, the plates were washed at least 5 times. Antigen-specific mouse antibodies were detected using 3,3=,5,5=-tetramethylbenzidine (TMB) substrate (Rockland, Gilbertsville, PA), with H2SO4 used as a stop solution. Plates were read at 450 nm using a Spectra Max M2 microplate reader. Unless otherwise stated, mean antibody titers are reported as the reciprocal dilution of the dilution that yields an absorbance value greater than the mean background + 2 standard deviations.

[0156] antibody binding antibody To simultaneously measure responses to both S1 and S2, MULTI-SPOT® 96-well, 2-spot plates (mesoscale instrument; MSD) were coated with SARS-CoV-2 antigen. The proteins were purchased from a source (Native Antigen Company) that produced the proteins in mammalian cells (293 cells). These were biotinylated and attached to their respective spots using individual U-PLEX linkers. To measure IgG antibodies, the plates were blocked with an MSD Blocker B for 1 hour with shaking, then washed three times, and the 1:4000 diluted sample was added. After incubation for 2 hours with shaking, the plates were washed three times. The plates were then incubated with a 1 μg / mL detection antibody (MSD SULFO-TAG™ anti-mouse IgG) for 1 hour. After three washes, reading buffer was added, and the plates were read with a Meso QuickPlex SQ 120.

[0157] SARS-CoV-2 neutralization assay Neutralizing antibodies were conventionally detected based on the SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT) kit (GenScript). This ELISA-based kit detects antibodies that interfere with the interaction between the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein and the ACE2 receptor on host cells, and correlates well with conventional viral neutralizing titers for SARS-CoV-2 infection in Vero cells (53). An advantage of this approach is that the assay can be performed in a BSL-2 laboratory. Serum from mice immunized with the candidate vaccine was diluted at 1:20, 1:100, 1:300, 1:500, 1:750, and 1:1000 using the provided sample dilution buffer. Serum from unimmunized mice was diluted at 1:20. Lung samples were diluted at 1:5, 1:20, and 1:100. Positive and negative controls were prepared in a 1:9 volume ratio according to the provided protocol. After dilution, serum or lung samples were individually incubated with HRP-RBD solution in a 1:1 ratio at 37°C for 30 minutes. Following incubation, 100 μl of each mixture of HRP-RBD and sample or control was added to the corresponding wells of a pre-coated capture plate with hACE2 and incubated again at 37°C for 15 minutes. The wells were then thoroughly washed, and 100 μl of the provided TMB (3,3=,5,5=-tetramethylbenzidine) solution was added to each well, and the plates were left to incubate at room temperature (20-25°C) for 15 minutes. Finally, 50 μl of stop solution was added to each well, and the plates were read using a 450 nm Spectra Max M2 microplate reader. The absorbance of a given sample is inversely correlated with the titer of the anti-SARS-CoV-2 RBD neutralizing antibody in the given sample. In accordance with the test kit protocol, the 20% inhibition cutoff for comparing the OD of a sample with that of a negative control was determined to be positive for the presence of a neutralizing antibody. Samples that were negative at the lowest dilution were set to be equal to half of the lowest dilution tested, which was either 10 for serum or 2.5 for lung samples.

[0158] Several studies using the cVNT assay at Visimederi under BSL3 conditions have measured further neutralizing antibody responses. The cVNT assay has readout information for cytopathic effects (CPE) to detect specific neutralizing antibodies against live SARS-CoV-2 in animal or human samples. The cVNT / CPE assay allows the virus to undergo multiple cycles of infection and release from cells; its exponential growth over several days (typically 72-hour incubation) causes partial or complete cellular monolayer detachment from the support surface, which can be clearly identified as CPE. Serum samples are heat-inactivated at 56°C for 30 minutes; 2-fold dilutions starting at 1:10 are performed and then mixed with an equal volume of virus solution containing 100 TCID50 SARS-CoV-2. The serum-virus mixture is incubated at 37°C for 1 hour in a humidified environment of 5% CO2. After incubation, 100 μL of the mixture at each dilution is added in two series to cell plates containing a semi-confluent Vero E6 monolayer. After incubation for 72 hours, the plates are examined in detail using an inverted optical microscope. The highest serum dilution that protects more than 50% of cells from CPE is considered the neutralizing titer.

[0159] Lung IgA ELISA Two weeks after final immunization (day 28 of the study), mice were sacrificed and bled via cardiac puncture. Lungs were removed and immediately frozen at -80°C. Upon thawing, the lungs were weighed. The lungs were homogenized in 150 μl of DPBS using a pellet pestle (Sigma Z359947). The homogenates were centrifuged at 1300 rpm for 3 minutes, and the supernatant was frozen. The total protein content of the lung homogenates was assessed using the Bradford assay to ensure that there was an equal amount of tissue in all samples before assessing the IgA content. Antigen-specific IgA titers in the lungs were detected using a mouse IgA ELISA kit (Mabtech) and pNPP substrate (Mabtech). In short, MaxiSorp plates (Nunc) were coated with S1 or S2 (The Native Antigen Company; 50 ng / well) in PBS for overnight adsorption at 4°C, then blocked for 1 hour in PBS + 0.05% Tween 20 (PBST) (PBS / T / B) solution with 0.1% BSA, followed by washing. Lung homogenates were serially diluted in PBS / T / B, starting with a 1:30 dilution. After incubation for 2 hours and washing, conjugated IgA was detected using MT39A-ALP conjugated antibody (1:1000) according to the manufacturer's protocol. Plates were read at 415 nm. The endpoint titer was considered as the x-intercept of the dilution curve at an absorbance value 3 × standard deviations greater than the absorbance of naive mouse serum. For non-responsive animals, a titer of 15 or half the value of the lowest dilution tested was set.

[0160] T cell response The spleen was removed and placed in 5 ml of Hanks equilibrium salt solution (containing 1 M HEPES and 5% FBS), then passed through a sterile strainer using a 5 ml syringe. After RBC lysis (Ebiosolutions), resuspension, and counting, the cells were ready for analysis. To stimulate the cells, they were cultured overnight at 5e5 cells / well with two peptide pools (Genscript) equivalent to 1 μg / ml of full-length S protein. The culture medium consisted of RPMI medium (Lonza) containing 0.01 M HEPES, 1 × L-glutamine, 1 × MEM basic amino acids, 1 × penicillin-streptomycin (penstrep), 10% FBS, and 5.5e-5 mol / l β-mercaptoethanol. Antigen-specific IFN-γ ELISPOT was measured using a Mabtech kit. After staining with appropriate antibodies, flow cytometry analysis was performed using an Attune flow cytometer and Flow Jo version 10.7.1. For flow cytometry, 2e6 splenocytes per well were incubated at 37°C for 18 hours with a peptide pool equivalent to either 1 or 5 ug / ml of full-length S, and brefelzin A (ThermoFisher) was added during the last 4 hours of incubation. The antibodies used were CD4 conjugated with APC-H7, CD8 conjugated with FITC, CD3 conjugated with BV650, IFN-y conjugated with PerCP-Cy5.5, IL-2 conjugated with BV421, TNFa conjugated with PE-Cy7, IL-4 conjugated with APC, CD44 conjugated with Alexa Fluor, and CD62L conjugated with PE (BD biosciences).

[0161] References for Example 5 TIFF2026086500000003.tif47160TIFF2026086500000004.tif237160TIFF2026086500000005.tif241160 TIFF2026086500000006.tif227160TIFF2026086500000007.tif230160TIFF2026086500000008.tif198161

[0162] Example 6 Study VXA-COV2-101 was a phase 1 open-label dose-ranging trial to evaluate the safety and immunogenicity of the SARS-CoV-2 oral tablet vaccine (rAd-SN, SEQ ID NO:10), which was referred to as VXA-CoV2-1 in Examples 6 and 7 and administered to healthy adult subjects aged 18–55 years.

[0163] The objective of this study was to evaluate the safety and immunogenicity of an enteric-coated oral VXA-CoV2-1 vaccine.

[0164] Participants were enrolled in a single Phase 1 unit in Southern California. After completing eligibility screening and confirmation, 35 participants were enrolled in this trial; sentinel participants (n=5) in Cohort 1 received vaccination on day 1 and a repeat dose on day 29. Participants in Cohorts 2 and 3 received a single dose on day 1. The study design is shown in the table below.

[0165] Research design for VXA-COV2-101 TIFF2026086500000009.tif42145

[0166] Sentinel subjects in Cohort 1 received a second dose (boost) on day 29 at the same dose level as the first dose.

[0167] B cell / antibody analysis The ability of VXA-CoV2-1 to promote B cells with high antibody-producing capacity was evaluated using both flow cytometry-based measurements and ELISPOT antibody-secreting cell (ASC) assays. It is well-established that B cells responding to vaccination become activated in the administration site and local inflow area lymph nodes, where they differentiate into plasmablasts after germinal center response. Between 6 and 8 days after immunization, a significant proportion of plasmablasts leave the germinal centers and transiently appear in the peripheral circulation, where they can be found to be highly rich in vaccine antigen-specific antibody-secreting cells (ASCs). Thus, in the VXA-CoV2-101 study, flow cytometry analysis of fixed whole blood samples collected before and after vaccination revealed a significant expansion of the overall CD27++CD38++ plasmablast population on day 8 post-vaccination, with approximately 70% (24 / 35) of vaccinated individuals showing a more than twofold increase in plasmablast frequency compared to baseline levels (Figure 9A-B). Further studies have shown upregulation of both IgA and the mucosal homing receptor α4β7 on the surface of circulating plasmablasts after vaccination, particularly in cohorts given higher dose levels of VXA-CoV2-1 (Figure 9C), thus suggesting vaccine-induced migration of this IgA-producing B cell population to mucosal tissue (Mora and von Andrian, 2008). Overall, these results are consistent with previous data published by the company in a Phase II influenza A attack infection study in humans, where the generation of IgA plasmablasts with similar mucosal features after oral influenza vaccination was found to be a strong indicator of vaccine-induced protection (Liebowitz et al., 2020).

[0168] Furthermore, the ELISpot assay was used to measure the ability of VXA-CoV2-1 to induce circulating antibody-secreting B cells capable of recognizing and binding to the S1 domain of the SARS-CoV-2 spike (S) antigen. This analysis demonstrated significant vaccine-inducible generation of S1-responsive IgA-secreting ASCs 8 days after initial immunization (Wilcoxon test, p=0.0002), with an overall median increase of fourfold compared to baseline levels (Figure 9D). More specifically, 8 / 12 (67%) of subjects in the lower-dose vaccine group for whom ASC measurements were available on both day 1 and day 8 were classified as “responders” as indicated by a median increase of more than twofold (2.67-fold increase in median IgA-secreting ASC counts per million cells) at day 8 post-vaccination compared to pre-vaccination levels (95% CI: 1.0–13.32). A slightly higher percentage of respondents (11 / 15 of subjects, 73%) were recorded in the higher vaccine dose cohort (a four-fold increase from the median; 95% CI: 1.3–13.32).

[0169] Using the Meso Scale Discovery (MSD) platform, levels of IgA antibodies specific to different SARS-CoV-2 antigens were measured in serum, saliva, and nasal samples before and after immunization. Consistent with the mucosal features of B-cell responses observed by flow cytometry and ELISPOT, IgA antibodies targeting the SARS-CoV-2 spike (S), nucleoprotein (N), and spike receptor-binding domain (RBD) could be found in both serum and mucosal compartments. Overall, 23% (8 / 35) of vaccinated subjects showed a vaccine-specific IgA increase of ≥50% in serum by day 29, and 6 / 8 of these subjects produced IgA targeting all three SARS-CoV-2 antigens in analysis. Consistent with the measurement of IgA+ B7+ plasmablasts, subjects in the higher-dose cohort showed a higher IgA antibody response specific to S in serum (Figure 9). As expected, considering the unique characteristics of the VXA-CoV2-1 oral vaccine candidate, a higher percentage of vaccinated individuals initiated a SARS-CoV-2 specific IgA antibody response in the mucosal compartment compared to serum, with 54% (19 / 35) of vaccinated individuals achieving a more than twofold increase in mucosal IgA in either saliva or nasal samples (Figure 9F). More specifically, 10 / 35 (29%) of vaccinated individuals had a more than twofold increase in IgA antibodies in saliva, while 12 / 35 (35%) reached the same threshold in the nasal compartment by 29 days post-vaccination. No significant difference in vaccine-specific IgA response in saliva or nasal samples was observed between the two dose groups (Figure 9F). Due to limitations in mucosal samples, measuring the neutralizing ability of IgA is difficult, but preliminary findings suggest that subjects with a twofold increase in specific nasal IgA also had the ability to neutralize in a surrogate neutralization assay that measured ACE2 binding to the spike protein (Figure 9F). As reported by Sterlin et al. (2021), secretory IgA has been shown to have a higher neutralizing ability against SARS-CoV-2 than IgG.

[0170] These findings are promising, as several reports highlight the potential of mucosal immunity and IgA antibody production to contribute to protection against COVID-19 (Ejemel et al., 2020; Russell et al., 2020; Sterlin et al., 2021). Notably, while injectable vaccines are not designed to effectively induce IgA antibodies in the respiratory mucosa, oral vaccination strategies may offer this advantage (Jeyanathan et al., 2020). Induction of a SARS-CoV-2 specific IgA response on key mucosal surfaces may also have a greater ability to block viral transmission, inducing bactericidal immunity and being a particularly desirable feature in scenarios where novel SARS-CoV-2 variants can replicate undetected in vaccinated subjects.

[0171] Analysis of IgG antibodies in serum after vaccination showed no increase in SARS-CoV-2 specific antibody response. Similarly, no significant SARS-CoV-2 antibody-mediated neutralization was observed in serum. The underlying reason for the absence of vaccine-specific IgG and antibody neutralization in serum is not currently defined, but it is possible that a single oral dose of VXA-CoV2-1 at the dose levels used in this study was insufficient to induce a robust vaccine-specific IgG neutralization response. Furthermore, it cannot be ruled out that the presence of the gene encoding N in the VXA-CoV2-1 construct may have distorted the immunogenicity profile of this vaccine candidate from serum neutralizing antibodies to T cell-mediated immunity.

[0172] T cell analysis In addition to B cells and antibodies, T cells also play a crucial role in the development of protective immune responses against many microbial infections. In COVID-19, T cells have been shown to target multiple SARS-CoV-2 proteins in convalescent subjects, while T cells appear to be less vulnerable to SARS-CoV-2 variants compared to antibodies (Grifoni et al., 2020; Ledford, 2021; Tarke et al., 2021).

[0173] The induction of SARS-CoV-2-specific T cells and Th1 / Th2 polarization after vaccination with VXA-CoV2-1 was measured using a restimulation assay and evaluated Th1 / Th2 cytokine response with peripheral blood mononuclear cells (PBMCs) from 26 pairs of samples collected pre-vaccination and 8 days post-vaccination and cultured with either S or N-derived SARS-CoV-2 peptides. PBMCs were thawed, rested overnight, and cultured with S or N peptide libraries (Miltenyi) in 1 × 10⁶ wells of Immunocult medium (Stemcell Technologies) in 96-well round-bottom plates in the presence of brefeldin A (Invitrogen) and monensin (Biolegend). 7 Cells were cultured at a concentration of cells / ml at 37°C for 5 hours. Cells were harvested and surface-stained with CD4-BV605, CD8-BV785, and zombie near-infrared viability dye (Biolegend). After fixation with 4% PFA (biotium) and permeabilization with Cytoperm (BD Biosciences), intracellular cytokine responses were evaluated using antibodies against cytokines: IFNγ-BV510 (Biolegend), TNFα-e450 (Thermofisher), IL-2-APC (Thermofisher), IL-4-PerCP (Biolegend), IL-5-PE (Biolegend), IL-13 (Biolegend), and CD107a-Alexa488 (Thermofisher). Analysis was performed using an Attune (Thermofisher) flow cytometer.

[0174] CD4 + A significant increase in the Th2 response, defined as intracellular production of IL5 / IL4 / IL13 cytokines by T cells, was not observed in any post-vaccination sample, in either the S or N response, compared to pre-vaccination levels. This is significant because earlier reports in this field assumed potential adverse events associated with antibody-dependent enhancement of infection (ADE) after Th2 polarization (Lee et al., 2020).

[0175] Notably, the majority of the vaccinated individuals in this study responded to restimulation with the S peptide, particularly CD8 + Not only from T cells (Figure 10A, C), but also from CD4 + T cells (Figure 10B) also showed a significant increase in the Th1 response, defined as intracellular production of IFNγ / TNFα cytokines and the degranulation marker CD107a, on day 8 after vaccination. In particular, 13 / 26 (50%) of subjects showed a more than twofold increase in Th1 cytokines in response to S, and 19 / 26 (73%) of subjects showed measurable cytokine production above baseline levels overall in CD8 + A T cell response was observed (Figure 10D). The increase in Th1 cytokine production after vaccination was on a smaller scale compared to the S-specific response, but was particularly pronounced in CD8 + T cells were also found to have increased levels of the Th1 cytokine CD8 after restimulation with SARS-CoV-2 N peptide (Figures 10E-F). More specifically, 9 / 26 (35%) of subjects showed increased levels of the Th1 cytokine CD8 after N restimulation on day 8 compared to baseline levels. + T cell production more than doubled. Overall, T cells induced after oral immunization with VXA-CoV2-1 showed markers of antiviral functionality in response to SARS-CoV-2 peptides, particularly IFNγ-producing CD8. +The percentage of T cells is noteworthy. This percentage of responsive CD8 T cells has not been reported for mRNA vaccines currently used against COVID-19. This could mean a very important advantage because CD8 T cells possess cytotoxic capabilities. + T cells are located in a unique position to eliminate virus-infected cells and may also help reduce transmission by decreasing the viral load in infected patients (Ledford, 2021). Follow-up analyses are expected to focus on a more direct comparison of vaccine-induced T cell immunity between VXA-CoV2-1 and SARS-CoV-2 mRNA vaccines (those that received EUA in the US).

[0176] In conclusion, VXA-CoV2-1 stimulates SARS-CoV-2 specific IgA antibodies and vaccine-specific T cells against both SARS-CoV-2 vaccine antigens S and N in key mucosal sites, particularly IFNγ-producing CD8 + By inducing T cells, it was involved in both the humoral and cellular arms of the immune system.

[0177] Another common goal of coronavirus vaccines is not only to protect against the current lineage, but also to protect against other circulating human coronavirus lineages and create a pan-coronavirus vaccine. To study this, we evaluated whether VXA-CoV2-1 induces T cells specific to four intrinsic human coronaviruses: 229E, HKU1, OC43, and NL63. For all four intrinsic human coronaviruses, we found increases above pre-vaccination levels (Figure 11), suggesting that the induced T cells are cross-reactive with circulating human coronaviruses.

[0178] Example 7 To compare the response induced by VXA-COV2-1 with that of current leading intramuscular COVID vaccines, subjects scheduled to receive mRNA vaccines were recruited and provided PBMCs (Patient-Based Microorganisms). PBMCs were collected at the same time points as our vaccinated subjects, i.e., pre-vaccination and 7 days post-vaccination, and T-cell activity was measured using the same in vitro assay as PBMCs derived from the VXA-COV1-101 clinical trial. Samples from all three vaccines were subjected to the same assay and analysis to control for assay variability.

[0179] Surprisingly, subjects who took VXA-COV2-1 tablets were found to have a T-cell response that was typically an order of magnitude higher than subjects who were intramuscularly vaccinated with either the Pfizer or Moderna vaccine, which are approved under emergency use authorization.

[0180] IFNγ and TNFα release from CD8 T cells was significantly increased (Figure 12A), and CD107a degranulation showed a small increase compared to baseline before vaccination. The mean percentage increase in IFNγ from CD8 T cells from day 1 onwards for vaccinated individuals was 0.4 / 0.09 / 2.3 for Pfizer / Moderna / Vaxart vaccinated individuals, respectively. This represents a >5-fold increase for those who took VXA-CoV-2 tablets compared to those who received intramuscular vaccines.

[0181] Because only a small subgroup of seven subjects was tested using the same assay as other vaccines, to account for potential bias in subject selection, the entire cohort measured previously was graphed together for comparison, and significance is still observed when the entire cohort is compared to the comparative experiment (Figure 12B). The mean of the entire cohort, including non-responders, was measured at 1.5%, and a >3.5-fold increase was still observed compared to those who received the intramuscular vaccine. The mean IFNγ response of the four convalescent subjects was 0.8. A representative facs plot showing the increase in VXA-CoV2-1 subjects 7 days post-vaccination is shown in Figure 12C. T-cell measurements reported from the intramuscular vaccine were taken at 7 days post-second vaccine administration. To explain this, PBMCs were also measured at 7 days post-second administration using the same assay, and it was found that PBMCs had responses of equal magnitude at both time points, with the exception of one subject who had a particularly good T-cell response with an increase at both time points (Figure 12D). This is similar to the data reported by Pfizer (Sahin et al. Nature 2021).

[0182] Further references cited in Examples 5-7 TIFF2026086500000010.tif118160TIFF2026086500000011.tif149160

[0183] Table of arrays TIFF2026086500000012.tif73159TIFF2026086500000013.tif241159TIFF2026086500000014.tif218159TIFF2026086500000015.tif243159TIFF2026086500000016.tif240159TIFF2026086500000017.tif242159TIFF2026086500000018.tif232159TIFF2026086500000019.tif240164TIFF2026086500000020.tif242159TIFF2026086500000021.tif241159TIFF2026086500000022.tif242160TIFF2026086500000023.tif240159TIFF2026086500000024.tif242159TIFF2026086500000025.tif242159TIFF2026086500000026.tif242159TIFF2026086500000027.tif242159TIFF2026086500000028.tif242159TIFF2026086500000029.tif242159TIFF2026086500000030.tif242159TIFF2026086500000031.tif242159TIFF2026086500000032.tif242159TIFF2026086500000033.tif242159TIFF2026086500000034.tif242159TIFF2026086500000035.tif242159TIFF2026086500000036.tif237159TIFF2026086500000037.tif240164TIFF2026086500000038.tif243159TIFF2026086500000039.tif242159TIFF2026086500000040.tif242159TIFF2026086500000041.tif242159TIFF2026086500000042.tif242160TIFF2026086500000043.tif242159TIFF2026086500000044.tif242159TIFF2026086500000045.tif242160TIFF2026086500000046.tif242159TIFF2026086500000047.tif242159TIFF2026086500000048.tif242159TIFF2026086500000049.tif242159TIFF2026086500000050.tif240159TIFF2026086500000051.tif242159TIFF2026086500000052.tif242159TIFF2026086500000053.tif242159TIFF2026086500000054.tif242159TIFF2026086500000055.tif242160TIFF2026086500000056.tif242159TIFF2026086500000057.tif242159TIFF2026086500000058.tif242159TIFF2026086500000059.tif242160TIFF2026086500000060.tif242159TIFF2026086500000061.tif242159TIFF2026086500000062.tif242159TIFF2026086500000063.tif237159TIFF2026086500000064.tif244159TIFF2026086500000065.tif225159TIFF2026086500000066.tif241159TIFF2026086500000067.tif241161TIFF2026086500000068.tif232159.

[0184] Additional references TIFF2026086500000069.tif223159TIFF2026086500000070.tif208159

[0185] The examples and embodiments described herein are for illustrative purposes only, and it is understood that various modifications or alterations will be suggested to those skilled in the art in view thereof, and that such modifications or alterations will fall within the scope of the intent and scope of this application and the appended claims. All publications, patents and patent applications referenced herein are incorporated herein by reference in their entirety for all purposes.

[0186] Sequence information SEQUENCE LISTING <110> VAXART, INC. <120> Chimeric Adenoviral Vectors <150> US 63 / 144,339 <151> 2021-02-01 <150> US 63 / 074,954 <151> 2020-09-04 <150> US 63 / 045,710 <151> 2020-06-29 <150> US 63 / 035,490 <151> 2020-06-05 <160> twenty four <170> PatentIn version 3.5 <210> 1 <211> 1273 <212> PRT <213> Severe acute respiratory syndrome coronavirus 2 <400> 1 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 705 710 715 720 Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln 980 985 990 Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005 Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 1010 1015 1020 Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1025 1030 1035 Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065 Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1070 1075 1080 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095 Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 1100 1105 1110 Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125 Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140 Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155 His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170 Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185 Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1190 1195 1200 Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu 1205 1210 1215 Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met 1220 1225 1230 Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro 1250 1255 1260 Val Leu Lys Gly Val Lys Leu His Tyr Thr 1265 1270 <210> 2 <211> 419 <212> PRT <213> Severe acute respiratory syndrome coronavirus 2 <400> 2 Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr 1 5 10 15 Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg 20 25 30 Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn 35 40 45 Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu 50 55 60 Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro 65 70 75 80 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 85 90 95 Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr 100 105 110 Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln 145 150 155 160 Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser 165 170 175 Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn 180 185 190 Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala 195 200 205 Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu 210 215 220 Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln 225 230 235 240 Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys 245 250 255 Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln 260 265 270 Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp 275 280 285 Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile 290 295 300 Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile 305 310 315 320 Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala 325 330 335 Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu 340 345 350 Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro 355 360 365 Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln 370 375 380 Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu 385 390 395 400 Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser 405 410 415 Thr Gln Ala <210> 3 <211> 3840 <212> DNA <213> Severe acute respiratory syndrome coronavirus 2 <400> 3 ggtaccgcca ccatgtttgt ttttctcgta ctcctgcccc tggtttcctc ccaatgtgtc 60 aatctgacta cccggaccca acttcctccc gcctacacca attcctttac ccgaggtgtt 120 tactacccag acaaagtgtt caggtcatcc gtcctccata gtacccaaga cctcttcctc 180 cctttttttt ctaacgttac ctggtttcac gctattcacg ttagcggcac caacggcacc 240 aaaagattcg ataaccccgt actgccgttc aacgacgggg tatattttgc ctctactgaa 300 aaatcaaaca tcatacgcgg atggatcttt gggactaccc tggactcaaa aactcagtcc 360 ctgctgattg tgaataacgc taccaacgtg gtgatcaaag tctgtgaatt ccagttttgc 420 aacgatcctt ttctcggcgt ttattatcac aaaaataaca aatcctggat ggagagcgag 480 ttccgggtgt actcctccgc gaataattgc accttcgaat atgtgtctca gccattcctc 540 atggacctcg aggggaagca gggcaatttt aagaatctgc gagaattcgt gttcaagaat 600 atagacggtt acttcaagat ttactccaaa cacaccccga ttaacctggt tagggacttg 660 cctcagggct tttctgcatt ggagcccctc gtggacctcc caatcggcat aaacattaca 720 agatttcaga ctttgcttgc attgcacagg agctatttga cacccggcga ttcttcttcc 780 ggatggaccg ctggagcagc tgcttattac gtgggctatc tgcagcctcg aacctttctt 840 ttgaagtaca acgaaaatgg aactatcacc gatgcagttg actgcgccct ggaccccctg 900 tccgaaacta agtgcacgct caaaagtttc acagtagaga aggggatata ccagactagc 960 aatttccgcg ttcagccaac cgaaagtata gtgcgctttc ctaatataac taacctgtgt 1020 cctttcgggg aagtgtttaa cgccactaga ttcgcttccg tctacgcctg gaatagaaag 1080 aggatctcaa attgcgttgc tgactatagt gttttgtaca attccgcctc tttctcaacc 1140 ttcaaatgtt acggggtgag ccctaccaaa ctgaacgacc tgtgctttac aaacgtatac 1200 gccgacagct ttgttatcag aggagacgag gttcgccaga ttgctccggg tcagacaggc 1260 aagattgctg attataatta caaactgccc gacgacttta caggatgtgt gatcgcgtgg 1320 aacagtaaca atcttgactc aaaggttggg ggtaattata attatcttta ccggctgttc 1380 agaaaaagca atttgaaacc cttcgaaagg gacatatcca ccgagatcta tcaggccggg 1440 tccactccat gcaatggtgt ggaaggtttt aattgctact tcccattgca gtcttatgga ttccaaccaa ccaatggcgt aggctaccag ccgtatcgcg ttgtcgtgct cagcttcgag ctgctccacg cccccgcgac cgtatgcggt cctaagaagt ccaccaatct tgttaagaac aagtgtgtaa actttaactt taacggggctg accgggaccg gcgttctgac tgaatctaac aaaaaattcc tgcctttcca gcagttcggc cgcgatattg ctgacaccac tgacgctgta 1740. agagaccctc agacccttga aattctcgat atcacacctt gcagctttgg gggcgtgtcc gtcatcactc caggactaa cacaagcaac caggtggcag tgttgtacca ggatgttaat tgtaccgagg tgccagtggc catccacgcc gatcaattga cacctacctg gagggttc agcacagggt ccaatgtttt tcagacaaga gccggatgtc tgatcggtgc cgagcatgtc aacaattcct acgagtgtga tatccccatt ggtgcggga tttgtgcatc atatcagacc cagactaata gcccaagaag agctagatcc gtcgctagtc aatccatcat tgcatataca atgtccctgg gagctgagaa ttcagtcgcg tattcaaaca attccattgc tattcctact aatttcacta tctccgtcac gaccgagatc ctgccagttt ccatgactaa gacttctgtt 2220 gactgcacca tgtatatctg tggcgatagc accgagtgca gtaatctgct tctgcagtac 2280 ggctccttct gcacacaact caatcgagca ctgaccggta ttgcagttga gcaggacaag 2340 aacacacagg aggtctttgc acaggtcaaa caaatttaca aaaccccccc cataaaagac 2400 tttggtgggt tcaacttcag ccaaatcctc ccagatccca gcaagccctc caaaagatcc 2460 ttcatcgaag accttttgtt caataaggta accctggccg acgcaggctt catcaaacaa 2520 tatggcgatt gccttggaga cattgctgcg cgcgatttga tctgtgctca gaaatttaac 2580 ggtttgaccg tgctgccccc acttctgact gatgagatga tagcacagta tacttctgct 2640 cttctggcag gaacaatcac ttccgggtgg acctttggcg ctggtgcagc actgcaaatc 2700 cccttcgcaa tgcaaatggc ctaccgattc aatggtattg gtgttaccca gaacgtgctc 2760 tatgagaatc agaaactcat cgccaatcag ttcaatagcg ctattggcaa gattcaggat 2820 tccctcagct ctaccgccag cgctctgggg aagctccagg acgtggtgaa ccaaaatgct 2880 caagcgctca atacccttgt gaaacagctc agctccaatt ttggcgcaat tagcagcgtt 2940 ctgaatgata ttctgtcccg gctggcaag gtagaagcag aagtccagat cgacaggctg 3000 atcaccgggc ggttgcagag tctccagacc tatgtcacac aacagctgat ccgcgccgcc 3060 gagatcaggg cttccgctaa cctggccgcc actaagatgt ccgaatgcgt gttggggcag 3120 agtaagcggg tcgacttttg cgggaaggga taccatctga tgagcttccc tcagtctgca 3180 ccccacggag tagtgttcct ccacgtcaca tatgtgcccg ctcaggaaaa gaatttcaca 3240 accgcacctg ctatctgtca cgacggcaag gccactttc ctagagaagg agttttcgta 3300 tctaacggca cccactggtt cgtgacacag cggaactttt acgagcctca gattataact 3360 aggcaaca ctttcgtgtc aggcaactgt gacgtggtga ttgggatcgt gaacaacaca 3420 gtctacgacc cattgcagcc cgagttggac tccttcaaag aggagcttga tagtatttc 3480 aagaaccata cctctcccga cgtggacctg ggggacatta gcggcatcaa tgcatccgtt 3540 gtgaatatcc agaagaaat cgataggctg aatgaggtcg caaaaaatct taatgagtca 3600 ctgattgatc tgcaggaact cggcaaatat gagcagtata ttaagtggcc gtggtacata 3660 tggctcggct ttatcgccgg tctgattgcc atcgtgatgg tgaccattat gctgtgttgt 3720 atgacaagct gctgttcatg tctcaaagga tgctgctcct gcggtagctg ctgtaagttc 3780 gatgaagacg acagtgagcc cgtgctcaaa ggagtgaaac tccactacac ataacgatcg 3840 <210> 4 <211> 1278 <212> DNA <213> Severe acute respiratory syndrome coronavirus 2 <400> 4 ggtaccgcca ccatgtccga taacggcccc cagaatcaga gaaacgctcc ccgcatcacg 60 ttcggcggac caagtgacag cacaggcagt aaccagaacg gagaacgctc cggtgctcgc 120 tccaagcagc gacggccgca agggcttccc aacaataccg ccagctggtt tacggctctg 180 acccaacacg ggaaagaaga tcttaaattc cccaggggcc agggcgtccc tatcaatact 240 aactccagcc cggatgatca gataggctac tatagacgcg ctacccgacg gatacgaggg 300 ggggacggca aaatgaagga cctttccccc cggtggtatt tctattactt gggcaccgga 360 ccagaagccg gactgcctta cggcgctaac aaagacggaa taatctgggt tgcgacggag 420 ggcgccctga atacacctaa agaccatatc ggcacaagaa atcctgctaa caatgccgcg 480 attgtgctcc agctgcctca gggaaccacg ctgcctaaag gttttaccc tgaggggtca 540 aggggggga gtcaagcgtc tagtaggtca tcctctcgct ctcgcaatag tcccggac 600 tcaccccag gcagcagcag aggaacctct cccgcacgga tggctggcaa tggggcat 660 gctgcccttg ctctccttct gctggatcgc cttaaccagc tcgaatcaa gatgtctgga 720 aaaggtcagc agcagcaagg ccagaccgtg aaagaga gtgcagctga agctagtaaa 780 aaagccacgcc aaaaacggac cgcaactaag gcatatacg taacacaggc cttcggcaga 840 agaggtccag aaaaacaca gggaacttt ggcgatcaag agctgattag acaggcaca 900 gattacaaac actggccaca gatcgcgcag tttgcaccaa gcgccctctgc attctcggg 960 atgagtcgga ttgggatgga agtcactcca tccgggacct ggcttaccta cacaggggca 1020 aaaactcg acgaacaaga cccaacttt aagatcagg tcatcctgct gaataacac 1080 atcgatgcct acaaaacttt ccccccaacc gaaccaaaga aagacaagaa aaaaaaggca 1140 gacgaaacgc aagcgctccc tcagcgccag aagaagcagc agaccgttac actgttgcca 1200 gcagcagatc tggatgattt ttccaagcag cttcaacaga gtatgtcaag cgctgacagc 1260 actcaggctt gacgatcg 1278 <210> 5 <211> 3369 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 5 ggtaccgcca ccatgtttgt ttttctcgta ctcctgcccc tggtttcctc ccaatgtgtc 60 aatctgacta cccggaccca acttcctccc gcctacacca attcctttac ccgaggtgtt 120 tactacccag acaaagtgtt caggtcatcc gtcctccata gtacccaaga cctcttcctc 180 cctttttttt ctaacgttac ctggtttcac gctattcacg ttagcggcac caacggcacc 240 aaaagattcg ataaccccgt actgccgttc aacgacgggg tatattttgc ctctactgaa 300 aaatcaaaca tcatacgcgg atggatcttt gggactaccc tggactcaaa aactcagtcc 360 ctgctgattg tgaataacgc taccaacgtg gtgatcaaag tctgtgaatt ccagttttgc 420 aacgatcctt ttctcggcgt ttattatcac aaaaataaca aatcctggat ggagagcgag 480 ttccgggtgt actcctccgc gaataattgc accttcgaat atgtgtctca gccattcctc 540 atggacctcg agggggaagca gggcaatttt aagaatctgc gagaattcgt gttcaagaat 600 atagacggtt acttcaagat ttactccaaa cacaccccga ttaacctggt tagggacttg 660 cctcagggct tttctgcatt ggagcccctc gtggacctcc caatcggcat aaacattaca 720 agatttcaga ctttgcttgc attgcacagg agctatttga cacccggcga ttcttcttcc 780 ggatggaccg ctggagcagc tgcttattac gtgggctatc tgcagcctcg aacctttctt 840 ttgaagtaca acgaaaatgg aactatcacc gatgcagttg actgcgccct ggaccccctg 900 tccgaaacta agtgcacgct caaaagtttc acagtagaga agggatata ccagactagc 960 aatttccgcg ttcagccaac cgaaagtata gtgcgctttc ctaatataac taacctgtgt 1020 cctttcgggg aagtgtttaa cgccactaga ttcgcttccg tctacgcctg gaatagaaag aggatctcaa attgcgttgc tgactatagt gttttgtaca attccgcctc tttctcaacc ttcaaatgtt acggggtgag ccctaccaaa ctgaacgacc tgtgctttac aaacgtatac gccgacagct ttgttatcag aggagacgag gttcgccaga ttgctccggg tcagacaggc 1260 aagattgctg attack caaactgccc gacgacttta caggatgtgt gatcgcgtgg aacagtaaca atcttgactc aaaggttggg ggtaattata attatcttta ccggctgttc agaaaaagca atttgaaacc cttcgaaagg gacatatcca ccgagatcta tcaggccggg tccactccat gcaatggtgt ggaaggtttt aattgctact tcccattgca gtcttatgga ttccaaccaa ccaatggcgt aggctaccag ccgtatcgcg ttgtcgtgct cagcttcgag ctgctccacg cccccgcgac cgtatgcggt cctaagaagt ccaccaatct tgttaagaac aagtgtgtaa actttaactt taacggggctg accgggaccg gcgttctgac tgaatctaac aaaaaattcc tgcctttcca gcagttcggc cgcgatattg ctgacaccac tgacgctgta 1740. agagaccctc agacccttga aattctcgat atcacacctt gcagctttgg gggcgtgtcc 1800 gtcatcactc caggaactaa cacaagcaac caggtggcag tgttgtacca ggatgttaat 1860 tgtaccgagg tgccagtgc catccacgcc catcattga cactacctg gagggtttac 1920 agcacagggt ccaatgtttt tcagacaaga gccggatgtc tgatcggtgc cgagcatgtc 1980 aacaattcct acgagtgtga tatccccatt ggtgcgggaa ttgtgcatc atatcagacc 2040 cagactaata gcccaagaag agctagatcc gtcgctagtc atccatcat tgcatataca 2100 atgatgtccg ataacggccc ccagaatcag agaacgctc cccgcatcac gttcggcgga 2160 ccaagtgaca gcacaggcag taaccagac ggagaacgct ccggtgctcg ctccaagcag 2220 cgacggccgc aagggctcc gccacatacc gccagctggt tacggctct gacccacac 2280 gggaaagaag atctttaatt ccccaggggc cagggcgtcc ctacaatac taactccagc 2340 ccggatgatc agataggcta ctatagacgc gctacccgac ggatacgagg gggggacggc 2400 aaaatgaagg accttcccc ccggtggtat ttctattact tggcaccgg accagaagcc 2460 ggactgcctt acggcgctaa caagacgga ataatctgggg ttgcgacgga gggcgccctg 2520 atacaccta agaccatat cggcacaaga aatcctgcta acatgccgc gattgtgctc 2580 cagctgcctc agggaccac gctgcctaaa gggttttacg ctgaggggtc aagggggggg 2640 agtcaagcgt ctagtaggtc atcctctcgc tctcgcaata gttcccggaa ctcacccca 2700 ggcagcagca gaggaacctc tcccgcacgg atggctggca atgggggaga tgctgccctt 2760 gctctccttc tgctggatcg ccttaaccag ctcgaatcaa agatgtctgg aaaggtcag 2820 cagcagcaag gccagaccgt gapagaag agtgcagctg aagctagtaa aaagccacgc 2880 caaaaacgga ccgcaacta ggcatataac gtaacacagg ccttcggcag agaggtcca 2940 gaaacac aggaactt tggcgatcaa gagctgatta gaggggcac agattacaa 3000 cactggccac agatcgcgca gtttgcacca agcgccctg cattctcgg gatgagtcgg 3060 attgggatgg aagtcactcc atccgggacc tggcttacct acacaggggc aaaaactc 3120 gacgacaag acccaactt windowcag gtcatcctgc tgaataaca catcgatgcc 3180 tacaaaactt tccccccaac cgaaccaaag aaagacaaga aaaaaaaggc agacgaaacg 3240 caagcgctcc ctcagcgcca gaagaagcag cagaccgtta cactgttgcc agcagcagat 3300 ctggatgatt tttccaagca gcttcaacag agtatgtcaa gcgctgacag cactcaggct 3360 tgacgatcg 3369 <210> 6 <211> 5752 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 6 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa 60 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata 120 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag 180 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc 240 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta 300 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg 360 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt 420 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca 480 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag 540 gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc 600 tgttttgacc tccatagaag acaccgggac cgatccagcc tgactctagc ctagctctga 660 agttggtggt gaggccctgg gcaggttggt atcaaggtta caagacaggt ttaaggagac 720 caatagaaac tgggcatgtg gagacagaga agactcttgg gtttctgata ggcactgact 780 ctctctgcct attggtctat tttcccaccc ttaggctgct ggtctgagcc taggagatct 840 ctcgaggtcg acggtatcga tgggtaccgc caccatgttt gtttttctcg tactcctgcc 900 cctggtttcc tcccaatgtg tcaatctgac tacccggacc caacttcctc ccgcctacac 960 caattccttt acccgaggtg tttactaccc agacaaagtg ttcaggtcat ccgtcctcca 1020 tagtacccaa gacctcttcc tccctttttt ttctaacgtt acctggtttc acgctattca 1080 cgttagcggc accaacggca ccaaaagatt cgataacccc gtactgccgt tcaacgacgg 1140 ggtatatttt gcctctactg aaaaatcaaa catcatacgc ggatggatct ttgggactac 1200 cctggactca aaaactcagt ccctgctgat tgtgaataac gctaccaacg tggtgatcaa 1260 agtctgtgaa ttccagtttt gcaacgatcc ttttctcggc gtttattatc acaaaaataa 1320 caaatcctgg atggagagcg agttccgggt gtactcctcc gcgaataatt gcaccttcga 1380 atatgtgtct cagccattcc tcatggacct cgaggggaag cagggcaatt ttaagaatct 1440 gcgagaattc gtgttcaaga atatagacgg ttacttcaag atttactcca aacacacccc 1500 gattaacctg gttagggact tgcctcaggg cttttctgca ttggagcccc tcgtggacct 1560 cccaatcggc ataaacatta caagatttca gactttgctt gcattgcaca ggagctattt 1620 gacacccggc gattcttctt ccggatggac cgctggagca gctgcttatt acgtgggcta 1680 tctgcagcct cgaacctttc ttttgaagta caacgaaaat ggaactatca ccgatgcagt 1740 tgactgcgcc ctggaccccc tgtccgaaac taagtgcacg ctcaaaagtt tcacagtaga 1800 gaagggata taccagacta gcaatttccg cgttcagcca accgaagta tagtgcgctt 1860 tcctaatata actaacctgt gtccttcgg ggaagtgttt aacgccacta gattcgctc 1920 cgtctacgcc tggaatagaa agaggatctc aaattgcgtt gctgactata gtgttttgta 1980 cavattccgcc tctttctca ccttcaatg ttacggggtg agccctacca aactgaacga 2040 cctgtgcttt acaacgtat acgccgacag ctttgttac agaggagacg aggttcgcca 2100 gattgctccg gtcagacag gcaagattgc tgattataat tacaactgc ccgacgactt 2160 tacaggatgt gtgatcgcgt ggaacagtaa caatctgac tcaaggttg ggggtatta 2220 taattatctt taccggctgt tcagaaaag caatttgaa cccttcgaaa gggacatatc 2280 caccgagatc tatcaggccg gtccactc atgcaatggt gtggaaggtt ttaattgcta 2340 cttcccattg cagtcttatg gattccacc aaccaatggc gtaggctacc agccgtatcg 2400 cgttgtcgtg ctcagctcg agctgctcca cgcccccgcg accgtagcg gtcctaagaa 2460 gtccaccaat cttgttaaga acagtgtgt aaactttaac tttaacgggc tgaccgggac 2520 cggcgttctg actgaatcta acaaaaaatt cctgcctttc cagcagttcg gccgcgatat 2580 tgctgacacc actgacgctg taagagaccc tcagaccctt gaaattctcg atatcacacc 2640 ttgcagcttt gggggcgtgt ccgtcatcac tccaggaact aacacaagca accaggtggc 2700 agtgttgtac caggatgtta attgtaccga ggtgccagtg gccatccacg ccgatcaatt 2760 gacacctacc tggagggttt acagcacagg gtccaatgtt tttcagacaa gagccggatg 2820 tctgatcggt gccgagcatg tcaacaattc ctacgagtgt gatatcccca ttggtgcggg 2880 aatttgtgca tcatatcaga cccagactaa tagcccaaga agagctagat ccgtcgctag 2940 tcaatccatc attgcatata caatgtccct gggagctgag aattcagtcg cgtattcaaa 3000 caattccatt gctattccta ctaatttcac tatctccgtc acgaccgaga tcctgccagt 3060 ttccatgact aagacttctg ttgactgcac catgtatatc tgtggcgata gcaccgagtg 3120 cagtaatctg cttctgcagt acggctcctt ctgcacacaa ctcaatcgag cactgaccgg 3180 tattgcagtt gagcaggaca agaacaca ggaggtcttt gcacaggtca aacaaattta 3240 caaaaccccc cccataaaag actttggtgg gttcaacttc agccaaatcc tcccagatcc 3300 cagcaagccc tccaaaagat ccttcatcga agaccttttg ttcaataagg taaccctggc 3360 cgacgcaggc ttcatcaaac aatatggcga ttgccttgga gacattgctg cgcgcgattt 3420 gatctgtgct cagaaattta acggtttgac cgtgctgccc ccacttctga ctgatgagat 3480 gatagcacag tatacttctg ctcttctggc aggaacaatc acttccgggt ggacctttgg 3540 cgctggtgca gcactgcaaa tccccttcgc aatgcaaatg gcctaccgat tcaatggtat 3600 tggtgttacc cagaacgtgc tctatgagaa tcagaaactc atcgccaatc agttcaatag 3660 cgctattggc aagattcagg attccctcag ctctaccgcc agcgctctgg ggaagctcca 3720 ggacgtggtg aaccaaaatg ctcaagcgct caataccctt gtgaaacagc tcagctccaa 3780 ttttggcgca attagcagcg ttctgaatga tattctgtcc cggctggaca aggtagaagc 3840 agaagtccag atcgacaggc tgatcaccgg gcggttgcag agtctccaga cctatgtcac 3900 acaacagctg atccgcgccg ccgagatcag ggcttccgct aacctggccg ccactaagat 3960 gtccgaatgc gtgttggggc agagtaagcg ggtcgacttt tgcgggaagg gataccatct 4020 gatgagcttc cctcagtctg caccccacgg agtagtgttc ctccacgtca catatgtgcc 4080 cgctcaggaa aagaatttca caaccgcacc tgctatctgt cacgacggca aggcccactt 4140 tcctagagaa ggagttttcg tatctaacgg cacccactgg ttcgtgacac agcggaactt 4200 ttacgagcct cagattataa ctacggacaa cactttcgtg tcaggcaact gtgacgtggt 4260 gattgggatc gtgaacaaca cagtctacga cccattgcag cccgagttgg actccttcaa 4320 agaggagctt gataagtatt tcaagaacca tacctctccc gacgtggacc tgggggacat 4380 tagcggcatc aatgcatccg ttgtgaatat ccagaaagaa atcgataggc tgaatgaggt 4440 cgcaaaaaat cttaatgagt cactgattga tctgcaggaa ctcggcaaat atgagcagta 4500 tattaagtgg ccgtggtaca tatggctcgg ctttatcgcc ggtctgattg ccatcgtgat 4560 ggtgaccatt atgctgtgtt gtatgacaag ctgctgttca tgtctcaaag gatgctgctc 4620 ctgcggtagc tgctgtaagt tcgatgaaga cgacagtgag cccgtgctca aaggagtgaa 4680 actccactac acataacgat cggatatcgc tagcgtaccg gcggccgccc tattctatag 4740 tgtcacctaa atgctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca 4800 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 4860 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 4920 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct 4980 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccaaagctta cgcgttagtt 5040 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta 5100 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt 5160 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg 5220 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta 5280 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga 5340 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attachtgg 5400 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc 5460 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact 5520 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt 5580 gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag agatatcggg 5640 ccactgcagg aaacgatatg ggctgaatac ggatccgtat tcagcccata tcgtttctct 5700 agaaataaaa tatctttatt ttcattacat ctgtgtgttg gttttttgtg tg 5752 <210> 7 <211> 7310 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 7 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa 60 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata 120 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag 180 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc 240 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta 300 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg 360 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt 420 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca 480 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag 540 gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc 600 tgttttgacc tccatagaag acaccgggac cgatccagcc tgactctagc ctagctctga 660 agttggtggt gaggccctgg gcaggttggt atcaaggtta caagacaggt ttaaggagac 720 caatagaaac tgggcatgtg gagacagaga agactcttgg gtttctgata ggcactgact 780 ctctctgcct attggtctat tttcccaccc ttaggctgct ggtctgagcc taggagatct 840 ctcgaggtcg acggtatcga tgggtaccgc caccatgttt gtttttctcg tactcctgcc 900 cctggtttcc tcccaatgtg tcaatctgac tacccggacc caacttcctc ccgcctacac 960 caattccttt acccgaggtg tttactaccc agacaaagtg ttcaggtcat ccgtcctcca 1020 tagtacccaa gacctcttcc tccctttttt ttctaacgtt acctggtttc acgctattca 1080 cgttagcggc accaacggca ccaaaagatt cgataacccc gtactgccgt tcaacgacgg 1140 ggtatatttt gcctctactg aaaaatcaaa catcatacgc ggatggatct ttgggactac 1200 cctggactca aaaactcagt ccctgctgat tgtgaataac gctaccaacg tggtgatcaa 1260 agtctgtgaa ttccagtttt gcaacgatcc ttttctcggc gtttattatc acaaaaataa 1320 caaatcctgg atggagagcg agttccgggt gtactcctcc gcgaataatt gcaccttcga 1380 atatgtgtct cagccattcc tcatggacct cgaggggaag cagggcaatt ttaagaatct 1440 gcgagaattc gtgttcaaga atatagacgg ttacttcaag atttactcca aacacacccc 1500 gattaacctg gttagggact tgcctcaggg cttttctgca ttggagcccc tcgtggacct 1560 cccaatcggc ataaacatta caagatttca gactttgctt gcattgcaca ggagctattt 1620 gandacccggc gattctctt ccggatggac cgctggagca gctgcttt acgtgggcta 1680 tctgcagcct cgaacctttc tttgaagta caacgaaat ggaactatca ccgatgcagt 1740 tgactgcgcc ctggaccccc tgtccgaac taagtgcacg ctcaagtt tcacagtaga 1800 gaagggata taccagacta gcaatttccg cgttcagcca accgaagta tagtgcgctt 1860 tcctaatata actaacctgt gtccttcgg ggaagtgttt aacgccacta gattcgctc 1920 cgtctacgcc tggaatagaa agaggatctc aaattgcgtt gctgactata gtgttttgta 1980 cavattccgcc tctttctca ccttcaatg ttacggggtg agccctacca aactgaacga 2040 cctgtgcttt acaacgtat acgccgacag ctttgttac agaggagacg aggttcgcca 2100 gattgctccg gtcagacag gcaagattgc tgattataat tacaactgc ccgacgactt 2160 tacaggatgt gtgatcgcgt ggaacagtaa caatctgac tcaaggttg ggggtatta 2220 taattatctt taccggctgt tcagaaaag caatttgaa cccttcgaaa gggacatatc 2280 caccgagatc tatcaggccg gtccactc atgcaatggt gtggaaggtt ttaattgcta 2340 cttcccattg cagtcttatg gattccaacc aaccaatggc gtaggctacc agccgtatcg 2400 cgttgtcgtg ctcagcttcg agctgctcca cgcccccgcg accgtatgcg gtcctaagaa 2460 gtccaccaat cttgttaaga acaagtgtgt aaactttaac tttaacgggc tgaccgggac 2520 cggcgttctg actgaatcta acaaaaaatt cctgcctttc cagcagttcg gccgcgatat 2580 tgctgacacc actgacgctg taagagaccc tcagaccctt gaaattctcg atatcacacc 2640 ttgcagcttt gggggcgtgt ccgtcatcac tccaggaact aacacaagca accaggtggc 2700 agtgttgtac caggatgtta attgtaccga ggtgccagtg gccatccacg ccgatcaatt 2760 gacacctacc tggagggttt acagcacagg gtccaatgtt tttcagacaa gagccggatg 2820 tctgatcggt gccgagcatg tcaacaattc ctacgagtgt gatatcccca ttggtgcggg 2880 aatttgtgca tcatatcaga cccagactaa tagcccaaga agagctagat ccgtcgctag 2940 tcaatccatc attgcatata caatgtccct gggagctgag aattcagtcg cgtattcaaa 3000 caattccatt gctattccta ctaatttcac tatctccgtc acgaccgaga tcctgccagt 3060 ttccatgact aagacttctg ttgactgcac catgtatatc tgtggcgata gcaccgagtg 3120 cagtaatctg cttctgcagt acggctcctt ctgcacacaa ctcaatcgag cactgaccgg 3180 tattgcagtt gagcaggaca agaacaca ggaggtcttt gcacaggtca aacaaattta 3240 caaaaccccc cccataaaag actttggtgg gttcaacttc agccaaatcc tcccagatcc 3300 cagcaagccc tccaaaagat ccttcatcga agaccttttg ttcaataagg taaccctggc 3360 cgacgcaggc ttcatcaaac aatatgggga ttgccttgga gacattgctg cgcgcgattt 3420 gatctgtgct cagaaattta acggtttgac cgtgctgccc ccacttctga ctgatgagat 3480 gatagcacag tatacttctg ctcttctggc aggaacaatc acttccgggt ggacctttgg 3540 cgctggtgca gcactgcaaa tccccttcgc aatgcaaatg gcctaccgat tcaatggtat 3600 tggtgttacc cagaacgtgc tctatgagaa tcagaaactc atcgccaatc agttcaatag 3660 cgctattggc aagattcagg attccctcag ctctaccgcc agcgctctgg ggaagctcca 3720 ggacgtggtg aaccaaaatg ctcaagcgct caataccctt gtgaaacagc tcagctccaa 3780 ttttggcgca attagcagcg ttctgaatga tattctgtcc cggctggaca aggtagaagc 3840 agaagtccag atcgacaggc tgatcaccgg gcggttgcag agtctccaga cctatgtcac 3900 acaacagctg atccgcgccg ccgagatcag ggcttccgct aacctggccg ccactaagat 3960 gtccgaatgc gtgttggggc agagtaagcg ggtcgacttt tgcgggaagg gataccatct 4020 gatgagcttc cctcagtctg caccccacgg agtagtgttc ctccacgtca catatgtgcc 4080 cgctcaggaa aagaatttca caaccgcacc tgctatctgt cacgacggca aggcccactt 4140 tcctagagaa ggagttttcg tatctaacgg cacccactgg ttcgtgacac agcggaactt 4200 ttacgagcct cagattataa ctacggacaa cactttcgtg tcaggcaact gtgacgtggt 4260 gattgggatc gtgaacaaca cagtctacga cccattgcag cccgagttgg actccttcaa 4320 agaggagctt gataagtatt tcaagaacca tacctctccc gacgtggacc tgggggacat 4380 tagcggcatc aatgcatccg ttgtgaatat ccagaaagaa atcgataggc tgaatgaggt 4440 cgcaaaaaat cttaatgagt cactgattga tctgcaggaa ctcggcaaat atgagcagta 4500 tattaagtgg ccgtggtaca tatggctcgg ctttatcgcc ggtctgattg ccatcgtgat 4560 ggtgaccatt atgctgtgtt gtatgacaag ctgctgttca tgtctcaaag gatgctgctc 4620 ctgcggtagc tgctgtaagt tcgatgaaga cgacagtgag cccgtgctca aaggagtgaa 4680 actccactac acataacgat cgacgcgtag agctcgctga tcagcctcga ctgtgccttc 4740 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 4800 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg 4860 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa 4920 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccaaagc 4980 ttgcggccgc gcccagcacc ccaaggcggc caacgccaaa actctccctc ctcctcttcc 5040 tcaatctcgc tctcgctctt tttttttttc gcaaaaggag gggagagggg gtaaaaaaat 5100 gctgcactgt gcggcgaagc cggtgagtga gcggcgcggg gccaatcagc gtgcgccgtt 5160 ccgaaagttg ccttttatgg ctcgagcggc cgcggcggcg ccctataaaa cccagcggcg 5220 cgacgcgcca ccaccgccga gaccctgcag gccgccacca tgtccgataa cggcccccag 5280 aatcagagaa acgctccccg catcacgttc ggcggaccaa gtgacagcac aggcagtaac 5340 cagaacggag aacgctccgg tgctcgctcc aagcagcgac ggccgcaagg gcttcccaac 5400 aataccgcca gctggtttac ggctctgacc caacacggga aagaagatct taaattcccc 5460 aggggccagg gcgtccctat caatactaac tccagcccgg atgatcagat aggctactat 5520 agacgcgcta cccgacggat acgagggggg gacggcaaaa tgaaggacct ttccccccgg 5580 tggtatttct attacttggg caccggacca gaagccggac tgccttacgg cgctaacaaa 5640 gacggaataa tctgggttgc gacggagggc gccctgaata cacctaaaga ccatatcggc 5700 acaagaaatc ctgctaacaa tgccgcgatt gtgctccagc tgcctcaggg aaccacgctg 5760 cctaaagggt tttacgctga ggggtcaagg ggggggagtc aagcgtctag taggtcatcc 5820 tctcgctctc gcaatagttc ccggaactca accccaggca gcagcagagg aacctctccc 5880 gcacggatgg ctggcaatgg gggagatgct gcccttgctc tccttctgct ggatcgcctt 5940 aaccagctcg aatcaaagat gtctggaaaa ggtcagcagc agcaaggcca gaccgtgaca 6000 aagaaggtg cagctgaagc tagtaaaaag ccacgccaaa aacggaccgc aactaaggca 6060 tataacgtaa cacaggcctt cggcagaaga ggtccagaac aaacacaggg aaactttggc 6120 gatcaagagc tgattagaca gggcacagat tacaaacact ggccacagat cgcgcagttt 6180 gcaccaagcg cctctgcatt cttcgggatg agtcggattg ggatggagagt cactccatcc 6240 gggacctggc ttacctacac aggggcaata aaactcgacg acaaagaccc aaactttaaa 6300 gatcaggtca tcctgctgaa taaacacatc gatgcctaca aaactttccc cccaaccgaa 6360 ccaaagaaag acaagaaaaa aaaggcagac gaacgcaag cgctccctca gcgccagaag 6420 aagcagcaga ccgttacact gttgccagca gcagatctgg atgatttttc caagcagctt 6480 6540 taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgacg cgttagttat 6600 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca 6660 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca 6720 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg 6780 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg 6840 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc 6900 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg 6960 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca 7020 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt 7080 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg 7140 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagag atatcgggcc 7200 actgcaggaa acgatatggg ctgaatacgg atccgtattc agcccatatc gtttctctag 7260 aaataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg 7310 <210> 8 <211> 5281 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 8 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa 60 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata 120 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag 180 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc 240 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta 300 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg 360 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt 420 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca 480 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag 540 gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc 600 tgttttgacc tccatagaag acaccgggac cgatccagcc tgactctagc ctagctctga 660 agttggtggt gaggccctgg gcaggttggt atcaaggtta caagacaggt ttaaggagac 720 caatagaaac tgggcatgtg gagacagaga agactcttgg gtttctgata ggcactgact 780 ctctctgcct attggtctat tttcccaccc ttaggctgct ggtctgagcc taggagatct 840 ctcgaggtcg acggtatcga tgggtaccgc caccatgttt gtttttctcg tactcctgcc 900 cctggtttcc tcccaatgtg tcaatctgac tacccggacc caacttcctc ccgcctacac 960 caattccttt acccgaggtg tttactaccc agacaaagtg ttcaggtcat ccgtcctcca 1020 tagtacccaa gacctcttcc tccctttttt ttctaacgtt acctggtttc acgctattca 1080 cgttagcggc accaacggca ccaaaagatt cgataacccc gtactgccgt tcaacgacgg 1140 ggtatatttt gcctctactg aaaaatcaaa catcatacgc ggatggatct ttgggactac 1200 cctggactca aaaactcagt ccctgctgat tgtgaataac gctaccaacg tggtgatcaa 1260 agtctgtgaa ttccagtttt gcaacgatcc ttttctcggc gtttattatc acaaaaataa 1320 caaatcctgg atggagagcg agttccgggt gtactcctcc gcgaataatt gcaccttcga 1380 atatgtgtct cagccattcc tcatggacct cgaggggaag cagggcaatt ttaagaatct 1440 gcgagaattc gtgttcaaga atatagacgg ttacttcaag atttactcca aacacacccc 1500 gattaacctg gttagggact tgcctcaggg cttttctgca ttggagcccc tcgtggacct 1560 cccaatcggc ataaacatta caagatttca gactttgctt gcattgcaca ggagctattt 1620 gacacccggc gattcttctt ccggatggac cgctggagca gctgcttatt acgtgggcta 1680 tctgcagcct cgaacctttc ttttgaagta caacgaaaat ggaactatca ccgatgcagt 1740 tgactgcgcc ctggaccccc tgtccgaaac taagtgcacg ctcaaaagtt tcacagtaga 1800 gaaggggata taccagacta gcaatttccg cgttcagcca accgaaagta tagtgcgctt 1860 tcctaatata actaacctgt gtcctttcgg ggaagtgttt aacgccacta gattcgcttc 1920 cgtctacgcc tggaatagaa agaggatctc aaattgcgtt gctgactata gtgttttgta 1980 caattccgcc tctttctcaa ccttcaaatg ttacggggtg agccctacca aactgaacga 2040 cctgtgcttt acaacgtat acgccgacag ctttgttac agaggagacg aggttcgcca 2100 gattgctccg gtcagacag gcaagattgc tgattataat tacaactgc ccgacgactt 2160 tacaggatgt gtgatcgcgt ggaacagtaa caatctgac tcaaggttg ggggtatta 2220 taattatctt taccggctgt tcagaaaag caatttgaa cccttcgaaa gggacatatc 2280 caccgagatc tatcaggccg gtccactc atgcaatggt gtggaaggtt ttaattgcta 2340 cttcccattg cagtcttatg gattccacc aaccaatggc gtaggctacc agccgtatcg 2400 cgttgtcgtg ctcagctcg agctgctcca cgcccccgcg accgtagcg gtcctaagaa 2460 gtccaccaat cttgttaaga acagtgtgt aaactttaac tttaacgggc tgaccgggac 2520 cggcgttctg actgaatcta aaaaaatt cctgccttc cagcagttcg gccgcgatat 2580 tgctgacacc actgacgctg taagagaccc tcagacctt gaaattctcg atcacacc 2640 ttgcagcttt gggggcgtgt ccgtcatcac tccaggact aacacaagca accaggtggc 2700 agtgttgtac caggatgtta attgtaccga ggtgccagtg gccatccacg ccgatcaatt 2760 gacacctacc tggagggttt acagcacagg gtccaatgtt tttcagacaa gagccggatg 2820 tctgatcggt gccgagcatg tcaacaattc ctacgagtgt gatatcccca ttggtgcggg 2880 aatttgtgca tcatatcaga cccagactaa tagcccaaga agagctagat ccgtcgctag 2940 tcaatccatc attgcatata caatgatgtc cgataacggc ccccagaatc agagaaacgc 3000 tccccgcatc acgttcggcg gaccaagtga cagcacaggc agtaaccaga acggagaacg 3060 ctccggtgct cgctccaagc agcgacggcc gcaagggctt cccaacaata ccgccagctg 3120 gttacggct ctgacccaac acggggaaaga agatcttaaa ttccccaggg gccagggcgt 3180 ccctatcaat actaactcca gcccggatga tcagataggc tactatagac gcgctacccg 3240 acggatacga gggggggacg gcaaaatgaa ggacctttcc ccccggtggt atttctatta 3300 cttgggcacc ggaccagaag ccggactgcc ttacggcgct aacaaagacg gaataatctg 3360 ggttgcgacg gagggcgcc tgaatacacc taaagaccat atcggcacaa gaaatcctgc 3420 taacaatgcc gcgattgtgc tccagctgcc tcagggaacc acgctgccta aagggtttta 3480 cgctgagggg tcaagggggg ggagtcaagc gtctagtagg tcatcctctc gctctcgcaa 3540 tagttcccgg aactcaaccc caggcagcag cagaggaacc tctcccgcac ggatggctgg 3600 caatggggga gatgctgccc ttgctctcct tctgctggat cgccttaacc agctcgaatc 3660 aaagatgtct ggaaaaggtc agcagcagca aggccagacc gtgacaaaga agagtgcagc 3720 tgaagctagt aaaaagccac gccaaaaacg gaccgcaact aaggcatata acgtaacaca 3780 ggccttcggc agaagaggtc cagaacaaac acagggaaac tttggcgatc aagagctgat 3840 tagacagggc acagattaca aacactggcc acagatcgcg cagtttgcac caagcgcctc 3900 tgcattcttc gggatgagtc ggattgggat ggaagtcact ccatccggga cctggcttac 3960 ctacacaggg gcaataaaac tcgacgacaa agacccaaac tttaaagatc aggtcatcct 4020 gctgaataaa cacatcgatg cctacaaaac tttcccccca accgaaccaa agaaagacaa 4080 gaaaaaaaag gcagacgaaa cgcaagcgct ccctcagcgc cagaagaagc agcagaccgt 4140 tacactgttg ccagcagcag atctggatga tttttccaag cagcttcaac agagtatgtc 4200 aagcgctgac agcactcagg cttgacgatc ggatatcgct agcgtaccgg cggccgccct 4260 attctatagt gtcacctaaa tgctagagct cgctgatcag cctcgactgt gccttctagt 4320 tgccagccat ctgttgttg cccctcccc gtgccttcct tgaccctgga aggtgccact 4380 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 4440 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacatagc 4500 agcatgctg gggatgcggt gggctctatg gcttctgagg cggaagaac caaagcttac 4560 gcgttagtta tcatagtaa tcaattacgg ggtcattagt tcatagccca tatagta 4620 tccgcgttac attackcg gtaaatggcc cgcctggctg acccccac gaccccgcc 4680 cattgacgtc ataatgacg tatgttccca tagtaacgcc atagggact ttccattgac 4740 gtcaatgggt ggagtattta cggtaactg cccacttggc agtacatca gtgtatcata 4800 tgccaagtac gccccctatt gacgtcaatg acggtaatg gcccgcctgg cattatgccc 4860 agtacatgac cttatgggac ttcctactt ggcagtacat ctacgtatta gtcatcgcta 4920 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac 4980 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc 5040 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc 5100 gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctaga 5160 gatatcgggc cactgcagga aacgatatgg gctgaatacg gatccgtatt cagcccatat 5220 cgtttctcta gaaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt 5280 g 5281 <210> 9 <211> 36177 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 9 taaggatccc atcatcaata atatacctta ttttggattg aagccaatat gataatgagg 60 gggtggagtt tgtgacgtgg cgcggggcgt gggaacgggg cgggtgacgt agtagtgtgg 120 cggaagtgtg atgttgcaag tgtggcggaa cacatgtaag cgacggatgt ggcaaaagtg 180 acgttttgg tgtgcgccgg tgtacacagg aagtgacaat ttcgcgcgg tttaggcgg 240 atgttgtagt aaatttgggc gtaccgagt aagatttggc catttcgcg ggaaaactga 300 ataagaggaa gtgaaatctg ataatttg tgttactcat agcgcgtaat actgctagag 360 atctggcgaa agggggatgt gctgcaggc gattaagttg gggtaacgcca gggttttccc 420 agtcacgacg ttgtaaaacg acggccagtg aattgtata cgactcacta taggcgaat 480 tgggtactgg ccacaggagc ttggccatt gcatacgttg tatccatatc atatatgta 540 catttatatt ggctcatgtc caattacc gccatgttga cattgattat tgactagtta 600 ttaatagtaa tcattacgg gtcattagt tcatagccca tattagt tccgcgttac 660 attackcg gtaaatggcc cgcctggctg acccccac gaccccgcc cattgacgtc 720 aaatgacg tatgttccca tagtaacgcc atagggact ttccattgac gtcaatgggt 780 ggagtattta cggtaactg cccacttggc agtacatca gtgtatcata tgccaagtac 840 gccccctatt gacgtcaatg acggtaatg gcccgcctgg cattatgccc agtacatgac 900 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt 960 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc 1020 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt 1080 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg 1140 ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc 1200 acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctgactc tagcctagct 1260 ctgaagttgg tggtgaggcc ctgggcaggt tggtatcaag gttacaagac aggtttaagg 1320 agaccaatag aaactgggca tgtggagaca gagaagactc ttgggtttct gataggcact 1380 gactctctct gcctattggt ctattttccc acccttaggc tgctggtctg agcctaggag 1440 atctctcgag gtcgacggta tcgatgggta ccgccaccat gtttgttttt ctcgtactcc 1500 tgcccctggt ttcctcccaa tgtgtcaatc tgactacccg gacccaactt cctcccgcct 1560 acaccaattc ctttacccga ggtgtttact acccagacaa agtgttcagg tcatccgtcc 1620 tccatagtac ccaagacctc ttcctccctt tttttctaa cgttacctgg tttcacgcta 1680 ttcacgttag cggcaccaac ggcaccaaaa gattcgataa ccccgtactg ccgttcaacg 1740 acggggtata ttttgcctct actgaaaaat caaacatcat acgcggatgg atctttggga 1800 ctaccctgga ctcaaaaact cagtccctgc tgattgtgaa taacgctacc aacgtggtga 1860 tcaaagtctg tgaattccag ttttgcaacg atccttttct cggcgtttat tatcacaaaa 1920 ataacaaatc ctggatggag agcgagttcc gggtgtactc ctccgcgaat aattgcacct 1980 tcgaatatgt gtctcagcca ttcctcatgg acctcgaggg gaagcagggc aattttaaga 2040 atctgcgaga attcgtgttc aagaatatag acggtactt caagatttac tccaaacaca 2100 ccccgattaa cctggttagg gacttgcctc agggcttttc tgcattggag cccctcgtgg 2160 acctcccaat cggcataaac attacaagat ttcagacttt gcttgcattg cacaggagct 2220 atttgacacc cggcgattct tcttcggat ggaccgctgg agcagctgct tattacgtgg 2280 gctatctgca gcctcgaacc tttcttttga agtacaacga aaatggaact atcaccgatg 2340 cagttgactg cgccctggac cccctgtccg aaactaagtg cacgctcaa agttcacag 2400 tagagaaggg gatataccag actagcaatt tccgcgttca gccaaccgaa agtatagtgc 2460 gctttcctaa tataactaac ctgtgtcctt tcgggagt gtttaacgcc actagattcg 2520 cttccgtcta cgcctggaat agaaagagga tctcaattg cgttgctgac tatagtgttt 2580 tgtacaatc cgccctttc tcaccttca aatgttacgg ggtgagccct accaactga 2640 acgacctgtg ctttacaaac gtatacgccg acagctttgt tatcagagga gacgaggttc 2700 gccagattgc tccgggtcag acaggcaga tgctgatta taattacaaa ctgcccgacg 2760 actttacagg atgtgtgatc gcgtggaaca gtaacaatct tgactcaag gttggggta 2820 attataatta tctttaccgg ctgttcagaa aaagcaattt gaacccttc gaaagggaca 2880 tatccaccga gatctatcag gccggggtcca ctccatgcaa tggtgtggaa ggttttaatt 2940 gctacttccc attgcagtct tatggattcc aaccaaccaa tggcgtaggc taccagccgt 3000 atcgcgttgt cgtgctcagc ttcgagctgc tccacgcccc cgcgaccgta tgcggtccta 3060 agaagtccac caatcttgtt aagaacaagt gtgtaaactt taactttaac gggctgaccg 3120 ggaccggcgt tctgactgaa tctaacaaaa aattcctgcc tttccagcag ttcggccgcg 3180 atattgctga caccactgac gctgtaagag accctcagac ccttgaaatt ctcgatatca 3240 caccttgcag ctttgggggc gtgtccgtca tcactccagg aactaacaca agcaaccagg 3300 tggcagtgtt gtaccaggat gttaattgta ccgaggtgcc agtggccatc cacgccgatc 3360 aattgacacc tacctggagg gtttacagca cagggtccaa tgtttttcag acaagagccg 3420 gatgtctgat cggtgccgag catgtcaaca attcctacga gtgtgatatc cccattggtg 3480 cgggaatttg tgcatcatat cagacccaga ctaatagccc aagaagagct agatccgtcg 3540 ctagtcaatc catcattgca tatacaatgt ccctgggagc tgagaattca gtcgcgtatt 3600 caaacaattc cattgctatt cctactaatt tcactatctc cgtcacgacc gagatcctgc 3660 cagtttccat gactaagact tctgttgact gcaccatgta tatctgtggc gatagcaccg 3720 agtgcagtaa tctgcttctg cagtacggct ccttctgcac acaactcaat cgagcactga 3780 ccggtattgc agttgagcag gacaagaaca cacaggaggt ctttgcacag gtcaaacaaa 3840 tttacaaaac cccccccata aaagactttg gtgggttcaa cttcagccaa atcctcccag 3900 atcccagcaa gccctccaaa agatccttca tcgaagacct tttgttcaat aaggtaaccc 3960 tggccgacgc aggcttcatc aaacaatatg gcgattgcct tggagacatt gctgcgcgcg 4020 atttgatctg tgctcagaaa tttaacggtt tgaccgtgct gcccccactt ctgactgatg 4080 agatgatagc acagtatact tctgctcttc tggcaggaac aatcacttcc gggtggacct 4140 ttggcgctgg tgcagcactg caaatcccct tcgcaatgca aatggcctac cgattcaatg 4200 gtattggtgt tacccagaac gtgctctatg agaatcagaa actcatcgcc aatcagttca 4260 atagcgctat tggcaagatt caggattccc tcagctctac cgccagcgct ctggggaagc 4320 tccaggacgt ggtgaaccaa aatgctcaag cgctcaatac ccttgtgaaa cagctcagct 4380 ccaattttgg cgcaattagc agcgttctga atgatattct gtcccggctg gacaaggtag 4440 aagcagaagt ccagatcgac aggctgatca ccgggcggtt gcagagtctc cagacctatg 4500 tcacacaaca gctgatccgc gccgccgaga tcagggcttc cgctaacctg gccgccacta 4560 agatgtccga atgcgtgttg gggcagagta agcgggtcga cttttgcggg aagggatacc 4620. atctgatgag cttccctcag tctgcacccc acggagtagt gttcctccac gtcacatatg 4680 tgcccgctca ggaaaagaat ttcacaaccg cacctgctat ctgtcacgac ggcaaggccc actttcctag agaaggagtt ttcgtatcta acggcaccca ctggttcgtg acacagcgga acttttacga gcctcagatt ataactacgg acaacacttt cgtgtcaggc aactgtgacg tggtgattgg gatcgtgaac aacacagtct acgacccatt gcagcccgag ttggactcct tcaaagagga gcttgataag tatttcaaga accatacctc tcccgacgtg gacctggggg acattagcgg catcaatgca tccgttgtga atatccagaa agaaatcgat aggctgaatg aggtcgcaaa aaatcttaat gagtcactga ttgatctgca ggaactcggc aaatatgagc agtatattaa gtggccgtgg tacatatggc tcggctttat cgccggtctg attgccatcg tgatggtgac cattatgctg tgttgtatga caagctgctg ttcatgtctc aaaggatgct gctcctgcgg tagctgctgt aagttcgatg aagacgacag tgagcccgtg ctcaaaggag 5280 tgaaactcca ctacacataa cgatcggata tcgctagcgt accggcggcc gccctattct 5340 atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca 5400 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 5460 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 5520 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca 5580 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccaaag cttacgcgtt 5640 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc 5700 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg 5760 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa 5820 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca 5880 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac 5940 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc 6000 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga 6060 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg 6120 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta 6180 cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatccg ctagagatat 6240 cgggccactg caggaaacga tatgggctga atacggatcc gtattcagcc catatcgttt 6300 ctctagaaat aaaatatctt tattttcatt acatctgtgt gttggttttt tgtgtgaatc 6360 gatagtacta acatacgctc tccatctcga gcctaagctt gtcgactcga agatctgggc 6420 gtggttaagg gtgggaaaga atatataagg tgggggtctt atgtagtttt gtatctgttt 6480 tgcagcagcc gccgccgcca tgagcaccaa ctcgtttgat ggaagcattg tgagctcata 6540 tttgacaacg cgcatgcccc catgggccgg ggtgcgtcag aatgtgatgg gctccagcat 6600 tgatggtcgc cccgtcctgc ccgcaaactc tactaccttg acctacgaga ccgtgtctgg 6660 aacgccgttg gagactgcag cctccgccgc cgcttcagcc gctgcagcca ccgcccgcgg 6720 gattgtgact gactttgctt tcctgagccc gcttgcaagc agtgcagctt cccgttcatc 6780 cgcccgcgat gacaagttga cggctctttt ggcacaattg gattctttga cccgggaact 6840 taatgtcgtt tctcagcagc tgttggatct gcgccagcag gtttctgccc tgaaggcttc 6900 ctcccctccc aatgcggttt aaaacataaa taaaaaacca gactctgttt ggatttggat 6960 caagcaagtg tcttgctgtc tttatttagg ggttttgcgc gcgcggtagg cccgggacca 7020 gcggtctcgg tcgttgaggg tcctgtgtat tttttccagg acgtggtaaa ggtgactctg 7080 gatgttcaga tacatgggca taagcccgtc tctggggtgg aggtagcacc actgcagagc 7140 ttcatgctgc ggggtggtgt tgtagatgat ccagtcgtag caggagcgct gggcgtggtg 7200 cctaaaaatg tctttcagta gcaagctgat tgccaggggc aggcccttgg tgtaagtgtt 7260 tacaaagcgg ttaagctgggg atgggtgcat acgtggggat atgagatgca tcttggactg 7320 tatttttagg ttggctatgt tcccagccat atccctccgg ggattcatgt tgtgcagaac 7380 caccagcaca gtgtatccgg tgcacttggg aaatttgtca tgtagcttag aaaggaaatgc 7440 gtggaagaac ttggagacgc ccttgtgacc tccaagattt tccatgcatt cgtccataat 7500 gatggcaatg ggcccacggg cggcggcctg ggcgaagata tttctgggat cactaacgtc 7560 atagttgtgt tccaggatga gatcgtcata ggccattttt aaaagcgcg ggcggagggt 7620 gccagactgc ggtataatgg ttccatccgg cccaggggcg tagttaccct cacagatttg 7680 catttcccac gctttgagtt cagatgggg gatcatgtct acctgcgggg cgatgaagaa 7740 aacggtttcc ggggtagggg agatcagctg ggaagaaagc aggttcctga gcagctgcga 7800 cttaccgcag ccggtgggcc cgtaaatcac acctattacc ggctgcaact ggtagttaag 7860 agagctgcag ctgccgtcat ccctgagcag gggggccact tcgttaagca tgtccctgac 7920 tcgcatgttt tccctgacca aatccgccag aaggcgctcg ccgcccagcg atagcagttc 7980 ttgcaaggaa gcaaagtttt tcaacggttt gagaccgtcc gccgtaggca tgcttttgag 8040 cgtttgacca agcagttcca ggcggtccca cagctcggtc acctgctcta cggcatctcg 8100 atccagcata tctcctcgtt tcgcgggttg gggcggcttt cgctgtacgg cagtagtcgg 8160 tgctcgtcca gacgggccag ggtcatgtct ttccacgggc gcagggtcct cgtcagcgta 8220 gtctgggtca cggtgaaggg gtgcgctccg ggctgcgcgc tggccagggt gcgcttgagg 8280 ctggtcctgc tggtgctgaa gcgctgccgg tcttcgccct gcgcgtcggc caggtagcat 8340 ttgaccatgg tgtcatagtc cagcccctcc gcggcgtggc ccttggcgcg cagcttgccc 8400 ttggaggagg cgccgcacga ggggcagtgc agacttttga gggcgtagag cttgggcgcg 8460 agaaataccg attccgggga gtaggcatcc gcgccgcagg ccccgcagac ggtctcgcat 8520 tccacgagcc aggtgagctc tggccgttcg gggtcaaaaa ccaggtttcc cccatgcttt 8580 ttgatgcgtt tcttacctct ggtttccatg agccggtgtc cacgctcggt gacgaaaagg 8640 ctgtccgtgt ccccgtatac agacttgaga ggcctgtcct cgagcggtgt tccgcggtcc 8700 tcctcgtata gaaactcgga ccactctgag acaaaggctc gcgtccaggc cagcacgaag 8760 gaggctaagt gggaggggta gcggtcgttg tccactaggg ggtccactcg ctccagggtg 8820 tgaagacaca tgtcgccctc ttcggcatca aggaaggtga ttggtttgta ggtgtaggcc 8880 acgtgaccgg gtgttcctga aggggggcta taaaaggggg tgggggcgcg ttcgtcctca 8940 ctctcttccg catcgctgtc tgcgagggcc agctgttggg gtgagtactc cctctgaaaa 9000 gcgggcatga cttctgcgct aagattgtca gtttccaaaa acgaggagga tttgatattc 9060 acctggcccg cggtgatgcc tttgagggtg gccgcatcca tctggtcaga aaagacaatc 9120 tttttgttgt caagcttggt ggcaaacgac ccgtagaggg cgttggacag caacttggcg 9180 atggagcgca gggtttggtt tttgtcgcga tcggcgcgct ccttggccgc gatgtttagc 9240 tgcacgtatt cgcgcgcaac gcaccgccat tcgggaaaga cggtggtgcg ctcgtcgggc 9300 accaggtgca cgcgccaacc gcggttgtgc agggtgacaa ggtcaacgct ggtggctacc 9360 tctccgcgta ggcgctcgtt ggtccagcag aggcggccgc ccttgcgcga gcagaatggc 9420 ggtagggggt ctagctgcgt ctcgtccggg gggtctgcgt ccacggtaaa gaccccgggc 9480 agcaggcgcg cgtcgaagta gtctatcttg catccttgca agtctagcgc ctgctgccat 9540 gcgcgggcgg caagcgcgcg ctcgtatggg ttgagtgggg gaccccatgg catggggtgg 9600. gtgagcgcgg aggcgtacat gccgcaaatg tcgtaaacgt agaggggctc tctgagtatt ccaagatatg tagggtagca tcttccaccg cggatgctgg cgcgcacgta atcgtatagt tcgtgcgagg gagcgaggag gtcgggaccg aggttgctac gggcgggctg ctctgctcgg 9780 aagactatct gcctgaagat ggcatgtgag ttggatgata tggttggacg ctggaagacg ttgaagctgg cgtctgtgag acctaccgcg tcacgcacga aggaggcgta ggagtcgcgc agcttgttga ccagctcggc ggtgacctgc acgtctaggg cgcagtagtc caggttttcc 9960 ttgatgatgt catacttatc ctgtcccttt tttttccaca gctcgcggtt gaggacaaac tcttcgcggt ctttccagta ctcttggatc ggaaacccgt cggcctccga acggtaagag 10080 cctagcatgt agaactggtt gacggcctgg tagcgcagc atcccttttc tagggtagc 10140 gcgtatgcct gcgcggcctt ccggagcgag gtgtgggtga gcgcaaaggt gtccctgacc 10200 atgactttga ggtactggta tttgaagtca gtgtcgtcgc atccgccctg ctcccagagc 10260. aaaaagtccg tgcgcttttt ggaacgcgga tttggcaggg cgaaggtgac atcgttgaag 10320 agtatctttc ccgcgcgagg cataaagttg cgtgtgatgc ggaagggtc cggcacctcg 10380 gaacggttgt taattacctg ggcggcgagc acgatctcgt caaagccgtt gatgttgtgg 10440 cccacaatgt aaagttccaa gaagcgcggg atgcccttga tggaaggcaa ttttttaagt 10500 tcctcgtagg tgagctcttc aggggagctg agcccgtgct ctgaaagggc ccagtctgca 10560 agatgagggt tggaagcgac gaatgagctc cacaggtcac gggccattag catttgcagg 10620 tggtcgcgaa aggtcctaaa ctggcgacct atggccatttt tttctggggt gatgcagtag 10680 aaggtaagcg ggctttgttc ccagcggtc catccaaggt tcgcggctag gtctcgcgcg 10740 gcagtcacta gaggctcatc tccgccgaac ttcatgacca gcatgaaggg cacgagctgc 10800 ttcccaaagg cccccatcca agtataggtc tctacatcgt aggtgacaaa gagacgctcg 10860 gtgcgaggat gcgagccgat cgggaagaac tggatctccc gccaccaatt ggaggagtgg 10920 ctattgatgt ggtgaaagta gaagtccctg cgacgggccg aacactcgtg ctggcttttg 10980 taaaaacgtg cgcagtactg gcagcggtgc acgggctgta catcctgcac gaggttgacc 11040 tgacgaccgc gcacaaggaa gcagagtggg aatttgagcc cctcgcctgg cgggtttggc 11100 tggtggtctt ctacttcggc tgcttgtcct tgaccgtctg gctgctcgag gggagttacg 11160 gtggatcgga ccaccacgcc gcgcgagccc aaagtccaga tgtccgcgcg cggcggtcgg 11220 agcttgatga caacatcgcg cagatgggag ctgtccatgg tctggagctc ccgcggcgtc 11280 aggtcaggcg ggagctcctg caggtttacc tcgcatagac gggtcagggc gcgggctaga 11340 tccaggtgat acctaatttc caggggctgg ttggtggcgg cgtcgatggc ttgcaagagg 11400 ccgcatcccc gcggcgcgac tacggtaccg cgcggcgggc ggtgggccgc gggggtgtcc 11460 ttggatgatg catctaaaag cggtgacgcg ggcgagcccc cggaggtagg gggggctccg 11520 gacccgccgg gagagggggc aggggcacgt cggcgccgcg cgcgggcagg agctggtgct 11580 gcgcgcgtag gttgctggcg aacgcgacga cgcggcggtt gatctcctga atctggcgcc 11640 tctgcgtgaa gacgacgggc ccggtgagct tgaacctgaa agagagttcg acagaatcaa 11700 tttcggtgtc gttgacggcg gcctggcgca aaatctcctg cacgtctcct gagttgtctt 11760 gataggcgat ctcggccatg aactgctcga tctcttcctc ctggagatct ccgcgtccgg 11820 ctcgctccac ggtggcggcg aggtcgttgg aaatgcgggc catgagctgc gagaaggcgt 11880 tgaggcctcc ctcgttccag acgcggctgt agaccacgcc cccttcggca tcgcgggcgc 11940 gcatgaccac ctgcgcgaga ttgagctcca cgtgccgggc gaagacggcg tagtttcgca 12000 ggcgctgaaa gaggtagttg agggtggtgg cggtgtgttc tgccacgaag aagtacataa 12060 cccagcgtcg caacgtggat tcgttgatat cccccaaggc ctcaaggcgc tccatggcct 12120 cgtagaagtc cacggcgaag ttgaaaaact gggagttgcg cgccgacacg gttaactcct 12180 cctccagaag acggatgagc tcggcgacag tgtcgcgcac ctcgcgctca aaggctacag 12240 gggcctcttc ttcttcttca atctcctctt ccataagggc ctccccttct tcttcttctg 12300 gcggcggtgg gggagggggg acacggcggc gacgacggcg caccgggagg cggtcgacaa 12360 agcgctcgat catctccccg cggcgacggc gcatggtctc ggtgacggcg cggccgttct 12420 cgcgggggcg cagttggaag acgccgcccg tcatgtcccg gttatgggtt ggcggggggc 12480 tgccatgcgg cagggatacg gcgctaacga tgcatctcaa caattgttgt gtaggtactc 12540 cgccgccgag ggacctgagc gagtccgcat cgaccggatc ggaaaacctc tcgagaaagg 12600 cgtctaacca gtcacagtcg caaggtaggc tgagcaccgt ggcgggcggc agcgggcggc 12660 ggtcggggtt gtttctggcg gaggtgctgc tgatgatgta attaaagtag gcggtcttga 12720 gacggcggat ggtcgacaga agcaccatgt ccttgggtcc ggcctgctga atgcgcaggc 12780 ggtcggccat gccccaggct tcgttttgac atcggcgcag gtctttgtag tagtcttgca 12840 tgagcctttc taccggcact tcttcttctc cttcctcttg tcctgcatct cttgcatcta 12900 tcgctgcggc ggcggcggag tttggccgta ggtggcgccc tcttcctccc atgcgtgtga 12960 ccccgaagcc cctcatcggc tgaagcaggg ctaggtcggc gacaacgcgc tcggctaata 13020 tggcctgctg cacctgcgtg agggtagact ggaagtcatc catgtccaca aagcggtggt atgcgcccgt gttgatggtg taagtgcagt tggccataac ggaccagtta acggtctggt 13140. gacccggctg cgagagctcg gtgtacctga gacgcgagta agccctcgag tcaataacgt 13200. agtcgttgca agtccgcacc aggtactggt atcccacca aaagtgcggc ggcggctggc ggtagagggg ccagcgtagg gtggccgggg ctccggggggc gagatcttcc aacataaggc 13320 gatgatatcc gtagatgtac ctggacatcc aggtgatgcc ggcggcggtg gtggaggcgc 13380 gcggaagtc gcggacgcgg ttccagatgt tgcgcagcgg caaaaagtgc tccatggtcg 13440 ggacgctctg gccggtcagg cgcgcgcaat cgttgacgct ctagcgtgca aaaggagagc 13500. ctgtaagcgg gcactcttcc gtggtctggt ggataattc gcaagggtat catggcggac 13560 gaccggggtt cgagccccgt atccggccgt ccgccgtgat ccatgcggtt accgcccgcg 13620 13680. tgtcgaaccc aggtgtgcga cgtcagacaa cgggggagtg ctccttttgg cttccttcca ggcgcggcgg ctgctgcgct agctttttg gccactggcc gcgcgcagcg tagcggtta 13740 ggctggaag cgaaagcatt aagtggctcg ctccctgtag ccggagggtt attttccaag ggttgagtcg cgggacccc ggttcgagtc tcggaccggc cggactgcgg cgaacgggggg 13860 tttgcctccc cgtcatgcaa gaccccgctt gcaaattcct ccggaaacag ggacgagccc cttttttgct tttcccagat gcatccggtg ctgcggcaga tgcgcccccc tcctcagcag 13980 cggcaagagc aagagcagcg gcagacatgc agggcaccct cccctcctcc taccgcgtca ggaggggcga catccgcggt tgacgcggca gcagatggtg attackcc cccgcggcgc 14100. cgggcccggc actacctgga cttggaggag ggcgagggcc tggcgcggct aggagcgccc 14160 tctcctgagc ggcacccaag ggtgcagctg aagcgtgata cgcgtgaggc gtacgtgccg cggcagaacc tgtttcgcga ccgcgaggga gaggagcccg aggagatgcg ggatcgaaag ttccacgcag ggcgcgagct gcggcatggc ctgaatcgcg agcggttgct gcgcgaggag gactttgagc ccgacgcgcg aaccggggatt agtcccgcgc gcgcacacgt ggcggccgcc 14400. gacctggtaa ccgcatacga gcagacggtg aaccaggaga ttaactttca aaaaagcttt 14460 aacaaccacg tgcgtacgct tgtggcgcgc gaggaggtgg ctataggact gatgcatctg 14520 tgggactttg taagcgcgct ggagcaaaac ccaaatagca agccgctcat ggcgcagctg 14580 ttccttatag tgcagcacag cagggacaac gaggcattca gggatgcgct gctaaacata 14640 gtagagcccg agggccgctg gctgctcgat ttgataaaca tcctgcagag catagtggtg 14700 caggagcgca gcttgagcct ggctgacaag gtggccgcca tcaactattc catgcttagc 14760 ctgggcaagt tttacgcccg caagatatac catacccctt acgttcccat agacaaggag 14820 gtaaagatcg aggggttcta catgcgcatg gcgctgaagg tgcttacctt gagcgacgac 14880 ctgggcgttt atcgcaacga gcgcatccac aaggccgtga gcgtgagccg gcggcgcgag 14940 ctcagcgacc gcgagctgat gcacagcctg caaagggccc tggctggcac gggcagcggc 15000 gatagagagg ccgagtccta ctttgacgcg ggcgctgacc tgcgctgggc cccaagccga 15060 cgcgccctgg aggcagctgg ggccggacct gggctggcgg tggcacccgc gcgcgctggc 15120 aacgtcggcg gcgtggagga atatgacgag gacgatgagt acgagccaga ggacggcgag 15180 tactaagcgg tgatgtttct gatcagatga tgcaagacgc aacggacccg gcggtgcggg 15240 cggcgctgca gagccagccg tccggcctta actccacgga cgactggcgc caggtcatgg 15300 accgcatcat gtcgctgact gcgcgcaatc ctgacgcgtt ccggcagcag ccgcaggcca 15360 accggctctc cgcaattctg gaagcggtgg tcccggcgcg cgcaaacccc acgcacgaga 15420 aggtgctggc gatcgtaaac gcgctggccg aaaacagggc catccggccc gacgaggccg 15480 gcctggtcta cgacgcgctg cttcagcgcg tggctcgtta caacagcggc aacgtgcaga 15540 ccaacctgga ccggctggtg ggggatgtgc gcgaggccgt ggcgcagcgt gagcgcgcgc 15600 agcagcaggg caacctgggc tccatggttg cactaaacgc cttcctgagt acacagcccg 15660 ccaacgtgcc gcggggacag gaggactaca ccaactttgt gagcgcactg cggctaatgg 15720 tgactgagac accgcaaagt gaggtgtacc agtctgggcc agactatttt ttccagacca 15780 gtagacaagg cctgcagacc gtaaacctga gccaggcttt caaaaacttg caggggctgt 15840 ggggggtgcg ggctcccaca ggcgaccgcg cgaccgtgtc tagcttgctg acgcccaact 15900 cgcgcctgtt gctgctgcta atagcgccct tcacggacag tggcagcgtg tcccgggaca 15960 catacctagg tcacttgctg acactgtacc gcgaggccat aggtcaggcg catgtggacg 16020 agcatacttt ccaggagatt acaagtgtca gccgcgcgct ggggcaggag gacacgggca 16080 gcctggaggc aaccctaaac tacctgctga ccaaccggcg gcagaagatc ccctcgttgc 16140 acagtttaaa cagcgaggag gagcgcattt tgcgctacgt gcagcagagc gtgagcctta 16200 acctgatgcg cgacggggta acgcccagcg tggcgctgga catgaccgcg cgcaacatgg 16260 aaccgggcat gtatgcctca aaccggccgt ttatcaaccg cctaatggac tacttgcatc 16320 gcgcggccgc cgtgaacccc gagtatttca ccaatgccat cttgaacccg cactggctac 16380 cgccccctgg tttctacacc gggggattcg aggtgcccga gggtaacgat ggattcctct 16440 gggacgacat agacgacagc gtgttttccc cgcaaccgca gaccctgcta gagttgcaac 16500 agcgcgagca ggcagaggcg gcgctgcgaa aggaaagctt ccgcaggcca agcagcttgt 16560 ccgatctagg cgctgcggcc ccgcggtcag atgctagtag cccatttcca agcttgatag 16620 ggtctcttac cagcactcgc accacccgcc cgcgcctgct gggcgaggag gagtacctaa 16680 acaactcgct gctgcagccg cagcgcgaaa aaaacctgcc tccggcattt cccaacaacg 16740 ggatagagag cctagtggac aagatgagta gatggaagac gtacgcgcag gagcacaggg 16800 acgtgccagg cccgcgcccg cccacccgtc gtcaaaggca cgaccgtcag cggggtctgg 16860 tgtgggagga cgatgactcg gcagacgaca gcagcgtcct ggatttggga gggagtggca 16920 acccgtttgc gcaccttcgc cccaggctgg ggagaatgtt ttaaaaaaaa aaaagcatga 16980 tgcaaaataa aaaactcacc aaggccatgg caccgagcgt tggttttctt gtattcccct 17040 tagtatgcgg cgcgcggcga tgtatgagga aggtcctcct ccctctcag agagtgtggt 17100 gagcgcggcg ccagtggcgg cggcgctggg ttctcccttc gatgctcccc tggacccgcc 17160 gtttgtgcct ccgcggtacc tgcggcctac cggggggaga aacagcatcc gttactctga 17220 gttggcaccc ctattcgaca ccacccgtgt gtacctggtg gacaacaagt caacggatgt 17280 ggcatccctg aactaccaga acgaccacag caactttctg accacggtca ttcaaaacaa 17340 tgactacagc ccgggggagg caagcacaca gaccatcaat cttgacgacc ggtcgcactg 17400 gggcggcgac ctgaaaacca tcctgcatac caacatgcca aatgtgaacg agttcatgtt 17460 taccaataag tttaaggcgc gggtgatggt gtcgcgcttg cctactaagg acaatcaggt 17520 ggagctgaaa tacgagtggg tggagttcac gctgcccgag ggcaactact ccgagaccat 17580 gaccatagac cttatgaaca acgcgatcgt ggagcactac ttgaaagtgg gcagacagaa 17640 cggggttctg gaaagcgaca tcggggtaaa gtttgacacc cgcaacttca gactggggtt 17700 tgaccccgtc actggtcttg tcatgcctgg ggtatataca aacgaagcct tccatccaga 17760 catcattttg ctgccaggat gcggggtgga cttcacccac agccgcctga gcaacttgtt 17820 gggcatccgc aagcggcaac ccttccagga gggctttagg atcacctacg atgatctgga 17880 gggtggtaac attcccgcac tgttggatgt ggacgcctac caggcgagct tgaaagatga 17940 caccgaacag ggcgggggtg gcgcaggcgg cagcaacagc agtggcagcg gcgcggaaga 18000 gaactccaac gcggcagccg cggcaatgca gccggtggag gacatgaacg atcatgccat 18060 tcgcggcgac acctttgcca cacgggctga ggagaagcgc gctgaggccg aagcagcggc 18120 cgaagctgcc gcccccgctg cgcaacccga ggtcgagaag cctcagaaga aaccggtgat 18180 caaacccctg acagaggaca gcaagaaacg cagttacaac ctaataagca atgacagcac 18240 cttcacccag taccgcagct ggtaccttgc atacaactac ggcgaccctc agaccggaat 18300 ccgctcatgg accctgcttt gcactcctga cgtaacctgc ggctcggagc aggtctactg 18360 gtcgttgcca gacatgatgc aagaccccgt gaccttccgc tccacgcgcc agatcagcaa 18420 ctttccggtg gtgggcgccg agctgttgcc cgtgcactcc aagagcttct acaacgacca 18480 ggccgtctac tcccaactca tccgccagtt tacctctctg acccacgtgt tcaatcgctt 18540 tcccgagaac cagattttgg cgcgcccgcc agcccccacc atcaccaccg tcagtgaaaa 18600 cgttcctgct ctcacagatc acgggacgct accgctgcgc aacagcatcg gaggagtcca 18660 gcgagtgacc attactgacg ccagacgccg cacctgcccc tacgtttaca aggccctggg 18720 catagtctcg ccgcgcgtcc tatcgagccg cactttttga gcaagcatgt ccatccttat 18780 atcgcccagc aataacacag gctggggcct gcgcttccca agcaagatgt ttggcggggc 18840 caagaagcgc tccgaccaac acccagtgcg cgtgcgcggg cactaccgcg cgccctgggg 18900 cgcgcacaaa cgcggccgca ctgggcgcac caccgtcgat gacgccatcg acgcggtggt 18960 ggaggaggcg cgcaactaca cgcccacgcc gccaccagtg tccacagtgg acgcggccat 19020 tcagaccgtg gtgcgcggag cccggcgcta tgctaaaatg aagagacggc ggaggcgcgt 19080 agcacgtcgc caccgccgcc gacccggcac tgccgcccaa cgcgcggcgg cggccctgct 19140 taaccgcgca cgtcgcaccg gccgacgggc ggccatgcgg gccgctcgaa ggctggccgc 19200 gggtattgtc actgtgcccc ccaggtccag gcgacgagcg gccgccgcag cagccgcggc 19260 cattagtgct atgactcagg gtcgcagggg caacgtgtat tgggtgcgcg actcggttag 19320 cggcctgcgc gtgcccgtgc gcacccgcc cccgcgcaac tagattgcaa gaaaaaacta 19380 cttagactcg tactgttgta tgtatccagc ggcggcggcg cgcaacgaag ctatgtccaa 19440 gcgcaaaatc aaagaagaga tgctccaggt catcgcgccg gagatctatg gccccccgaa 19500 gaaggaagag caggattaca agccccgaaa gctaaagcgg gtcaaaaaga aaaagaaaga 19560 tgatgatgat gaacttgacg acgaggtgga actgctgcac gctaccgcgc ccaggcgacg 19620 ggtacagtgg aaaggtcgac gcgtaaaacg tgttttgcga cccggcacca ccgtagtctt 19680 tacgccccggt gagcgctcca cccgcaccta caagcgcgtg tatgatgagg tgtacggcga 19740 cgaggacctg cttgagcagg ccaacgagcg cctcggggag tttgcctacg gaaagcggca 19800 taaggacatg ctggcgttgc cgctggacga gggcaaccca acacctagcc taaagcccgt 19860 aacactgcag caggtgctgc ccgcgcttgc accgtccgaa gaaaagcgcg gcctaaagcg 19920 cgagtctggt gacttggcac ccaccgtgca gctgatggta cccaagcgcc agcgactgga 19980 agatgtcttg gaaaaaatga ccgtggaacc tgggctggag cccgaggtcc gcgtgcggcc 20040 aatcaagcag gtggcgccgg gactgggcgt gcagaccgtg gacgttcaga tacccactac 20100 cagtagcacc agtattgcca ccgccacaga gggcatggag acacaaacgt ccccggttgc 20160 ctcagcggtg gcggatgccg cggtgcaggc ggtcgctgcg gccgcgtcca agacctctac 20220 ggaggtgcaa acggacccgt ggatgtttcg cgtttcagcc ccccggcgcc cgcgccgttc 20280 gaggaagtac ggcgccgcca gcgcgctact gcccgaatat gccctacatc cttccattgc 20340 gcctaccccc ggctatcgtg gctacaccta ccgccccaga agacgagcaa ctacccgacg 20400 ccgaaccacc actggaaccc gccgccgccg tcgccgtcgc cagcccgtgc tggccccgat 20460 ttccgtgcgc agggtggctc gcgaaggagg caggaccctg gtgctgccaa cagcgcgcta 20520 ccaccccagc atcgtttaaa agccggtctt tgtggttctt gcagatatgg ccctcacctg 20580 ccgcctccgt ttcccggtgc cgggattccg aggaagaatg caccgtagga ggggcatggc 20640 cggccacggc ctgacgggcg gcatgcgtcg tgcgcaccac cggcggcggc gcgcgtcgca 20700 ccgtcgcatg cgcggcggta tcctgcccct ccttattcca ctgatcgccg cggcgattgg 20760 cgccgtgccc ggaattgcat ccgtggcctt gcaggcgcag agacactgat taaaaacaag 20820 ttgcatgtgg aaaaatcaaa ataaaaagtc tggactctca cgctcgcttg gtcctgtaac 20880 tattttgtag aatggagaac atcaactttg cgtctctggc cccgcgacac ggctcgcgcc 20940 cgttcatggg aaactggcaa gatatcggca ccagcaatat gagcggtggc gccttcagct 21000 ggggctcgct gtggagcggc attaaaatt tcggttccac cgttaagaac tatggcagca 21060 aggcctggaa cagcagcaca ggccagatgc tgagggataa gttgaaagag caaaatttcc 21120 aacaaaaggt ggtagatggc ctggcctctg gcattagcgg ggtggtggac ctggccaacc 21180 aggcagtgca aaataagatt aacagtaagc ttgatccccg ccctcccgta gaggagcctc 21240 caccggccgt ggagacagtg tctccagagg ggcgtggcga aaagcgtccg cgccccgaca 21300 gggaagaaac tctggtgacg caaatagacg agcctccctc gtacgaggag gcactaaagc 21360 aaggcctgcc caccacccgt cccatcgcgc ccatggctac cggagtgctg ggccagcaca 21420 cacccgtaac gctggacctg cctccccccg ccgacaccca gcagaaacct gtgctgccag 21480 gcccgaccgc cgttgttgta acccgtccta gccgcgcgtc cctgcgccgc gccgccagcg 21540 gtccgcgatc gttgcggccc gtagccagtg gcaactggca aagcacactg aacagcatcg 21600 tgggtctggg ggtgcaatcc ctgaagcgcc gacgatgctt ctgatagcta acgtgtcgta 21660 tgtgtgtcat gtatgcgtcc atgtcgccgc cagaggagct gctgagccgc cgcgcgcccg 21720 ctttccaaga tggctacccc ttcgatgatg ccgcagtggt cttacatgca catctcgggc 21780 caggacgcct cggagtacct gagccccggg ctggtgcagt ttgcccgcgc caccgagacg 21840 tacttcagcc tgaataacaa gtttagaaac cccacggtgg cgcctacgca cgacgtgacc 21900 acagaccggt cccagcgttt gacgctgcgg ttcatccctg tggaccgtga ggatactgcg 21960 tactcgtaca aggcgcggtt caccctagct gtgggtgata accgtgtgct ggacatggct 22020 tccacgtact ttgacatccg cggcgtgctg gandaggggcc ctacttta gccctactct 22080 ggcactgcct acacgccct ggctcccaag ggtgccccaa atccttgcga atgggatgaa 22140 gctgctactg ctcttgaaat aaacctagaa gagaggacg atgacaacga aggagaagta 22200 gacgagcaag ctgagcagca aaaaactcac gtatttgggc aggccctta ttctgtata 22260 atattacaa aggaggtat tcaataggt gtcgaaggtc aaacacctaa atatgccgat 22320 aaaacatttc aacctgacc tcaatagga gatctcagt ggtacgaac agaattaat 22380 catgcagctg ggagtcct aaaaaagact acccaatga aaccatgtta cggttcatat 22440 gcaaaaccca aaatgaaaa tggaggcaa ggcattcttg taaagcaca aaatggaaag 22500 ctagaaagtc aagtggaat gcaattttc tcaactactg aggcagccgc agcaatggt 22560 gataacttga ctcctaaagt ggtattgtac agtgagatg tagatataga aaccccagac 22620 actcatattt cttacatgcc cactattag gaagtact cacgagaact aatgggccaa 22680 caatctatgc ccaacaggcc taattacatt gctttagg acaatttat tggtctaatg 22740 tattacaaca gcacgggtaa tatgggtgtt ctggcgggcc aagcatcgca gttgaatgct 22800 gttgtagatt tgcaagacag aaacacagag ctttcatacc agctttgct tgattccatt 22860 ggtgatagaa ccaggtactt ttctgtgg aatcaggctg ttgacagcta tgatccagat 22920 gttagaatta ttgaaaatca tggaactgaa gatgaacttc caattactg ctttccactg 22980 ggaggtgtga ttaatacaga gactcttacc aaggtaaaac ctaaaacagg tcaggaaat 23040 ggatgggaa aagatgctac agatttca gataaaaatg aaataaggt tggaataat 23100 tttgccatgg aaatcaatct aaatgccaac ctgtggagaa atttcctgta ctccaacata 23160 gcgctgtatt tgcccgacaa gctaaagtac agtccttcca acgtaaaaat ttctgataac 23220 ccaaacacct acgactacat gaacaagcga gtggtggctc ccggctagt ggactgctac 23280 attackaccttg gagcacgctg gtcccttgac tatatggaca acgtcaaccc attack 23340 caccgcaatg ctggcctgcg ctaccgctca atgttgctg gcaatgtcg ctagtgccc 23400 ttccacatcc aggtgcctca gaagttcttt gccattaaaa acctccttct cctgccgggc 23460 tcatacacct acgagtggaa cttcaggaag gatgttaaca tggttctgca gagctcccta 23520 ggaaatgacc taagggttga cggagccagc attaagtttg atagcatttg cctttacgcc 23580 accttcttcc ccatggccca caacaccgcc tccacgcttg aggccatgct tagaaacgac 23640 accaacgacc agtcctttaa cgactatctc tccgccgcca acatgctcta ccctataccc 23700 gccaacgcta ccaacgtgcc catatccatc cctcccgca actgggcggc tttccgcggc 23760 tgggccttca cgcgccttaa gactaaggaa accccatcac tgggctcggg ctacgaccct 23820 tattacacct actctggctc tataccctac ctagatggaa ccttttacct caaccacacc 23880 tttaagaagg tggccattac ctttgactct tctgtcagct ggcctggcaa tgaccgcctg 23940 cttaccccca acgagtttga aattaagcgc tcagttgacg gggagggtta caacgttgcc 24000 cagtgtaaca tgaccaaaga ctggttcctg gtacaaatgc tagctaacta taacattggc 24060 taccagggct tctatatccc agagagctac aaggaccgca tgtactcctt ctttagaaac 24120 ttccagccca tgagccgtca ggtggtggat gatactaaat acaaggacta ccaacaggtg 24180 ggcatcctac accaacacaa caactctgga tttgttggct accttgcccc caccatgcgc 24240 gaaggacagg cctaccctgc taacttcccc tatccgctta taggcaagac cgcagttgac 24300 agcattaccc agaaaaagtt tctttgcgat cgcacccttt ggcgcatccc attctccagt 24360 aactttatgt ccatgggcgc actcacagac ctgggccaaa accttctcta cgccaactcc 24420 gcccacgcgc tagacatgac ttttgaggtg gatcccatgg acgagcccac ccttctttat 24480 gttttgtttg aagtctttga cgtggtccgt gtgcaccagc cgcaccgcgg cgtcatcgaa 24540 accgtgtacc tgcgcacgcc cttctcggcc ggcaacgcca caacataaag aagcaagcaa 24600 catcaacaac agctgccgcc atgggctcca gtgagcagga actgaaagcc attgtcaaag 24660 atcttggttg tgggccatat tttttgggca cctatgacaa gcgctttcca ggctttgttt 24720 ctccacacaa gctcgcctgc gccatagtca atacggccgg tcgcgagact gggggcgtac 24780 actggatggc ctttgcctgg aacccgcact caaaaacatg ctacctcttt gagccctttg 24840 gcttttctga ccagcgactc aagcaggttt accagtttga gtacgagtca ctcctgcgcc 24900 gtagcgccat tgcttcttcc cccgaccgct gtataacgct ggaaaagtcc acccaaagcg 24960 tacaggggcc caactcggcc gcctgtggac tattctgctg catgtttctc cacgcctttg 25020 ccaactggcc ccaaactccc atggatcaca accccaccat gaaccttatt accggggtac 25080 ccaactccat gctcaacagt ccccaggtac agcccaccct gcgtcgcaac caggaacagc 25140 tctacagctt cctggagcgc cactcgccct acttccgcag ccacagtgcg cagattagga 25200 gcgccacttc tttttgtcac ttgaaaaaca tgtaaaaata atgtactaga gacactttca 25260 ataaaggcaa atgcttttat ttgtacactc tcgggtgatt atttaccccc acccttgccg 25320 tctgcgccgt ttaaaaatca aaggggttct gccgcgcatc gctatgcgcc actggcaggg 25380 acacgttgcg atactggtgt ttagtgctcc acttaaactc aggcacaacc atccgcggca 25440 gctcggtgaa gttttcactc cagaggctc gcaccatcac caacgcgttt agcaggtcgg 25500 gcgccgatat cttgaagtcg cagttggggc ctccgcccctg cgcgcgcgag ttgcgataca 25560 cagggttgca gcactggaac actatcagcg ccgggtggtg cacgctggcc agcacgctct 25620 tgtcggagat cagatccgcg tccaggtcct ccgcgttgct cagggcgaac ggagtcaact 25680 ttggtagctg ccttcccaaa aagggcgcgt gcccaggctt tgagttgcac tcgcaccgta 25740 gtggcatcaa aaggtgaccg tgcccggtct gggcgttagg atacagcgcc tgcataaaaag 25800 ccttgatctg cttaaaagcc acctgagcct ttgcgccttc agagaagaac atgccgcaag 25860 acttgccgga aaactgattg gccggacagg ccgcgtcgtg cacgcagcac cttgcgtcgg 25920 tgttggagat ctgcaccaca tttcggcccc accggttctt cacgatcttg gccttgctag 25980 actgctcctt cagcgcgcgc tgcccgttt cgctcgtcac atccatttca atcacgtgct 26040 ccttattat cataatgctt ccgtgtagac acttaagctc gccttcgatc tcagcgcagc 26100 ggtgcagcca caacgcgcag cccgtgggct cgtgatgctt gtaggtcacc tctgcaaacg 26160 actgcaggta cgcctgcagg aatcgcccca tcatcgtcac aaaggtcttg ttgctggtga 26220 aggtcagctg caacccgcgg tgctcctcgt tcagccaggt cttgcatacg gccgccagag 26280 cttccacttg gtcaggcagt agtttgaagt tcgcctttag atcgttatcc acgtggtact 26340 tgtccatcag cgcgcgcgca gcctccatgc ccttctccca cgcagacacg atcggcacac 26400 tcagcgggtt catcaccgta atttcacttt ccgcttcgct gggctcttcc tcttcctctt 26460 gcgtccgcat accacgcgcc actgggtcgt cttcattcag ccgccgcact gtgcgcttac 26520 ctcctttgcc atgcttgatt agcaccggtg ggttgctgaa acccaccatt tgtagcgcca 26580 catcttctct ttcttcctcg ctgtccacga ttacctctgg tgatggcggg cgctcgggct 26640 tgggagaagg gcgcttcttt ttcttcttgg gcgcaatggc caaatccgcc gccgaggtcg 26700 atggccgcgg gctgggtgtg cgcggcacca gcgcgtcttg tgatgagtct tcctcgtcct 26760 cggactcgat acgccgcctc atccgctttt ttgggggcgc ccggggaggc ggcggcgacg 26820 gggacgggga cgacacgtcc tccatggttg ggggacgtcg cgccgcaccg cgtccgcgct 26880 cgggggtggt ttcgcgctgc tcctcttccc gactggccat ttccttctcc tataggcaga 26940 aaaagatcat ggagtcagtc gagaagaagg acagcctaac cgccccctct gagttcgcca 27000 ccaccgcctc caccgatgcc gccaacgcgc ctaccacctt ccccgtcgag gcacccccgc 27060 ttgaggagga ggaagtgatt atcgagcagg acccaggttt tgtaagcgaa gacgacgagg 27120 accgctcagt accaacagag gataaaaagc aagaccagga caacgcagag gcaaacgagg 27180 aacaagtcgg gcggggggac gaaaggcatg gcgactacct agatgtggga gacgacgtgc 27240 tgttgaagca tctgcagcgc cagtgcgcca ttatctgcga cgcgttgcaa gagcgcagcg 27300 atgtgcccct cgccatagcg gatgtcagcc ttgcctacga acgccaccta ttctcaccgc 27360 gcgtaccccc caaacgccaa gaaaacggca catgcgagcc caacccgcgc ctcaacttct 27420 accccgtatt tgccgtgcca gaggtgcttg ccacctatca catctttttc caaaactgca 27480 agatacccct atcctgccgt gccaaccgca gccgagcgga caagcagctg gccttgcggc 27540 agggcgctgt catacctgat atcgcctcgc tcaacgaagt gccaaaaaatc tttgagggtc 27600 ttggacgcga cgagaagcgc gcggcaaacg ctctgcaaca ggaaaacagc gaaaatgaaa 27660 gtcactctgg agtgttggtg gaactcgagg gtgacaacgc gcgcctagcc gtactaaac 27720 gcagcatcga ggtcacccac tttgcctacc cggcacttaa cctacccccc aaggtcatga 27780 gcacagtcat gagtgagctg atcgtgcgcc gtgcgcagcc cctggagagg gatgcaaatt 27840 tgcaagaaca aacagaggag ggcctacccg cagttggcga cgagcagcta gcgcgctggc 27900 ttcaaacgcg cgagcctgcc gacttggagg agcgacgcaa actaatgatg gccgcagtgc 27960 tcgttaccgt ggagcttgag tgcatgcagc ggttctttgc tgacccggag atgcagcgca 28020 agctagagga aacattgcac tacacctttc gacagggcta cgtacgccag gcctgcaaga 28080 tctccaacgt ggagctctgc aacctggtct cctaccttgg aattttgcac gaaaaccgcc 28140 ttgggcaaaa cgtgcttcat tccacgctca agggcgaggc gcgccgcgac tacgtccgcg 28200 actgcgttta cttatttcta tgctacacct ggcagacggc catgggcgtt tggcagcagt 28260 gcttggagga gtgcaacctc agaagctgc agaaactgct aaagcaaaac ttgaaggacc 28320 tatggacggc cttcaacgag cgctccgtgg ccgcgcacct ggcggacatc attttccccg 28380 aacgcctgct taaaaccctg caacagggtc tgccagactt caccagtcaa agcatgttgc 28440 agaactttag gaactttatc ctagagcgct caggaatctt gcccgccacc tgctgtgcac 28500 ttcctagcga ctttgtgccc attaagtacc gcgaatgccc tccgccgctt tggggccact 28560 gctaccttct gcagctagcc aactaccttg cctaccactc tgacataatg gaagacgtga 28620 gcggtgacgg tctactggag tgtcactgtc gctgcaacct atgcaccccg caccgctccc 28680 tggtttgcaa ttcgcagctg cttaacgaaa gtcaaattat cggtaccttt gagctgcagg 28740 gtccctcgcc tgacgaaaag tccgcggctc cggggttgaa actcactccg gggctgtgga 28800 cgtcggctta ccttcgcaaa tttgtacctg aggactacca cgcccacgag attaggttct 28860 acgaagacca atcccgcccg cctaatgcgg agcttaccgc ctgcgtcatt acccagggcc 28920 acattcttgg ccaattgcaa gccatcaaca aagcccgcca agagtttctg ctacgaaagg 28980 gacggggggt ttacttggac ccccagtccg gcgaggagct caacccaatc cccccgccgc 29040 cgcagcccta tcagcagcag ccgcgggccc ttgcttccca ggatggcacc caaaaagaag 29100 ctgcagctgc cgccgccacc cacggacgag gaggaatact gggacagtca ggcagaggag 29160 gttttggacg aggaggagga ggacatgatg gaagactggg agagcctaga cgaggaagct 29220 tccgaggtcg aagaggtgtc agacgaaaca ccgtcaccct cggtcgcatt cccctcgccg 29280 gcgccccaga aatcggcaac cggttccagc atggctacaa cctccgctcc tcaggcgccg 29340 ccggcactgc ccgttcgccg acccaaccgt agatgggaca ccactggaac cagggccggt 29400 aagtccaagc agccgccgcc gttagcccaa gagcaacaac agcgccaagg ctaccgctca 29460 tggcgcgggc acaagaacgc catagttgct tgcttgcaag actgtggggg caacatctcc 29520 ttcgcccgcc gctttcttct ctaccatcac ggcgtggcct tcccccgtaa catcctgcat 29580 tactaccgtc atctctacag cccatactgc accggcggca gcggcagca cagcagcggc 29640 cacacagaag aaggcgac cggatagca gactctgaca aagcccaag aatccacagc 29700 ggcggcagca gcaggaggag gagcgctgcg tctggcgccc aacgaacccg tcgacccg 29760 cgagcttaga aacaggattt ttcccactct gtatgctata ttcaacaga gcaggggcca 29820 agaacaagag ctgaaaataa aaaacaggtc tctgcgatcc ctcacccgca gctgcctgta 29880 tcacaaagc gagatcagc ttcggcgcac gctggaagc gcggaggctc tctcagtaa 29940 atactgcgcg ctgactctta aggactagtt tcgcgcctt tctcaattt aagcgcgaaa 30000 actacgtcat ctccagcggc cacacccggc gccagcacct gttgtcagcg ccattatgag 30060 CAggaaatt cccacgccct acatgtggag ttaccagcca CAATgggac ttgcggctgg 30120 agctgcccaa gactactcaa cccgataaa ctacatgagc gcgggacccc acatgatatc 30180 ccgggtcaac ggaatacgcg cccaccgaaa ccgaattctc ctggaacagg cggctattac 30240 caccacacct cgtaatacc ttaatccccg tagttgccc gctgccctgg tgtaccagga 30300 aagtcccgct cccaccactg tggtacttcc cagagacgcc caggccgaag ttcagatgac 30360 taactcaggg gcgcagcttg cgggcggctt tcgtcacagg gtgcggtcgc ccgggcaggg 30420 tataactcac ctgacaatca gagggcgagg tattcagctc aacgacgagt cggtgagctc 30480 ctcgcttggt ctccgtccgg acgggacatt tcagatcggc ggcgccggcc gctcttcatt 30540 cacgcctcgt caggcaatcc taactctgca gacctcgtcc tctgagccgc gctctggagg 30600 cattggaact ctgcaattta ttgaggagtt tgtgccatcg gtctacttta accccttctc 30660 gggacctccc ggccactatc cggatcaatt tattcctaac tttgacgcgg taaaggactc 30720 ggcggacggc tacgactgaa tgttaagtgg agaggcagag caactgcgcc tgaaacacct 30780 ggtccactgt cgccgccaca agtgctttgc ccgcgactcc ggtgagtttt gctactttga 30840 attgcccgag gatcatatcg agggcccggc gcacggcgtc cggcttaccg cccagggaga 30900 gcttgcccgt agcctgattc gggagtttac ccagcgcccc ctgctagttg agcgggacag 30960 gggaccctgt gttctcactg tgatttgcaa ctgtcctaac cctggattac atcaagatcc 31020 tctagttaat gtcaggtcgc ctaagtcgat taactagagt acccggggat cttattccct 31080 ttaactaata aaaaaaaata ataaagcatc acttacttaa aatcagttag caaatttctg 31140 tccagtttat tcagcagcac ctccttgccc tcctcccagc tctggtattg cagcttcctc 31200 ctggctgcaa actttctcca caatctaaat ggaatgtcag tttcctcctg ttcctgtcca 31260 tccgcaccca ctatcttcat gttgttgcag atgaagcgcg caagaccgtc tgaagatacc 31320 ttcaaccccg tgtatccata tgacacggaa accggtcctc caactgtgcc ttttcttact 31380 cctccctttg tatcccccaa tgggtttcaa gagagtcccc ctggggtact ctctttgcgc 31440 ctatccgaac ctctagttac ctccaatggc atgcttgcgc tcaaaatggg caacggcctc 31500 tctctggacg aggccggcaa ccttacctcc caaaatgtaa ccactgtgag cccacctctc 31560 aaaaaaacca agtcaaacat aaacctggaa atatctgcac ccctcacagt tacctcagaa 31620 gccctaactg tggctgccgc cgcacctcta atggtcgcgg gcaacacact caccatgcaa 31680 tcacaggccc cgctaaccgt gcacgactcc aaacttagca ttgccaccca aggacccctc 31740 acagtgtcag aaggaaagct agccctgcaa acatcaggcc ccctcaccac caccgatagc 31800 agtaccctta ctatcactgc ctcaccccct ctaactactg ccactggtag cttgggcatt 31860 gacttgaaag agcccattta tacacaaaat ggaaaactag gactaaagta cggggctcct 31920 ttgcatgtaa cagacgacct aaacactttg accgtagcaa ctggtccagg tgtgactatt 31980 aataatactt ccttgcaaac taaagttact ggagccttgg gttttgattc acaaggcaat 32040 atgcaactta atgtagcagg aggactaagg attgattctc aaaacagacg ccttatactt 32100 gatgttagtt atccgtttga tgctcaaaac caactaaatc taagactagg acagggccct 32160 ctttttataa actcagccca caacttggat attaactaca acaaaggcct ttacttgttt 32220 acagcttcaa acaattccaa aaagcttgag gttaacctaa gcactgccaa ggggttgatg 32280 ttgacgcta cagccatagc cattaatgca ggagatgggc ttgaatttgg ttcacctaat 32340 gcaccaaacaaatcccct siaaaaaaaa attggccatg gcctagaatt tgattcaac 32400 aaggctatgg ttcctaact aggactggc cttagttttg acaccacagg tgccattaca 32460 gtaggaaaca aaaataatga taagctact ttgtggacca caccagctcc atctcctaac 32520 tgtagactaa atgcagagaa agatgctaaa ctcactttgg tcttacaa atgtggcagt 32580 caaatacttg ctacagtttc agttttggct gttaaaggca gttggctcc atatctgga 32640 acagttcaa gtgctcatct tattataga tttgacgaaa atggagtgct actaacaat 32700 tccttcctgg acccagaata ttggacttt agaatggag atcttactga aggcacagcc 32760 tatacaaacg ctgttggatt tatgcctaac ctcagctt atccaaatc tcacggtaaa 32820 actgccaaaa gtaacattgt cagtcaagtt tacttaacg gagacaaac taaacctgta 32880 acactaacca ttacactaaa cggtacacag gaaacaggag acacaactcc aagtgcatac 32940 tctatgtcat ttcatggga ctggtctggc cacaactaca ttaatgaat atttgccaca 33000 tcctcttaca ctttttcata cattgcccaa gaataagaa tcgtttgtgt tatgtttcaa 33060 cgtgtttatt tttcaattgc agaaaatttc aagtcatttt tcattcagta gtatagcccc 33120 accaccacat agcttataca gatcaccgta ccttaatcaa actcacagaa ccctagtatt 33180 caacctgcca cctccctccc aacacacaga gtacacagtc ctttctcccc ggctggcctt 33240 aaaaagcatc atatcatggg taacagacat attcttaggt gttatattcc acacggtttc 33300 ctgtcgagcc aaacgctcat cagtgatatt aataaactcc ccgggcagct cacttaagtt 33360 catgtcgctg tccagctgct gagccacagg ctgctgtcca acttgcggtt gcttaacggg 33420 cggcgaagga gaagtccacg cctacatggg ggtagagtca taatcgtgca tcaggatagg 33480 gcggtggtgc tgcagcagcg cgcgaataaa ctgctgccgc cgccgctccg tcctgcagga 33540 atacaacatg gcagtggtct cctcagcgat gattcgcacc gcccgcagca taaggcgcct 33600 tgtcctccgg gcacagcagc gcaccctgat ctcacttaaa tcagcacagt aactgcagca 33660 cagcaccaca atattgttca aaatcccaca gtgcaaggcg ctgtatccaa agctcatggc 33720 ggggaccaca gaacccacgt ggccatcata ccacaagcgc aggtagatta agtggcgacc 33780 cctcataaac acgctggaca taaacattac ctcttttggc atgttgtaat tcaccacctc 33840 ccggtaccat ataaacctct gattaaacat ggcgccatcc accaccatcc taaaccagct 33900 ggccaaaacc tgcccgccgg ctatacactg cagggaaccg ggactggaac aatgacagtg 33960 gagagcccag gactcgtaac catggatcat catgctcgtc atgatatcaa tgttggcaca 34020 acacaggcac acgtgcatac acttcctcag gattacaagc tcctcccgcg ttagaaccat 34080 atcccaggga acaacccatt cctgaatcag cgtaaatccc acactgcagg gaagacctcg 34140 cacgtaactc acgttgtgca ttgtcaaagt gttacattcg ggcagcagcg gatgatcctc 34200 cagtatggta gcgcgggttt ctgtctcaaa aggaggtaga cgatccctac tgtacggagt 34260 gcgccgagac aaccgagatc gtgttggtcg tagtgtcatg ccaaatggaa cgccggacgt 34320 agtcatattt cctgaagcaa aaccaggtgc gggcgtgaca aacagatctg cgtctccggt 34380 ctcgccgctt agatcgctct gtgtagtagt tgtagtatat ccactctctc aaagcatcca 34440 ggcgccccct ggcttcgggt tctatgtaaa ctccttcatg cgccgctgcc ctgataacat 34500 ccaccaccgc agaataagcc acacccagcc aacctacaca ttcgttctgc gagtcacaca 34560 cgggaggagc gggaagagct ggaagaacca tgtttttttt tttattccaa aagattatcc 34620 aaaacctcaa aatgaagatc tattaagtga acgcgctccc ctccggtggc gtggtcaaac 34680 tctacagcca aagaacagat aatggcattt gtaagatgtt gcacaatggc ttccaaaagg 34740 caaacggccc tcacgtccaa gtggacgtaa aggctaaacc cttcagggtg aatctcctct 34800 ataaacattc cagcaccttc aaccatgccc aaataattct catctcgcca ccttctcaat 34860 atatctctaa gcaaatcccg aatattaagt ccggccattg taaaaatctg ctccagagcg 34920 ccctccacct tcagcctcaa gcagcgaatc atgattgcaa aaattcaggt tcctcacaga 34980 cctgtataag attcaaaagc ggaacattaa caaaaatacc gcgatcccgt aggtcccttc 35040 gcagggccag ctgaacataa tcgtgcaggt ctgcacggac cagcgcggcc acttccccgc 35100 35160 ccagcgtagc cccgatgtaa gcttgttgca tgggcggcga tataaatgc aaggtgctgc 35220 tcaaaaaatc aggcaaagcc tcgcgcaaaa aagaaagcac atcgtagtca tgctcatgca 35280 gataaaggca ggtaagctcc ggaaccacca cagaaaaaga caccattttt ctctcaaaca 35340 tgtctgcggg tttctgcata aacacaaaat aaataacaa aaaaacattt aaacattaga 35400 agcctgtctt acaacaggaa aaacaaccct tataagcata agacggacta cggccatgcc 35460 ggcgtgaccg taaaaaaact ggtcaccgtg attaaaaaagc accaccgaca gctcctcggt 35520 catgtccgga gtcataatgt aagactcggt aaacacatca ggttgattca catcggtcag 35580 tgctaaaaag cgaccgaaat agcccggggg aatacatacc cgcaggcgta gagacaacat 35640 tacagccccc ataggaggta taacaaaatt aataggagag aaaaacacat aaacacctga 35700 aaaaccctcc tgcctaggca aaatagcacc ctcccgctcc agaacaacat acagcgcttc 35760 cacagcggca gccataacag tcagccttac cagtaaaaa gaaaacctat taaaaaaaca 35820 35880. ccctcgac cggcaccagc tcaatcagtc acagtgtaaa aaagggcca gtgcagagcg agtatatata ggactaaaa atgacgtac ggttaaagtc cacaaaaaac acccagaaaa ccgcacgcga acctacgccc agaacgaa gccaaaaaac ccacaacttc ctcaaatcgt cacttccgtt ttcccacgtt acgtcacttc ccattttaag aaaactacaa ttcccaacac atacaagtta ctccgcccta aaacctacgt cacccgcccc gttcccacgc cccgcgccac gtcacaaact ccaccccctc attack tggcttcaat ccaaataag gtatatt <210> 10 <211> 37735 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 10 tagccc atcatcaata atatacctta ttttggattg aagccaatat gataatgagg gggtggagtt tgtgacgtgg cgcggggcgt gggacgggg cgggtgacgt agtagtgtgg 120 cggaagtgtg atgttgcaag tgtggcggaa cacatgtaag cgacggatgt ggcaaaagtg acgttttgg tgtgcgccgg tgtacacagg aagtgacaat ttcgcgcgg tttaggcgg 240 atgttgtagt aaatttgggc gtaccgagt aagatttggc catttcgcg ggaaaactga 300 ataagaggaa gtgaaatctg ataatttg tgttactcat agcgcgtaat actgctagag 360 atctggcgaa agggggatgt gctgcaggc gattaagttg gggtaacgcca gggttttccc 420 agtcacgacg ttgtaaaacg acggccagtg aattgtata cgactcacta taggcgaat 480 tgggtactgg ccacaggagc ttggccatt gcatacgttg tatccatatc atatatgta 540 catttatatt ggctcatgtc caattacc gccatgttga cattgattat tgactagtta 600 ttaatagtaa tcattacgg gtcattagt tcatagccca tattagt tccgcgttac 660 attackcg gtaaatggcc cgcctggctg acccccac gaccccgcc cattgacgtc 720 aaatgacg tatgttccca tagtaacgcc atagggact ttccattgac gtcaatgggt 780 ggagtattta cggtaactg cccacttggc agtacatca gtgtatcata tgccaagtac 840 gccccctatt gacgtcaatg acggtaatg gcccgcctgg cattatgccc agtacatgac 900 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt 960 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc 1020 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt 1080 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg 1140 ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc 1200 acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctgactc tagcctagct 1260 ctgaagttgg tggtgaggcc ctgggcaggt tggtatcaag gttacaagac aggtttaagg 1320 agaccaatag aaactgggca tgtggagaca gagaagactc ttgggtttct gataggcact 1380 gactctctct gcctattggt ctattttccc acccttaggc tgctggtctg agcctaggag 1440 atctctcgag gtcgacggta tcgatgggta ccgccaccat gtttgttttt ctcgtactcc 1500 tgcccctggt ttcctcccaa tgtgtcaatc tgactacccg gacccaactt cctcccgcct 1560 acaccaattc ctttacccga ggtgtttact acccagacaa agtgttcagg tcatccgtcc 1620 tccatagtac ccaagacctc ttcctccctt tttttctaa cgttacctgg tttcacgcta 1680 ttcacgttag cggcaccaac ggcaccaaaa gattcgataa ccccgtactg ccgttcaacg 1740 acggggtata ttttgcctct actgaaaaat caaacatcat acgcggatgg atctttggga 1800 ctaccctgga ctcaaaaact cagtccctgc tgattgtgaa taacgctacc aacgtggtga 1860 tcaaagtctg tgaattccag ttttgcaacg atccttttct cggcgtttat tatcacaaaa 1920 ataacaaatc ctggatggag agcgagttcc gggtgtactc ctccgcgaat aattgcacct 1980 tcgaatatgt gtctcagcca ttcctcatgg acctcgaggg gaagcagggc aattttaaga 2040 atctgcgaga attcgtgttc aagaatatag acggtactt caagatttac tccaaacaca 2100 ccccgattaa cctggttagg gacttgcctc agggcttttc tgcattggag cccctcgtgg 2160 acctcccaat cggcataaac attacaagat ttcagacttt gcttgcattg cacaggagct 2220 atttgacacc cggcgattct tcttcggat ggaccgctgg agcagctgct tattacgtgg 2280 gctatctgca gcctcgaacc tttcttttga agtacaacga aaatggaact atcaccgatg 2340 cagttgactg cgccctggac cccctgtccg aaactaagtg cacgctcaa agttcacag 2400 tagagaaggg gatataccag actagcaatt tccgcgttca gccaaccgaa agtatagtgc 2460 gctttcctaa tataactaac ctgtgtcctt tcgggagt gtttaacgcc actagattcg 2520 cttccgtcta cgcctggaat agaaagagga tctcaattg cgttgctgac tatagtgttt 2580 tgtacaatc cgccctttc tcaccttca aatgttacgg ggtgagccct accaactga 2640 acgacctgtg ctttacaaac gtatacgccg acagctttgt tatcagagga gacgaggttc 2700 gccagattgc tccgggtcag acaggcaga tgctgatta taattacaaa ctgcccgacg 2760 actttacagg atgtgtgatc gcgtggaaca gtaacaatct tgactcaag gttggggta 2820 attataatta tctttaccgg ctgttcagaa aaagcaattt gaacccttc gaaagggaca 2880 tatccaccga gatctatcag gccggggtcca ctccatgcaa tggtgtggaa ggttttaatt 2940 gctacttccc attgcagtct tatggattcc aaccaaccaa tggcgtaggc taccagccgt 3000 atcgcgttgt cgtgctcagc ttcgagctgc tccacgcccc cgcgaccgta tgcggtccta 3060 agaagtccac caatcttgtt aagaacaagt gtgtaaactt taactttaac gggctgaccg 3120 ggaccggcgt tctgactgaa tctaacaaaa aattcctgcc tttccagcag ttcggccgcg 3180 atattgctga caccactgac gctgtaagag accctcagac ccttgaaatt ctcgatatca 3240 caccttgcag ctttgggggc gtgtccgtca tcactccagg aactaacaca agcaaccagg 3300 tggcagtgtt gtaccaggat gttaattgta ccgaggtgcc agtggccatc cacgccgatc 3360 aattgacacc tacctggagg gtttacagca cagggtccaa tgtttttcag acaagagccg 3420 gatgtctgat cggtgccgag catgtcaaca attcctacga gtgtgatatc cccattggtg 3480 cgggaatttg tgcatcatat cagacccaga ctaatagccc aagaagagct agatccgtcg 3540 ctagtcaatc catcattgca tatacaatgt ccctgggagc tgagaattca gtcgcgtatt 3600 caaacaattc cattgctatt cctactaatt tcactatctc cgtcacgacc gagatcctgc 3660 cagtttccat gactaagact tctgttgact gcaccatgta tatctgtggc gatagcaccg 3720 agtgcagtaa tctgcttctg cagtacggct ccttctgcac acaactcaat cgagcactga 3780 ccggtattgc agttgagcag gacaagaaca cacaggaggt ctttgcacag gtcaaacaaa 3840 tttacaaaac cccccccata aaagactttg gtgggttcaa cttcagccaa atcctcccag 3900 atcccagcaa gccctccaaa agatccttca tcgaagacct tttgttcaat aaggtaaccc 3960 tggccgacgc aggcttcatc aaacaatatg gcgattgcct tggagacatt gctgcgcgcg 4020 atttgatctg tgctcagaaa tttaacggtt tgaccgtgct gcccccactt ctgactgatg 4080 agatgatagc acagtatact tctgctcttc tggcaggaac aatcacttcc gggtggacct 4140 ttggcgctgg tgcagcactg caaatcccct tcgcaatgca aatggcctac cgattcaatg 4200 gtattggtgt tacccagaac gtgctctatg agaatcagaa actcatcgcc aatcagttca 4260 atagcgctat tggcaagatt caggattccc tcagctctac cgccagcgct ctggggaagc 4320 tccaggacgt ggtgaaccaa aatgctcaag cgctcaatac ccttgtgaaa cagctcagct 4380 ccaattttgg cgcaattagc agcgttctga atgatattct gtcccggctg gacaaggtag 4440 aagcagaagt ccagatcgac aggctgatca ccgggcggtt gcagagtctc cagacctatg 4500 tcacacaaca gctgatccgc gccgccgaga tcagggcttc cgctaacctg gccgccacta 4560 agatgtccga atgcgtgttg gggcagagta agcgggtcga cttttgcggg aagggatacc 4620. atctgatgag cttccctcag tctgcacccc acggagtagt gttcctccac gtcacatatg 4680 tgcccgctca ggaaaagaat ttcacaaccg cacctgctat ctgtcacgac ggcaaggccc actttcctag agaaggagtt ttcgtatcta acggcaccca ctggttcgtg acacagcgga acttttacga gcctcagatt ataactacgg acaacacttt cgtgtcaggc aactgtgacg tggtgattgg gatcgtgaac aacacagtct acgacccatt gcagcccgag ttggactcct tcaaagagga gcttgataag tatttcaaga accatacctc tcccgacgtg gacctggggg acattagcgg catcaatgca tccgttgtga atatccagaa agaaatcgat aggctgaatg aggtcgcaaa aaatcttaat gagtcactga ttgatctgca ggaactcggc aaatatgagc agtatattaa gtggccgtgg tacatatggc tcggctttat cgccggtctg attgccatcg tgatggtgac cattatgctg tgttgtatga caagctgctg ttcatgtctc aaaggatgct gctcctgcgg tagctgctgt aagttcgatg aagacgacag tgagcccgtg ctcaaaggag 5280 tgaaactcca ctacacataa cgatcgacgc gtagagctcg ctgatcagcc tcgactgtgc 5340 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 5400 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 5460 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 5520 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 5580 aagcttgcgg ccgcgcccag caccccaagg cggccaacgc caaaactctc cctcctcctc 5640 ttcctcaatc tcgctctcgc tctttttttt tttcgcaaaa ggaggggaga gggggtaaaa 5700 aaatgctgca ctgtgcggcg aagccggtga gtgagcggcg cggggccaat cagcgtgcgc 5760 cgttccgaaa gttgcctttt atggctcgag cggccgcggc ggcgccctat aaaacccagc 5820 ggcgcgacgc gccaccaccg ccgagaccct gcaggccgcc accatgtccg ataacggccc 5880 ccagaatcag agaaacgctc cccgcatcac gttcggcgga ccaagtgaca gcacaggcag 5940 taaccagaac ggagaacgct ccggtgctcg ctccaagcag cgacggccgc aagggcttcc 6000 siacaatacc gccagctggt ttacggctct gaccacac gggaagaag atctttaatt 6060 ccccaggggc cagggcgtcc ctatcaatac taactccagc ccggatgatc agataggcta 6120 ctatagacgc gctacccgac ggatacgagg gggggacggc aaatgaagg accttcccc 6180 ccggtggtat ttctattact tgggcaccgg accagaagcc ggactgcctt acggcgctaa 6240 caagacgga ataatctggg ttgcgacgga gggcgccctg atacaccta aagaccatat 6300 cggcacaaga aatcctgcta acatgccgc gattgtgctc cagctgccctc agggaaccac 6360 gctgcctaa gggttttacg ctgaggggtc aagggggggg agtcaagcgt ctagtaggtc 6420 atcctctcgc tctcgcaata gttcccggaa ctcacccca ggcagcagca gaggaacctc 6480 tcccgcacgg atggctggca atgggggaga tgctgccctt gctctccttc tgctggatcg 6540 ccttaaccag ctcgaatca agatgtgtgg aaaaggtcag cagcagcaag gccagaccgt 6600 ghaaagaag agtgcagctg aagctagtaa aaagccacgc haaaacgga ccgcaactaa 6660 ggcatataac gtaacacagg ccttcggcag agaggtcca gacaacac agggaaactt 6720 tggcgatcaa gagctgatta vakagggcac agattacaa cactggccac agatcgcgca 6780 gtttgcacca agcgccctg cattctcgg gatgagtcgg attgggatgg aagtcactcc 6840 atccgggacc tggcttacct acacaggggc aaaaaactc gacgacaag acccaaactt 6900 taaagatcag gtcatcctgc tgaataaca catcgatgcc tacaaactt tcccccac 6960 cgaaccaaag aagacaga aaaaaaaggc agacgaacg caagcgctcc ctcagcgcca 7020 gagaagcag cagaccgtta cactgttgcc agcagcagat ctggatgat ttccaagca 7080 gcttcacag agtatgtca gcgctgacag cactcaggct tgaggcgcgc cgctgaccga 7140 taaaayaat atcttttt tcattacatc tgtgtgttgg tttttgtgt gacgcgttag 7200 ttattatag taatcatta cggggtcatt agttcatagc ccatatatgg agttccgcgt 7260 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgacccc gcccattgac 7320 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg 7380 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag 7440 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat 7500 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat 7560 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt 7620 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga 7680 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg 7740 gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct agagatatcg 7800 ggccactgca ggaaacgata tgggctgaat acggatccgt attcagccca tatcgtttct 7860 ctagaataa aatatcttta tttcattac atctgtgtgt tggttttttg tgtgaatcga 7920 tagtactaac atacgctctc catctcgagc ctaagcttgt cgactcgaag atctgggcgt 7980 ggttaagggt gggaaagaat atataaggtg ggggtcttat gtagttttgt atctgttttg 8040 cagcagccgc cgccgccatg agcaccaact cgtttgatgg aagcattgtg agctcatatt 8100 tgacaacgcg catgccccca tgggccgggg tgcgtcagaa tgtgatgggc tccagcattg 8160 atggtcgccc cgtcctgccc gcaaactcta ctaccttgac ctacgagacc gtgtctggaa 8220 cgccgttgga gactgcagcc tccgccgccg cttcagccgc tgcagccacc gcccgcggga 8280 ttgtgactga ctttgctttc ctgagcccgc ttgcaagcag tgcagcttcc cgttcatccg 8340 cccgcgatga caagttgacg gctcttttgg cacaattgga ttctttgacc cgggaactta 8400 atgtcgtttc tcagcagctg ttggatctgc gccagcaggt ttctgccctg aaggcttcct 8460 cccctcccaa tgcggtttaa aacataaata aaaaaccaga ctctgtttgg atttggatca 8520 agcaagtgtc ttgctgtctt tatttagggg ttttgcgcgc gcggtaggcc cgggaccagc 8580 ggtctcggtc gttgagggtc ctgtgtattt tttccaggac gtggtaaagg tgactctgga 8640 tgttcagata catgggcata agcccgtctc tggggtggag gtagcaccac tgcagagctt 8700 catgctgcgg ggtggtgttg tagatgatcc agtcgtagca ggagcgctgg gcgtggtgcc 8760 taaaaatgtc tttcagtagc aagctgattg ccaggggcag gcccttggtg taagtgttta 8820 caaagcggtt aagctgggat gggtgcatac gtggggatat gagatgcatc ttggactgta 8880 tttttaggtt ggctatgttc ccagccatat ccctccgggg attcatgttg tgcagaacca 8940 ccagcacagt gtatccggtg cacttgggaa atttgtcatg tagcttagaa ggaaatgcgt 9000 ggaagaactt ggagacgccc ttgtgacctc caagattttc catgcattcg tccataatga 9060 tggcaatggg cccacgggcg gcggcctggg cgaagatatt tctgggatca ctaacgtcat 9120 agttgtgttc caggatgaga tcgtcatagg ccatttttac aaagcgcggg cggagggtgc 9180 cagactgcgg tataatggtt ccatccggcc caggggcgta gttaccctca cagatttgca 9240 tttcccacgc tttgagttca gatgggggga tcatgtctac ctgcggggcg atgaagaaaa 9300 cggtttccgg ggtaggggag atcagctggg aagaaagcag gttcctgagc agctgcgact 9360 taccgcagcc ggtgggcccg taaatcacac ctattaccgg ctgcaactgg tagttaagag 9420 agctgcagct gccgtcatcc ctgagcaggg gggccacttc gttaagcatg tccctgactc 9480 gcatgttttc cctgaccaaa tccgccagaa ggcgctcgcc gcccagcgat agcagttctt 9540 gcaaggaagc aaagtttttc aacggtttga gaccgtccgc cgtaggcatg cttttgagcg 9600 tttgaccaag cagttccagg cggtcccaca gctcggtcac ctgctctacg gcatctcgat 9660 ccagcatatc tcctcgtttc gcgggttggg gcggctttcg ctgtacggca gtagtcggtg 9720 ctcgtccaga cgggccaggg tcatgtcttt ccacgggcgc agggtcctcg tcagcgtagt 9780 ctgggtcacg gtgaaggggt gcgctccggg ctgcgcgctg gccagggtgc gcttgaggct 9840 ggtcctgctg gtgctgaagc gctgccggtc ttcgccctgc gcgtcggcca ggtagcattt 9900 gaccatggtg tcatagtcca gcccctccgc ggcgtggccc ttggcgcgca gcttgccctt 9960 ggaggaggcg ccgcacgagg ggcagtgcag acttttgagg gcgtagagct tgggcgcgag 10020 aaataccgat tccggggagt aggcatccgc gccgcaggcc ccgcagacgg tctcgcattc 10080 cacgagccag gtgagctctg gccgttcggg gtcaaaaacc aggtttcccc catgcttttt 10140 gatgcgtttc ttacctctgg tttccatgag ccggtgtcca cgctcggtga cgaaaaggct 10200 gtccgtgtcc ccgtatacag acttgagagg cctgtcctcg agcggtgttc cgcggtcctc 10260 ctcgtataga aactcggacc actctgagac aaaggctcgc gtccaggcca gcacgaagga 10320 ggctaagtgg gaggggtagc ggtcgttgtc cactaggggg tccactcgct ccagggtgtg 10380 aagacacatg tcgccctctt cggcatcaag gaaggtgatt ggtttgtagg tgtaggccac 10440 gtgaccgggt gttcctgaag gggggctata aaagggggtg ggggcgcgtt cgtcctcact 10500 ctcttccgca tcgctgtctg cgagggccag ctgttggggt gagtactccc tctgaaaagc 10560 gggcatgact tctgcgctaa gattgtcagt ttccaaaaac gaggaggatt tgatattcac 10620 ctggcccgcg gtgatgcctt tgagggtggc cgcatccatc tggtcagaaa agacaatctt 10680 tttgttgtca agcttggtgg caaacgaccc gtagagggcg ttggacagca acttggcgat 10740 ggagcgcagg gtttggtttt tgtcgcgatc ggcgcgctcc ttggccgcga tgtttagctg 10800 cacgtattcg cgcgcaacgc accgccattc gggaaagacg gtggtgcgct cgtcgggcac 10860 caggtgcacg cgccaaccgc ggttgtgcag ggtgacaagg tcaacgctgg tggctacctc 10920 tccgcgtagg cgctcgttgg tccagcagag gcggccgccc ttgcgcgagc agaatggcgg 10980 tagggggtct agctgcgtct cgtccggggg gtctgcgtcc acggtaaaga ccccgggcag 11040 caggcgcgcg tcgaagtagt ctatcttgca tccttgcaag tctagcgcct gctgccatgc 11100 gcgggcggca agcgcgcgct cgtatgggtt gagtggggga ccccatggca tggggtgggt 11160 gagcgcggag gcgtacatgc cgcaaatgtc gtaaacgtag aggggctctc tgagtattcc 11220 aagatatgta gggtagcatc ttccaccgcg gatgctggcg cgcacgtaat cgtatagttc 11280 gtgcgaggga gcgaggaggt cgggaccgag gttgctacgg gcgggctgct ctgctcggaa 11340 gactatctgc ctgaagatgg catgtgagtt ggatgatatg gttggacgct ggaagacgtt 11400 gaagctggcg tctgtgagac ctaccgcgtc acgcacgaag gaggcgtagg agtcgcgcag 11460 cttgttgacc agctcggcgg tgacctgcac gtctagggcg cagtagtcca gggtttcctt 11520 gatgatgtca tacttatcct gtcccttttt tttccacagc tcgcggttga ggacaaactc 11580 ttcgcggtct ttccagtact cttggatcgg aaacccgtcg gcctccgaac ggtaagagcc 11640 tagcatgtag aactggttga cggcctggta ggcgcagcat cccttttcta cgggtagcgc 11700 gtatgcctgc gcggccttcc ggagcgaggt gtgggtgagc gcaaaggtgt ccctgaccat 11760 gactttgagg tactggtatt tgaagtcagt gtcgtcgcat ccgccctgct cccagagcaa 11820 aaagtccgtg cgctttttgg aacgcggatt tggcagggcg aaggtgacat cgttgaagag 11880 tatctttccc gcgcgaggca taaagttgcg tgtgatgcgg aagggtcccg gcacctcgga 11940 acggttgtta attacctggg cggcgagcac gatctcgtca aagccgttga tgttgtggcc 12000 cacaatgtaa agttccaaga agcgcgggat gcccttgatg gaaggcaatt ttttaagttc 12060 ctcgtaggtg agctcttcag gggagctgag cccgtgctct gaaagggccc agtctgcaag 12120 atgagggttg gaagcgacga atgagctcca caggtcacgg gccattagca tttgcaggtg 12180 gtcgcgaaag gtcctaaact ggcgacctat ggccattttt tctggggtga tgcagtagaa 12240 ggtaagcggg tcttgttccc agcggtccca tccaaggttc gcggctaggt ctcgcgcggc 12300 agtcactaga ggctcatctc cgccgaactt catgaccagc atgaagggca cgagctgctt 12360 cccaaaggcc cccatccaag tataggtctc tacatcgtag gtgacaaaga gacgctcggt 12420 gcgaggatgc gagccgatcg ggaagaactg gatctcccgc caccaattgg aggagtggct 12480 attgatgtgg tgaagtaga agtccctgcg acgggccgaa cactcgtgct ggcttttgta 12540 aaaacgtgcg footactggc agcggtgcac gggctgtaca tcctgcacga ggttgacctg 12600 acgaccgcgc acaaggaagc agagtgggaa tttgagcccc tcgcctggcg ggtttggctg 12660 gtggtcttct acttcggctg cttgtccttg accgtctggc tgctcgaggg gagttacggt 12720 ggatcggacc accacgccgc gcgagcccaa agtccagatg tccgcgcgcg gcggtcggag 12780 cttgatgaca acatcgcgca gatgggagct gtccatggtc tggagctccc gcggcgtcag 12840 gtcaggcggg agctcctgca ggtttacctc catagacgg gtcagggcgc gggctagatc 12900 caggtgatac ctaatttcca ggggctggtt ggtggcggcg tcgatggctt caagaggcc 12960 gcatccccgc ggcgcgacta cggtaccgcg cggcgggcgg tgggccgcgg gggtgtcctt 13020 ggatgatgca tctaaaagcg gtgacgcggg cgagcccccg gagtagggg gggctccgga 13080 cccgccggga gagggggcag gggcacgtcg gcgccgcgcg cgggcaggag ctggtgctgc 13140 gcgcgtaggt tgctggcgaa cgcgacgacg cggcggttga tctcctgaat ctggcgctc 13200 tgcgtgaaga cgacgggccc ggtgagcttg aacctgaaag agagttcgac agaatcaatt 13260 tcggtgtcgt tgacggcggc ctggcgcaaa atctcctgca cgtctcctga gttgtcttga 13320 taggcgatct cggccatgaa ctgctcgatc tcttctctcct ggagatctcc gcgtccggct 13380 cgctccacgg tggcggcgag gtcgttggaa atgcgggcca tgagctgcga gaaggcgttg 13440 aggcctccct cgttccagac gcggctgtag accacgcccc cttcggcatc gcgggcgcgc 13500 atgaccacct gcgcgagatt gagctccacg tgccgggcga agacggcgta gtttcgcagg 13560 cgctgaaaga ggtagttgag ggtggtggcg gtgtgttctg ccacgaagaa gtacataacc 13620 cagcgtcgca acgtggattc gttgatatcc cccaaggcct caaggcgctc catggcctcg 13680 tagaagtcca cggcgaagtt gaaaaactgg gagttgcgcg ccgacacggt taactcctcc 13740 tccagaagac ggatgagctc ggcgacagtg tcgcgcacct cgcgctcaaa ggctacaggg 13800 gcctcttctt cttcttcaat ctcctcttcc ataagggcct ccccttcttc ttcttctggc 13860 ggcggtgggg gaggggggac acggcggcga cgacggcgca ccgggaggcg gtcgacaaag 13920 cgctcgatca tctccccgcg gcgacggcgc atggtctcgg tgacggcgcg gccgttctcg 13980 cgggggcgca gttggaagac gccgcccgtc atgtcccggt tatgggttgg cggggggctg 14040 ccatgcggca gggatacggc gctaacgatg catctcaaca attgttgtgt aggtactccg 14100 ccgccgaggg acctgagcga gtccgcatcg accggatcgg aaaacctctc gagaaaggcg 14160 tctaaccagt cacagtcgca aggtaggctg agcaccgtgg cgggcggcag cgggcggcgg 14220 tcggggttgt ttctggcgga ggtgctgctg atgatgtaat taaagtaggc ggtcttgaga 14280 cggcggatgg tcgacagaag caccatgtcc ttgggtccgg cctgctgaat gcgcaggcgg 14340 tcggccatgc cccaggcttc gttttgacat cggcgcaggt ctttgtagta gtcttgcatg 14400 agcctttcta ccggcacttc ttcttctcct tcctcttgtc ctgcatctct tgcatcttc 14460 gctgcggcgg cggcggagtt tggccgtagg tggcgccctc ttcctcccat gcgtgtgacc 14520 ccgaagcccc tcatcggctg aagcagggct aggtcggcga caacgcgctc ggctatatg 14640. gcctgctgca cctgcgtgag ggtagactgg aagtcatcca tgtccacaaa gcggtggtat gcgcccgtgt tgatggtgta agtgcagttg gccataacgg accagttaac ggtctggtga 14700. cccggctgcg agagctcggt gtacctgaga cgcgagtag ccctcgagtc aatacgtag 14760 tcgttgcaag tccgcaccag gtactggtat cccaccaaaa agtgcggcgg cggctggcgg tagggggcc agcgtagggt ggccggggct ccggggggcga gatcttccaa cataaggcga 14880. tgatatccgt agatgtacct ggacatccag gtgatgccgg cggcggtggt ggaggcgcgc 14940 ggaaagtcgc ggacgcggtt ccagatgttg cgcagcggca aaaagtgctc catggtcggg acgctctggc cggtcaggcg cgcgcaatcg ttgacgctct agcgtgcaaa aggagagcct 15060 gtaagcgggc actcttccgt ggtctggtgg ataattcgc aagggtatca tggcggacga ccggggttcg agccccgtat ccggccgtcc gccgtgatcc atgcggttac cgcccgcgtg 15180 tcgaacccag gtgtgcgacg tcagacaacg ggggagtgct ccttttggct tccttccagg 15240 cgcggcggct gctgcgctag cttttttggc cactggccgc gcgcagcgta agcggttagg 15300 ctggaaagcg aaagcattaa gtggctcgct ccctgtagcc ggagggttat tttccaaggg 15360 ttgagtcgcg ggacccccgg ttcgagtctc ggaccggccg gactgcggcg aacgggggtt 15420 tgcctccccg tcatgcaaga ccccgcttgc aaattcctcc ggaaacaggg acgagcccct 15480 tttttgcttt tcccagatgc atccggtgct gcggcagatg cgcccccctc ctcagcagcg 15540 gcaagagcaa gagcagcggc agacatgcag ggcaccctcc cctcctccta ccgcgtcagg 15600 aggggcgaca tccgcggttg acgcggcagc agatggtgat tacgaacccc cgcggcgccg 15660 ggcccggcac tacctggact tggaggaggg cgagggcctg gcgcggctag gagcgccctc 15720 tcctgagcgg cacccaaggg tgcagctgaa gcgtgatacg cgtgaggcgt acgtgccgcg 15780 gcagaacctg tttcgcgacc gcgagggaga ggagcccgag gagatgcggg atcgaaagtt 15840 ccacgcaggg cgcgagctgc ggcatggcct gaatcgcgag cggttgctgc gcgaggagga 15900 ctttgagccc gacgcgcgaa ccgggattag tcccgcgcgc gcacacgtgg cggccgccga 15960 cctggtaacc gcatacgagc agacggtgaa ccaggagatt aactttcaaa aaagctttaa 16020 caaccacgtg cgtacgcttg tggcgcgcga ggaggtggct ataggactga tgcatctgtg 16080 ggactttgta agcgcgctgg agcaaaaccc aaatagcaag ccgctcatgg cgcagctgtt 16140 ccttatagtg cagcacagca gggacaacga ggcattcagg gatgcgctgc taaacatagt 16200 agagcccgag ggccgctggc tgctcgattt gataaacatc ctgcagagca tagtggtgca 16260 ggagcgcagc ttgagcctgg ctgacaaggt ggccgccatc aactattcca tgcttagcct 16320 gggcaagttt tacgcccgca agatatacca taccccttac gttcccatag acaaggaggt 16380 aaagatcgag gggttctaca tgcgcatggc gctgaaggtg cttaccttga gcgacgacct 16440 gggcgtttat cgcaacgagc gcatccacaa ggccgtgagc gtgagccggc ggcgcgagct 16500 cagcgaccgc gagctgatgc acagcctgca aagggccctg gctggcacgg gcagcggcga 16560 tagagaggcc gagtcctact ttgacgcggg cgctgacctg cgctgggccc caagccgacg 16620 cgccctggag gcagctgggg ccggacctgg gctggcggtg gcacccgcgc gcgctggcaa 16680 cgtcggcggc gtggaggaat atgacgagga cgatgagtac gagccagagg acggcgagta 16740 ctaagcggtg atgtttctga tcagatgatg caagacgcaa cggacccggc ggtgcgggcg 16800 gcgctgcaga gccagccgtc cggccttaac tccacggacg actggcgcca ggtcatggac 16860 cgcatcatgt cgctgactgc gcgcaatcct gacgcgttcc ggcagcagcc gcaggccaac 16920 cggctctccg caattctgga agcggtggtc ccggcgcgcg caaaccccac gcacgagaag 16980 gtgctggcga tcgtaaacgc gctggccgaa aacagggcca tccggcccga cgaggccggc 17040 ctggtctacg acgcgctgct tcagcgcgtg gctcgttaca acagcggcaa cgtgcagacc 17100 aacctggacc ggctggtggg ggatgtgcgc gaggccgtgg cgcagcgtga gcgcgcgcag 17160 cagcagggca acctgggctc catggttgca ctaaacgcct tcctgagtac acagcccgcc aacgtgccgc ggggacagga ggactacacc aactttgtga gcgcactgcg gctaatggtg actgagacac cgcaaagtga ggtgtaccag tctgggccag actattttt ccagaccagt agacaaggcc tgcagaccgt aaacctgagc caggctttca aaaacttgca ggggctgtgg ggggtgcggg ctcccacagg cgaccgcgcg accgtgtcta gcttgctgac gcccaactcg 17460 cgcctgttgc tgctgctaat agcgcccttc acggacagtg gcagcgtgtc ccgggacaca 17520 tacctaggtc acttgctgac actgtaccgc gaggccatag gtcaggcgca tgtggacgag catactttcc aggagattac aagtgtcagc cgcgcgctgg ggcaggagga cacgggcagc ctggaggcaa ccctaaacta cctgctgacc aaccggcggc agaagatccc ctcgttgcac agtttaaca gcgaggagga gcgcattttg cgctacgtgc agcagagcgt gagccttaac ctgatgcgcg acggggtac gcccagcgtg gcgctggaca tgaccgcgcg caacatggaa 17820. ccgggcatgt atgcctcaaa ccggccgttt atcaaccgcc taatggacta cttgcatcgc gcggccgccg tgaaccccga gtatttcacc aatgccatct tgaacccgca ctggctaccg 17940 ccccctggtt tctacaccgg gggattcgag gtgcccgagg gtaacgatgg attcctctgg 18000 gacgacatag acgacagcgt gttttccccg caaccgcaga ccctgctaga gttgcaacag 18060 cgcgagcagg cagaggcggc gctgcgaaag gaaagctttcc gcaggccaag cagcttgtcc 18120 gatctaggcg ctgcggcccc gcggtcagat gctagtagcc catttccaag cttgataggg 18180 tctcttacca gcactcgcac caccgccg cgcctgctgg gcgaggagga gtacctaaac 18240 aactcgctgc tgcagccgca gcgcgaaaa aacctgcctc cggcatttcc caacaacggg 18300 atagagagcc tagtggacaa gatgagtaga tggaagacgt acgcgcagga gcacagggac 18360 gtgccaggcc cgcgcccgcc caccgtcgt caaaggcacg accgtcagcg gggtctggtg 18420 tgggaggacg atgactcggc agacgacagc agcgtcctgg atttgggagg gagtggcaac 18480 ccgtttgcgc accttcgccc caggctgggg agaatgtttt aaaaaaaaa aagcatgatg 18540 caaaataaaa aactcaccaa ggccatggca ccgagcgttg gttttcttgt attcccctta gtatgcggcg cgcggcgatg tatgaggag gtcctcctcc ctcctacgag agtgtggtga 18660 gcgcggcgcc agtggcggcg gcgctggggtt ctcccttcga tgctcccctg gacccgccgt 18720 ttgtgcctcc gcggtacctg cggcctaccg gggggagaaa cagcatccgt tactctgagt 18780 tggcacccct attcgacacc acccgtgtgt acctggtgga caacaagtca acggatgtgg catccctga ctaccagac gaccacagca actttctgac cacggtcatt caaaacaatg actacagccc gggggaggca agcacacaga ccatcaatct tgacgaccgg tcgcactggg gcggcgacct gaaaaccatc ctgcatacca acatgccaaa tgtgaacgag ttcatgttta ccaataagtt taaggcgcgg gtgatggtgt cgcgcttgcc tactaaggac aatcaggtgg agctgaaata cgagtgggtg gagttcacgc tgcccgaggg caactactcc gagaccatga ccatagacct tatgaacaac gcgatcgtgg agcactactt gaaagtgggc agcagaacg gggttctgga aagcgacatc ggggtaaagt ttgacacccg caacttcaga ctggggtttg accccgtcac tggtcttgtc atgcctgggg tatatacaa cgaagccttc catccagaca 19320 tcattttgct gccaggatgc gggtggact tcaccacag ccgcctgagc aacttgttgg 19380 gcatccgcaa gcggcaaccc ttccaggagg gctttaggat cacctacgat gatctggagg 19440 gtggtaacat tcccgcactg ttggatgtgg acgcctacca ggcgagcttg aagatgaca 19500 ccgaacagggg cgggggtggc gcaggcggca gcacagcag tggcagcggc gcggaagaga 19560 actccaacgc ggcagccgcg gcaatgcagc cggtggagga catgaacgat catgccattc 19620 gcggcgacac ctttgccaca cgggctgagg agaagcgcgc tgaggccgaa gcagcggccg 19680 aagctgccgc ccccgctgcg caacccgagg tcgagaagcc tcagagaaa ccggtgatca 19740 aacccctgac agaggacagc aagaaacgca gttacaacct ataagcaat gagacacct 19800 tcacccagta ccgcagctgg taccttgcat acactacgg cgaccctcag accggaatcc 19860 gctcatggac cctgctttgc actcctgacg taacctgcgg ctcggagcag gtctactggt 19920 cgttgccaga catgatgcaa gaccccgtga ccttccgctc cacgcgccag atcagcaact ttccggtggt gggcgccgag ctgttgcccg tgcactccaa gagcttctac aacgaccagg 20040 ccgtctactc ccctcatc cccgttta cctctctgac cccgtgttc aatcgctttc ccgagaacca gattttggcg cgcccgccag cccccaccat caccaccgtc agtgaaaacg ttcctgctct slightly slightly slightly slightly slightly slightly slightly gagtgaccat tactgacgcc agacgccgca cctgccccta cgtttacaag gccctgggca tagtctcgcc gcgcgtccta tcgagccgca ctttttgagc aagcatgtcc atccttatat 20340 cgcccagcaa taacacaggc tggggcctgc gcttcccaag caagatgttt ggcggggcca agaagcgctc cgaccaacac ccagtgcgcg tgcgcgggca ctaccgcgcg ccctggggcg 20520. cgcacaaacg cggccgcact gggcgcacca ccgtcgatga cgccatcgac gcggtggtgg aggaggcgcg caactaccg cccacgccgc cacagtgtc cacagtggac gcggccattc 20580 agaccgtggt gcgcggagcc cggcgctatg ctaaaatgaa gagacggcgg aggcgcgtag 20640 cacgtcgcca ccgccgccga cccggcactg ccgcccaacg cgcggcggcg gccctgctta 20700 accgcgcacg tcgcaccggc cgacgggcgg ccatgcgggc cgctcgaagg ctggccgcgg 20760 gtattgtcac tgtgcccccc aggtccaggc gacgagcggc cgccgcagca gccgcggcca 20820 ttagtgctat gactcagggt cgcaggggca acgtgtattg ggtgcgcgac tcggttagcg 20880 gcctgcgcgt gcccgtgcgc acccgccccc cgcgcaacta gattgcaaga aaaaactact 20940 tagactcgta ctgttgtatg tatccagcgg cggcggcgcg caacgaagct atgtccaagc 21000 gcaaaatcaa agaagagatg ctccaggtca tcgcgccgga gatctatggc cccccgaaga 21060 aggaagagca ggattacaag ccccgaaagc taaagcgggt caaaaagaaa aagaaagatg 21120 atgatgatga acttgacgac gaggtggaac tgctgcacgc taccgcgccc aggcgacggg 21180 tacagtggaa aggtcgacgc gtaaaacgtg ttttgcgacc cggcaccacc gtagtcttta 21240 cgcccggtga gcgctccacc cgcacctaca agcgcgtgta tgatgaggtg tacggcgacg 21300 aggacctgct tgagcaggcc aacgagcgcc tcggggagagtt tgcctacgga aagcggcata 21360 aggacatgct ggcgttgccg ctggacgagg gcaacccaac acctagccta aagcccgtaa 21420 cactgcagca ggtgctgccc gcgcttgcac cgtccgaaga aaagcgcggc ctaaagcgcg 21480 agtctggtga cttggcaccc accgtgcagc tgatggtacc caagcgccag cgactggaag 21540 atgtcttgga aaaaatgacc gtggaacctg ggctggagcc cgaggtccgc gtgcggccaa 21600 tcaagcaggt ggcgccggga ctgggcgtgc agaccgtgga cgttcagata cccactacca 21660 gtagcaccag tattgccacc gccacagagg gcatggagac acaaacgtcc ccggttgcct 21720 cagcggtggc ggatgccgcg gtgcaggcgg tcgctgcggc cgcgtccaag accctctacgg 21780 aggtgcaaac ggacccgtgg atgtttcgcg tttcagcccc ccggcgcccg cgccgttcga 21840 ggaagtacgg cgccgccagc gcgctactgc ccgaatatgc cctacatcct tccattgcgc 21900 ctacccccgg ctatcgtggc tacacctacc gccccagaag acgagcaact acccgacgcc 21960 gaaccaccac tggaacccgc cgccgccgtc gccgtcgcca gcccgtgctg gccccgattt 22020 ccgtgcgcag ggtggctcgc gaaggaggca ggaccctggt gctgccaaca gcgcgctacc 22080 accccagcat cgtttaaaag ccggtctttg tggttcttgc agatatggcc ctcacctgcc 22140 gcctccgttt cccggtgccg ggattccgag gaagaatgca ccgtaggagg ggcatggccg 22200 gccacggcct gacgggcggc atgcgtcgtg cgcaccaccg gcggcggcgc gcgtcgcacc 22260 gtcgcatgcg cggcggtatc ctgcccctcc gatcgccgcg gcgattggcg 22320 ccgtgcccgg aattgcatcc gtggccttgc aggcgcagag acactgatta aaaacaagtt 22380 gcatgtggaa aaatcaaaat aaaaagtctg gactctcacg ctcgcttggt cctgtaacta 22440 ttttgtagaa tggaagacat caactttgcg tctctggccc cgcgacacgg ctcgcgcccg 22500 ttcatgggaa actggcaaga tatcggcacc agcaatatga gcggtggcgc cttcagctgg 22560 ggctcgctgt ggagcggcat taaaaatttc ggttccaccg ttaagaacta tggcagcaag 22620 gcctggaaca gcagcacagg ccagatgctg agggataagt tgaaagagca aaatttccaa 22680 caaaaggtgg tagatggcct ggcctctggc attagcgggg tggtggacct ggccaaccag 22740 gcagtgcaaa ataagattaa cattaagctt gatccccgcc ctcccgtaga ggagcctcca 22800 ccggccgtgg agacagtgtc tccagagggg cgtggcgaaa agcgtccgcg ccccgacagg 22860 gaagaactc tggtgacgca aatagacgag cctccctcgt acgaggaggc actaaagcaa 22920 ggcctgccca ccacccgtcc catcgcgccc atggctaccg gagtgctgggg ccagcacaca 22980 cccgtaacgc tggacctgcc tccccccgcc gacacccagc agaaacctgt gctgccaggc 23040 ccgaccgccg ttgttgtaac ccgtcctagc cgcgcgtccc tgcgccgcgc cgccagcggt 23100 ccgcgatcgt tgcggcccgt agccagtggc aactggcaaa gcacactgaa cagcatcgtg 23160 ggtctgggg tgcaatccct gaagcgccga cgatgcttct gatagctaac gtgtcgtatg 23220 tgtgtcatgt atgcgtccat gtcgccgcca gaggagctgc tgagccgccg cgcgcccgct 23280 ttccaagatg gctacccctt cgatgatgcc gcagtggtct tacatgcaca tctcgggcca 23340 ggacgcctcg gagtacctga gccccgggct ggtgcagttt gcccgcgcca ccgagacgta 23400 cttcagcctg aataacaagt ttagaaaccc cacggtggcg cctacgcacg acgtgaccac 23460 agaccggtcc cagcgtttga cgctgcggtt catccctgtg gaccgtgagg atactgcgta 23520 ctcgtacaag gcgcggttca ccctagctgt gggtgataac cgtgtgctgg acatggcttc 23580 cacgtacttt gacatccgcg gcgtgctgga caggggccct acttttaagc cctactctgg 23640 cactgcctac aacgccctgg ctcccaaggg tgccccaaat ccttgcgaat gggatgaagc 23700 tgctactgct cttgaaataa acctagaaga agaggacgat gacaacgaag acgaagtaga 23760 cgagcaagct gagcagcaaa aaactcacgt atttgggcag gcgccttatt ctggtataaa 23820 tattacaaag gagggtattc aaataggtgt cgaaggtcaa acacctaaat atgccgataa 23880 aacatttcaa cctgaacctc aaataggaga atctcagtgg tacgaaacag aaattaatca 23940 tgcagctggg agagtcctaa aaaagactac cccaatgaaa ccatgttacg gttcatatgc 24000 aaaacccaca aatgaaaatg gagggcaagg cattcttgta aagcaacaaa atggaaagct 24060 agaaagtcaa gtggaaatgc aatttctc aactactgag gcagccgcag gcaatggtga 24120 taacttgact cctaaagtgg tattgtacag tgagatgta gatatagaaa cccagacac 24180 tcatatttct tacatgccca ctattaagga aggtactca cgagaacta tgggccaaca 24240 atctatgccc aacaggccta attacattgc tttagggac aattttattg gtctaatgta 24300 ttacacagc acgggtaata tgggtgttct ggcgggcca gcatcgcagt tgatgctgt 24360 tgtagatttg caacacagaa accagagct ttcataccag cttttgcttg attccattgg 24420 tgatagaacc aggtactttt ctatgtggaa tcaggctgttzgagctatg atccagatgt 24480 tagaattatt gaaaatcatg gaactgaga tgaacttcca aattactgct ttccactggg 24540 aggtgtgatt atacagaga ctcttaccaa ggtaaaacct aaaacaggtc aggaaatgg 24600 atgggaaaa gatgctacag aattttcaga taaaaatgaa atagagttg gaataattt 24660 tgccatggaa atcaatctaa atgccaacct gtggagaat ttcctgtact ccaacatagc 24720 gctgtatttg cccgacaagc taaagtacag tccttccac gtaaaaattt ctgataaccc 24780 aaacacctac gactacatga acaagcgagt ggtggctccc gggctagtgg actgctacat 24840 taaccttgga gcacgctggt cccttgacta tatggacaac gtcaacccat ttaaccacca 24900 ccgcaatgct ggcctgcgct accgctcaat gttgctgggc aatggtcgct atgtgccctt 24960 ccacatccag gtgcctcaga agttctttgc cattaaaaac ctccttctcc tgccgggctc 25020 atacacctac gagtggaact tcaggaagga tgttaacatg gttctgcaga gctccctagg 25080 aaatgaccta agggttgacg gagccagcat taagtttgat agcatttgcc tttacgccac 25140 cttcttcccc atggcccaca acaccgcctc cacgcttgag gccatgctta gaaacgacac 25200 caacgaccag tcctttaacg actatctctc cgccgccaac atgctctacc ctatacccgc 25260 caacgctacc aacgtgccca tatccatccc ctcccgcaac tgggcggctt tccgcggctg 25320 ggccttcacg cgccttaaga ctaaggaaac cccatcactg ggctcgggct acgaccctta 25380 ttacacctac tctggctcta taccctacct agatggaacc ttttacctca accacacctt 25440 taagaaggtg gccattacct ttgactcttc tgtcagctgg cctggcaatg accgcctgct 25500 tacccccaac gagtttgaaa ttaagcgctc agttgacggg gagggttaca acgttgccca 25560 gtgtaacatg accaaagact ggttcctggt acaaatgcta gctaactata acattggcta 25620 ccagggcttc tatatcccag agagctacaa ggaccgcatg tactccttct ttagaaactt 25680 ccagcccatg agccgtcagg tggtggatga tactaatac aggactacc aacaggtggg 25740 catcctacac caacaaca actctggatt tgttggctac cttgccccca ccatgcgcga 25800 aggacaggcc taccctgcta acttccccta tccgcttata ggcaagaccg cagttgacag 25860 cattacccag aaaaagtttc tttgcgatcg caccctttgg cgcatcccat tctccagataa 25920 ctttatgtcc atgggcgcac tcacagacct gggccaaaac cttctctacg ccaactccgc 25980 ccacgcgcta gacatgactt ttgaggtgga tcccatggac gagcccaccc ttctttatgt 26040 tttgtttgaa gtctttgacg tggtccgtgt gcaccagccg caccgcggcg tcatcgaaac 26100 cgtgtacctg cgcacgccct tctcggccgg caacgccaca acataaagaa cgaagcaaca 26160 tcaacaacag ctgccgccat gggctccagt gagcaggaac tgaaagccat tgtcaaagat 26220 cttggttgtg ggccatattt tttgggcacc tatgacaagc gctttccagg ctttgtttct 26280 ccacacaagc tcgcctgcgc catagtcaat acggccggtc gcgagactgg gggcgtacac 26340 tggatggcct ttgcctggaa cccgcactca aaaacatgct acctctttga gccctttggc 26400 ttttctgacc agcgactcaa gcaggtttac cagtttgagt acgagtcact cctgcgccgt 26460 agcgccattg cttcttcccc cgaccgctgt ataacgctgg aaaagtccac ccaaagcgta 26520 caggggccca actcggccgc ctgtggacta ttctgctgca tgtttctcca cgcctttgcc 26580 aactggcccc aaactcccat ggatcacaac cccaccatga accttattac cggggtaccc 26640 aactccatgc tcaacagtcc ccaggtacag cccaccctgc gtcgcaacca ggaacagctc 26700 tacagcttcc tggagcgcca ctcgccctac ttccgcagcc acagtgcgca gattaggagc 26760 gccacttctt tttgtcactt gaaaaacatg taaaaataat gtactagaga cactttcaat 26820 aaaggcaaat gcttttattt gtacactctc gggtgattat ttacccccac ccttgccgtc 26880 tgcgccgttt aaaaatcaaa ggggttctgc cgcgcatcgc tatgcgccac tggcagggac 26940 acgttgcgat actggtgttt agtgctccac ttaaactcag gcacaaccat ccgcggcagc 27000 tcggtgaagt tttcactcca caggctgcgc accatcacca acgcgtttag caggtcgggc 27060 gccgatatct tgaagtcgca gttggggcct ccgccctgcg cgcgcgagtt gcgatacaca 27120 gggttgcagc actggaacac tatcagcgcc gggtggtgca cgctggccag cacgctcttg 27180 tcggagatca gatccgcgtc caggtcctcc gcgttgctca gggcgaacgg agtcaacttt 27240 ggtagctgcc ttcccaaaaa gggcgcgtgc ccaggctttg agttgcactc gcaccgtagt 27300 ggcatcaaaa ggtgaccgtg cccggtctgg gcgttaggat acagcgcctg cataaaagcc 27360 ttgatctgct taaaagccac ctgagccttt gcgccttcag agaagaacat gccgcaagac 27420 ttgccggaaa actgattggc cggacaggcc gcgtcgtgca cgcagcacct tgcgtcggtg 27480 ttggagatct gcaccacatt tcggccccac cggttcttca cgatcttggc cttgctagac 27540 tgctccttca gcgcgcgctg cccgttttcg ctcgtcacat ccatttcaat cacgtgctcc 27600 ttatttatca taatgcttcc gtgtagacac ttaagctcgc cttcgatctc agcgcagcgg 27660 tgcagccaca acgcgcagcc cgtgggctcg tgatgcttgt aggtcacctc tgcaaacgac 27720 tgcaggtacg cctgcaggaa tcgccccatc atcgtcacaa aggtcttgtt gctggtgaag 27780 gtcagctgca acccgcggtg ctcctcgttc agccaggtct tgcatacggc cgccagagct 27840 tccacttggt caggcagtag tttgaagttc gcctttagat cgttatccac gtggtacttg 27900 tccatcagcg cgcgcgcagc ctccatgccc ttctcccacg cagacacgat cggcacactc 27960 agcgggttca tcaccgtaat ttcactttcc gcttcgctgg gctcttcctc ttcctcttgc 28020 gtccgcatac cacgcgccac tgggtcgtct tcattcagcc gccgcactgt gcgcttacct 28080 cctttgccat gcttgattag caccggtggg ttgctgaaac ccaccatttg tagcgccaca 28140 tcttctcttt cttcctcgct gtccacgatt acctctggtg atggcgggcg ctcgggcttg 28200 ggagaagggc gcttcttttt cttcttgggc gcaatggcca aatccgccgc cgaggtcgat 28260 ggccgcgggc tgggtgtgcg cggcaccagc gcgtcttgtg atgagtcttc ctcgtcctcg 28320 gactcgatac gccgcctcat ccgcttttttt gggggcgcccc ggggaggcgg cggcgacggg 28380 gacggggacg acacgtcctc catggttggg ggacgtcgcg ccgcaccgcg tccgcgctcg 28440 ggggtggttt cgcgctgctc ctcttccga ctggccattt ccttctccta taggcaagaa 28500 aagatcatgg agtcagtcga gaagaaggac agcctaaccg ccccctctga gttcgccacc 28560 accgcctcca ccgatgccgc caacgcgcct accaccttcc ccgtcgaggc acccccgctt 28620 gaggagg aagtgattat cgagcaggac ccaggttttg taaggaga cgacgaggac 28680 28740 caagtcgggc gggggacga aaggcatggc gactacctag atgtgggaga cgacgtgctg 28800 ttgaagcatc tgcagcgcca gtgcgccatt atctgcgacg cgttgcaaga gcgcagcgat 28860 gtgcccctcg ccatagcgga tgtcagcctt gcctacgaac gccacctatt ctcaccgcgc 28920 gtacccccca aacgccaaga aaacggcaca tgcgagccca acccgcgcct caacttctac 28980 cccgtatttg ccgtgccaga ggtgcttgcc acctatcaca tctttttcca aaactgcaag 29040 atacccctat cctgccgtgc caaccgcagc cgagcggaca agcagctggc cttgcggcag 29100 ggcgctgtca tacctgatat cgcctcgctc aacgaagtgc caaaaatctt tgagggtctt 29160 ggacgcgacg agaagcgcgc ggcaaacgct ctgcaacagg aaaacagcga aaatgaaagt 29220 cactctggag tgttggtgga actcgagggt gacaacgcgc gcctagccgt actaaaacgc 29280 agcatcgagg tcacccactt tgcctacccg gcacttaacc taccccccaa ggtcatgagc 29340 acagtcatga gtgagctgat cgtgcgccgt gcgcagcccc tggagaggga tgcaaatttg 29400 caagaacaaa cagaggaggg cctacccgca gttggcgacg agcagctagc gcgctggctt 29460 caaacgcgcg agcctgccga cttggaggag cgacgcaaac taatgatggc cgcagtgctc 29520 gttaccgtgg agcttgagtg catgcagcgg ttctttgctg acccggagat gcagcgcaag 29580 ctagaggaaa cattgcacta cacctttcga cagggctacg tacgccaggc ctgcaagatc 29640 tccaacgtgg agctctgcaa cctggtctcc taccttggaa ttttgcacga aaaccgcctt 29700 gggcaaaacg tgcttcattc cacgctcaag ggcgaggcgc gccgcgacta cgtccgcgac 29760 tgcgtttact tatttctatg ctacacctgg cagacggcca tgggcgtttg gcagcagtgc 29820 ttggaggagt gcaacctcaa ggagctgcag aaactgctaa agcaaaactt gaaggaccta 29880 tggacggcct tcaacgagcg ctccgtggcc gcgcacctgg cggacatcat tttccccgaa 29940 cgcctgctta aaaccctgca acagggtctg ccagacttca ccagtcaaag catgttgcag 30000 aactttagga actttatcct agagcgctca ggaatcttgc ccgccacctg ctgtgcactt 30060 cctagcgact ttgtgcccat taagtaccgc gaatgccctc cgccgctttg gggccactgc 30120 taccttctgc agctagccaa ctaccttgcc taccactctg acataatgga agacgtgagc 30180 ggtgacggtc tactggagtg tcactgtcgc tgcaacctat gcaccccgca ccgctccctg 30240 gtttgcaatt cgcagctgct taacgaaagt caaattatcg gtacctttga gctgcagggt 30300 ccctcgcctg acgaaaagtc cgcggctccg gggttgaaac tcactccggg gctgtggacg 30360 tcggcttacc ttcgcaaatt tgtacctgag gactaccacg cccacgagat taggttctac 30420 gaagaccaat cccgcccgcc taatgcggag cttaccgcct gcgtcattac ccagggccac 30480 attcttggcc aattgcaagc catcaacaaa gcccgccaag agtttctgct acgaaaggga 30540 cggggggttt acttggaccc ccagtccggc gaggagctca acccaatccc cccgccgccg 30600 cagccctatc agcagcagcc gcgggccctt gcttcccagg atggcaccca aaaagaagct 30660 gcagctgccg ccgccaccca cggacgagga ggaatactgg gacagtcagg cagaggaggt 30720 tttggacgag gaggaggagg acatgatgga agactgggag agcctagacg aggaagcttc 30780 cgaggtcgaa gaggtgtcag acgaaacacc gtcaccctcg gtcgcattcc cctcgccggc 30840 gccccagaaa tcggcaaccg gttccagcat ggctacaacc tccgctcctc aggcgccgcc 30900 ggcactgccc gttcgccgac ccaaccgtag atgggacacc actggaacca gggccggtaa 30960 gtccaagcag ccgccgccgt tagcccaaga gcaacaacag cgccaaggct accgctcatg 31080. gcgcgggcac aagaacgcca tagttgcttg cttgcaagac tgtgggggca acatctcctt cgcccgccgc tttcttctct accatcacgg cgtggccttc ccccgtaaca tcctgcatta 31140 ctaccgtcat ctctacagcc catactgcac cggcggcagc ggcagcaaca gcagcggcca cacagaagca aaggcgaccg gatagcaaga ctctgacaaa gcccaagaa tccacagcgg cggcagcagc rich gcgctgcgtc tggcgcccaa cgaacccgta tcgacccgcg agcttagaaa caggatttt cccactctgt atgctatatt tcaacagagc aggggccaag 31440. aacaagagct gaaaataaaa aacaggtctc tgcgatccct cacccgcagc tgcctgtatc acaaaagcga agatcagctt cggcgcacgc tggaagacgc ggaggctctc ttcagtaaat actgcgcgct gactcttaag gactagtttc gcgccctttc tcaaatttaa gcgcgaaaac tacgtcatct ccagcggcca cacccggcgc cagcacctgt tgtcagcgcc attatgagca aggaattcc cacgccctac atgtggagtt accagccaca aatgggactt gcggctggag ctgcccaaga ctactcaacc cgaataaact acatgagcgc gggaccccac atgatatccc 31740 gggtcaacgg aatacgcgcc caccgaaacc gaattctcct ggaacaggcg gctattacca 31800 ccacacctcg taataacctt aatccccgta gttggcccgc tgccctggtg taccaggaaa 31860 gtcccgctcc caccactgtg gtacttccca gagacgccca ggccgaagtt cagatgacta 31920 actcaggggc gcagcttgcg ggcggctttc gtcacagggt gcggtcgccc gggcagggta 31980 taactcacct gacaatcaga gggcgaggta ttcagctcaa cgacgagtcg gtgagctcct 32040 cgcttggtct ccgtccggac gggacatttc agatcggcgg cgccggccgc tcttcattca 32100 cgcctcgtca ggcaatccta actctgcaga cctcgtcctc tgagccgcgc tctggaggca 32160 ttggaactct gcaatttatt gaggagtttg tgccatcggt ctactttaac cccttctcgg 32220 gacctcccgg ccactatccg gatcaattta ttcctaactt tgacgcggta aaggactcgg 32280 cggacggcta cgactgaatg ttaagtggag aggcagagca actgcgcctg aaacacctgg 32340 tccactgtcg ccgccacaag tgctttgccc gcgactccgg tgagttttgc tactttgaat 32400 tgcccgagga tcatatcgag ggcccggcgc acggcgtccg gcttaccgcc cagggagagc 32460 ttgcccgtag cctgattcgg gagtttaccc agcgccccct gctagttgag cgggacaggg 32520 gaccctgtgt tctcactgtg atttgcaact gtcctaaccc tggattacat caagatcctc 32580 tagttaatgt caggtcgcct aagtcgatta actagagtac ccggggatct tattcccttt 32640 aactaataaa aaaaaataat aaagcatcac ttacttaaaa tcagttagca aatttctgtc 32700 cagtttattc agcagcacct ccttgccctc ctcccagctc tggtattgca gcttctctcct 32760 ggctgcaaac tttctccaca atctaaatgg aatgtcagtt tcctcctgtt cctgtccatc 32820 cgcacccact atcttcatgt tgttgcagat gaagcgcgca agaccgtctg aagatacctt 32880 caaccccgtg tatccatatg acacggaaac cggtcctcca actgtgcctt ttcttactcc 32940 tccctttgta tcccccaatg ggtttcaaga gagtccccct ggggtactct ctttgcgcct 33000 atccgaacct ctagttacct ccaatggcat gcttgcgctc aaaatgggca acggcctctc 33060 tctggacgag gccggcaacc ttacctccca aaatgtaacc actgtgagcc cacctctcaa 33120 aaaaaccaag tcaaacataa acctggaaat atctgcaccc ctcacagtta cctcagaagc 33180 cctaactgtg gctgccgccg cacctctaat ggtcgcgggc aacacactca ccatgcaatc 33240 acaggccccg ctaaccgtgc acgactccaa acttagcatt gccacccaag gacccctcac 33300 agtgtcagaa ggaaagctag ccctgcaaac atcaggcccc ctcaccacca ccgatagcag 33360 tacccttact atcactgcct caccccctct aactactgcc actggtagct tgggcattga 33420 cttgaaagag cccatttata cacaaaatgg aaaactagga ctaaagtacg gggctccttt 33480 gcatgtaaca gacgacctaa acactttgac cgtagcaact ggtccaggtg tgactattaa 33540 taatacttcc ttgcaaacta aagttactgg agccttgggt tttgattcac aaggcaatat 33600 gcaacttaat gtagcaggag gactaaggat tgattctcaa aacagacgcc ttatacttga 33660 tgttagttat ccgtttgatg ctcaaaacca actaaatcta agactaggac agggccctct 33720 ttttataaac tcagcccaca acttggatat taactacaac aaaggcctttt actgttttac 33780 agctcaac aattccaaa agcttgaggt taacctaagc actgccaagg ggttgatgtt 33840 tgacgctaca gccatagcca ttaatgcagg agatgggctt gatttggtt cacctaatgc 33900 accaacaca aatcccctca aaaaaaaaat tggccatggc ctagaatttg attcaacaa 33960 ggctatggtt cctaaactag gaactggcct tagtttgac agcacaggtg ccattacagt 34020 aggaaacaa aaatgata agctacttt gtggaccaca ccagctccat ctcctaactg 34080 tagactaaat gcagagaaag atgctaaact cactttggtc ttagtaaaat gtggcagtca 34140 atacttgct acagttcag tttggctgt taaggcagt tggctccaa tatctggac 34200 agttcaagt gctcatctta ttatagatt tgacgaaat ggagtgctac taacaattc 34260 cttcctggac ccagaatatt ggaacttag aaatggagat cttactgaag gcacagccta 34320 tacaacgct gttggatta tgcctaacct atcagcttat ccaaatctc acggtaaaac 34380 tgccaaaagt aacattgtca gtcaagttta cttaaacgga cakaaacta aacctgtaac 34440 actaaccatt acactaaacg gtacacagga aacaggagac acaactccaa gtgcatactc 34500 tatgtcattt tcatgggact ggtctggcca caactacatt aatgaaatat ttgccacatc 34560 ctcttacact ttttcataca ttgcccaaga ataaagaatc gtttgtgtta tgtttcaacg 34620 tgtttatttt tcaattgcag aaaatttcaa gtcatttttc attcagtagt atagccccac 34680 caccacatag cttatacaga tcaccgtacc ttaatcaaac tcacagaacc ctagtattca 34740 acctgccacc tccctcccaa cacacagagt acacagtcct ttctccccgg ctggccttaa 34800 aaagcatcat atcatgggta acagacatat tcttaggtgt tatattccac acggtttcct 34860 gtcgagccaa acgctcatca gtgatattaa taaactcccc gggcagctca cttaagttca 34920 tgtcgctgtc cagctgctga gccacaggct gctgtccaac ttgcggttgc ttaacgggcg 34980 gcgaaggaga agtccacgcc tacatggggg tagagtcata atcgtgcatc aggatagggc 35040 ggtggtgctg cagcagcgcg cgaataaact gctgccgccg ccgctccgtc ctgcaggaat 35100 acaacatggc agtggtctcc tcagcgatga ttcgcaccgc ccgcagcata aggcgccttg tcctccgggc acagcagcgc accctgatct cacttaaatc agcacagtaa ctgcagcaca 35280. gcaccacaat attgttcaaa atcccacagt gcaaggcgct gtatccaaag ctcatggcgg ggaccacaga acccacgtgg ccatcatacc acaagcgcag gtagattag tggcgacccc 35400. tcataacac gctggacata aacattacct cttttggcat gttgtaattc accacctccc ggtaccatat aaacctctga ttaaacatgg cgccatccac caccatccta aaccagctgg ccaaaacctg cccgccggct atacactgca gggaccggg actggaca tgacagtgga gagcccagga ctcgtaacca tggatcatca tgctcgtcat gatatcaatg ttggcacaac acaggcacac gtgcatacac ttcctcagga ttacaagctc ctcccgcgtt agaaccatat cccaggac aacccattcc tgaatcagcg taaatcccac actgcaggga agacctcgca 35760. cgtaactcac gttgtgcatt gtcaaagtgt tacattcggg cagcagcgga tgatcctcca gtatggtagc gcgggtttct gtctcaaaag gaggtagcg atccctactg tacggagtgc gccgagacaa ccgagatcgt gttggtcgta gtgtcatgcc aaatggaacg ccggacgtag 35880 35940. tcatatttcc tgaagcaaaa ccaggtgcgg gcgtgacaaa cagatctgcg tctccggtct cgccgcttag atcgctctgt gtagtagttg tagtatatcc actctctcaa agcatccagg cgccccctgg cttcgggttc tatgtaaact ccttcatgcg ccgctgccct gataacatcc 36120. accaccgcag grandfather acccagcca cctacacatt cgttctgcga gtcacacacg ggaggagcgg gaagagctgg aagaaccatg tttttttttt tattccaaaa gattatccaa aacctcaaaa tgaagatcta tgaagtgaac gcgctcccct ccggtggcgt ggtcaaactc 36300. tggcatttgt aagatgttgc acaatggctt ccaaaaggca aacggccctc acgtccaagt ggacgtaag gctaaccct tcagggtgaa tctcctctat aaacattcca gcaccttcaa ccatgcccaa atattctca tctcgccacc ttctcaatat atctctaagc aaatcccgaa tattaagtcc ggccattgta aaaatctgct ccagagcgcc ctccaccttc agcctcaagc agcgaatcat gattgcaaa attcaggttc ctcacagacc 36540 tgtataagat tcaaagcgg aacattaaca aaaataccgc gatcccgtag gtcccttcgc 36600 agggccagct gaacataatc gtgcaggtct gcacggacca gcgcggccac ttccccgcca 36660 ggaaccatga caaagaacc cacactgatt atgacacgca tactcggagc tatgctacc 36720 agcgtagccc cgatgtaagc ttgttgcatg ggcgggata taaatgcaa ggtgctgctc 36780 aaaaaatcag gcaagccctc gcgcaaaaaa gaagcacat cgtagtcatg ctcatgcaga 36840 taaaggcagg taagctccgg aaccaccaca gaaaaagaca ccattttct ctcaaacatg 36900 tctgcgggtt tctgcataaa cacaataa aaaaaaaaacttaa acattagaag 36960 cctgtcttac aacaggaaaaaaccctta taagcatag acggactacg gccatgccgg 37020 cgtgaccgta aaaaaactgg tcaccgtgat taaaaagcac caccgacagc tcctcggtca 37080 tgtccggagt cataatgtaa gactcggtaa acacatcagg ttgatcaca tcggtcagtg 37140 ctaaaaagcg accgaatag cccgggggaa tacatacccg caggcgtaga gaacatta 37200 cagcccccat aggagtata acaaattta taggagaa aaacacata acacctgaaa 37260 aaccctcctg cctaggcaaa atagcaccct cccgctccag aaaacatac agcgcttcca 37320 cagcggcagc catacagtc agccttacca gtaaaaaga aaacctatta aaaaaacacc 37380 actcgacacg gcaccagctc aatcagtcac agtgtaaaaa agggccaagt gcagagcgag 37440 tatatatagg actaaaaaat gacgtaacgg ttaaagtcca CAAAaaacac ccagaaaacc 37500 gcacgcgaac ctacgcccag aaacgaaagc caaaaaccc acacttcct caatcgtca 37560 cttccgtttt cccacgttac gtcactcccc attttagaa aactacaatt cccacacat 37620 acagttact ccgccctaaa acctacgtca cccgccccgt tccgccc cgcgccacgt 37680 cacaaactcc accccctcat tatcatattg gcttcaatcc aaaataggt atatt 37735 <210> 11 <211> 35706 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 11 taaggatccc atcatcaata atacctta ttttg aagccaatat gataatgagg 60 gggtggagtt tgtgacgtgg cgcggggcgt gggaacgggg cgggtgacgt agtagtgtgg 120 cggaagtgtg atgttgcaag tgtggcggaa cacatgtaag cgacggatgt ggcaaagtg 180 acgttttgg tgtgcgccgg tgtacacagg aagtgacaat ttcgcgcgg tttaggcgg 240 atgttgtagt aaatttgggc gtaccgagt aagatttggc catttcgcg ggaaaactga 300 ataagaggaa gtgaaatctg ataatttg tgttactcat agcgcgtaat actgctagag 360 atctggcgaa agggggatgt gctgcaggc gattaagttg gggtaacgcca gggttttccc 420 agtcacgacg ttgtaaaacg acggccagtg aattgtata cgactcacta taggcgaat 480 tgggtactgg ccacaggagc ttggccatt gcatacgttg tatccatatc atatatgta 540 catttatatt ggctcatgtc caattacc gccatgttga cattgattat tgactagtta 600 ttaatagtaa tcattacgg gtcattagt tcatagccca tattagt tccgcgttac 660 attackcg gtaaatggcc cgcctggctg acccccac gaccccgcc cattgacgtc 720 aataatgacg tatgttccca tagtaacgcc atagggact ttccattgac gtcaatgggt 780 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac 840 gcccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac 900 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt 960 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc 1020 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt 1080 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg 1140 ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc 1200 acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctgactc tagcctagct 1260 ctgaagttgg tggtgaggcc ctgggcaggt tggtatcaag gttacaagac aggtttaagg 1320 agaccaatag aaactgggca tgtggagaca gagaagactc ttgggtttct gataggcact 1380 gactctctct gcctattggt ctattttccc acccttaggc tgctggtctg agcctaggag 1440 atctctcgag gtcgacggta tcgatgggta ccgccaccat gtttgtttt ctcgtactcc 1500 tgcccctggt ttcctcccaa tgtgtcaatc tgactacccg gacccaactt cctcccgcct 1560 acaccaattc ctttacccga ggtgtttact acccagacaa agtgttcagg tcatccgtcc 1620 tccatagtac ccaagacctc ttcctccctt tttttctaa cgttacctgg tttcacgcta 1680 ttcacgttag cggcaccaac ggcaccaaaa gattcgataa ccccgtactg ccgttcaacg 1740 acggggtata ttttgcctct actgaaaaat caaacatcat acgcggatgg atctttggga 1800 ctaccctgga ctcaaaaact cagtccctgc tgattgtgaa taacgctacc aacgtggtga 1860 tcaaagtctg tgaattccag ttttgcaacg atccttttct cggcgtttat tatcacaaaa 1920 ataacaaatc ctggatggag agcgagttcc gggtgtactc ctccgcgaat aattgcacct 1980 tcgaatatgt gtctcagcca ttcctcatgg acctcgaggg gaagcagggc aattttaaga 2040 atctgcgaga attcgtgttc aagaatatag acggtactt caagatttac tccaaacaca 2100 ccccgattaa cctggttagg gacttgcctc agggcttttc tgcattggag cccctcgtgg 2160 acccccaat cggcataaac attacagat ttcagactt gcttgcattg cacaggagct 2220 atttgacacc cggcgattct tctccgat ggaccgctgg agcagctgct tattacgtgg 2280 gctatctgca gcctcgaacc tttctttga agtacaacga aaatggaact atcaccgatg 2340 cagttgactg cgccctggac cccctgtccg aaactaagtg cacgctcaa agttcacag 2400 tagagaaggg gatataccag actagcaatt tccgcgttca gccaaccgaa agtatagtgc 2460 gctttcctaa tataactaac ctgtgtcctt tcgggagt gtttaacgcc actagattcg 2520 cttccgtcta cgcctggaat agaaagagga tctcaattg cgttgctgac tatagtgttt 2580 tgtacaatc cgccctttc tcaccttca aatgttacgg ggtgagccct accaactga 2640 acgacctgtg ctttacaaac gtatacgccg acagctttgt tatcagagga gacgaggttc 2700 gccagattgc tccgggtcag acaggcaga tgctgatta taattacaaa ctgcccgacg 2760 actttacagg atgtgtgatc gcgtggaaca gtaacaatct tgactcaag gttggggta 2820 attataatta tctttaccgg ctgttcagaa aaagcaattt gaacccttc gaaagggaca 2880 tatccaccga gatctatcag gccgggtcca ctccatgcaa tggtgtggaa ggttttaatt 2940 gctacttccc attgcagtct tatggattcc aaccaaccaa tggcgtaggc taccagccgt 3000 atcgcgttgt cgtgctcagc ttcgagctgc tccacgcccc cgcgaccgta tgcggtcta 3060 agaagtccac caatcttgtt aagaacaagt gtgtaaactt taactttaac gggctgaccg 3120 ggaccggcgt tctgactgaa tctaacaaaa aattcctgcc tttccagcag ttcggccgcg 3180 atattgctga caccactgac gctgtaagag accctcagac ccttgaaatt ctcgatatca 3240 caccttgcag ctttgggggc gtgtccgtca tcactccagg aactaacaca agcaaccagg 3300 tggcagtgtt gtaccaggat gttaattgta ccgaggtgcc agtggccatc cacgccgatc 3360 aattgacacc tacctggagg gtttacagca cagggtccaa tgtttttcag acaagagccg 3420 gatgtctgat cggtgccgag catgtcaaca attcctacga gtgtgatatc cccattggtg 3480 cgggaatttg tgcatcatat cagacccaga ctaatagccc aagaagagct agatccgtcg 3540 ctagtcaatc catcattgca tatacaatga tgtccgataa cggcccccag aatcagagaa 3600 acgctccccg catcacgttc ggcggaccaa gtgacagcac aggcagtaac cagaacggag 3660 aacgctccgg tgctcgctcc aagcagcgac ggccgcaagg gcttcccaac aataccgcca 3720 gctggtttac ggctctgacc caacacggga aagaagatct taaattcccc aggggccagg 3780 gcgtccctat caatactaac tccagcccgg atgatcagat aggctactat agacgcgcta 3840 cccgacggat acgagggggg gacggcaaaa tgaaggacct ttccccccgg tggtatttct 3900 attacttggg caccggacca gaagccggac tgccttacgg cgctaacaaa gacggaataa 3960 tctgggttgc gacggagggc gccctgaata cacctaaaga ccatatcggc acaagaaatc 4020 ctgctaacaa tgccgcgatt gtgctccagc tgcctcaggg aaccacgctg cctaaagggt 4080 tttacgctga ggggtcaagg ggggggagtc aagcgtctag taggtcatcc tctcgctctc 4140 gcaatagttc ccggaactca accccaggca gcagcagagg aacctctccc gcacggatgg 4200 ctggcaatgg gggagatgct gcccttgctc tccttctgct ggatcgcctt aaccagctcg 4260 aatcaaagat gtctggaaaa ggtcagcagc agcaaggcca gaccgtgaca aagaagagtg 4320 cagctgaagc tagtaaaaag ccacgccaaa aacggaccgc aactaaggca tataacgtaa 4380 cacaggcctt cggcagaaga ggtccagaac aaacacaggg aaactttggc gatcaagagc 4440 tgattagaca gggcacagat tacaacact ggccacagat cgcgcagttt gcaccaagcg 4500 cctctgcatt cttcgggatg agtcggattg ggatggagagt cactccatcc gggacctggc 4560 ttacctacac aggggcaata aaactcgacg aaaagaccc aaactttaaa gatcaggtca 4620 tcctgctgaa taaacacatc gatgcctaca aaactttccc cccaaccgaa ccaaagaaag 4680 acaagaaaaa aaaggcagac gaacgcaag cgctccctca gcgccagaag aagcagcaga 4740 ccgttacact gttgccagca gcagatctgg atgatttttc caagcagctt caagagga 4800 tgtcaagcgc tgacagcact caggcttgac gatcggatat cgctagcgta ccggcggccg 4860 ccctattcta tagtgtcacc taaatgctag agctcgctga tcagcctcga ctgtgccttc 4920 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 4980 cactccact gtcctttcct ataaatga ggaaattgca tcgcattgtc tgagtaggtg 5040 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa 5100 tagcaggcat gctgggggatg cggtgggctc tatggcttct gaggcggaaa gaaccaaagc 5160 ttacgcgtta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg 5220 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc 5280 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat 5340 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat 5400 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat 5460 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc 5520 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac 5580 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa 5640 aatcaacggg actttccaaa atgtcgtaac aactccgcccc cattgacgca aatgggcggt 5700 aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc 5760 tagagagatatc gggccactgc aggaaacgat atgggctgaa tacggatccg tattcagccc 5820 atatcgtttc tctagaaata aaatatcttt attttcatta catctgtgtg ttggtttttt 5880 gtgtgaatcg atagtactaa catacgctct ccatctcgag cctaagcttg tcgactcgaa 5940 gatctgggcg tggttaaggg tgggaaagaa tatataaggt gggggtctta tgtagttttg 6000 tatctgtttt gcagcagccg ccgccgccat gagcaccaac tcgtttgatg gaagcattgt 6060 gagctcatat ttgacaacgc gcatgccccc atgggccggg gtgcgtcaga atgtgatggg 6120 ctccagcatt gatggtcgcc ccgtcctgcc cgcaaactct actaccttga cctacgagac 6180 cgtgtctgga acgccgttgg agactgcagc ctccgccgcc gcttcagccg ctgcagccac 6240 cgcccgcggg attgtgactg actttgcttt cctgagcccg cttgcaagca gtgcagcttc 6300 ccgttcatcc gcccgcgatg acaagttgac ggctcttttg gcacaattgg attctttgac 6360 ccgggaactt aatgtcgttt ctcagcagct gttggatctg cgccagcagg tttctgccct 6420 gaaggcttcc tcccctccca atgcggttta aaacataaat aaaaaaccag actctgtttg 6480 gatttggatc aagcaagtgt cttgctgtct ttatttaggg gttttgcgcg cgcggtaggc 6540 ccgggaccag cggtctcggt cgttgagggt cctgtgtatt ttttccagga cgtggtaaag 6600 gtgactctgg atgttcagat acatgggcat aagcccgtct ctggggtgga ggtagcacca 6660 ctgcagagct tcatgctgcg gggtggtgtt gtagatgatc cagtcgtagc aggagcgctg 6720 ggcgtggtgc ctaaaaatgt ctttcagtag caagctgatt gccaggggca ggcccttggt 6780 gtaagtgttt acaaagcggt taagctggga tgggtgcata cgtggggata tgagatgcat 6840 cttggactgt atttttaggt tggctatgtt cccagccata tccctccggg gattcatgtt 6900 gtgcagaacc accagcacag tgtatccggt gcacttggga aatttgtcat gtagcttaga 6960 aggaaatgcg tggaagaact tggagacgcc cttgtgacct ccaagatttt ccatgcattc 7020 gtccataatg atggcaatgg gcccacgggc ggcggcctgg gcgaagatat ttctgggatc 7080 actaacgtca tagttgtgtt ccaggatgag atcgtcatag gccattttta caaagcgcgg 7140 gcggagggtg ccagactgcg gtataatggt tccatccggc ccaggggcgt agttaccctc 7200 acagatttgc atttcccacg ctttgagttc agatgggggg atcatgtcta cctgcggggc 7260 7320. gatgaagaa acggtttccg gggtagggga gatcagctgg gagaaagca ggttcctgag 7380. gtaaatcaca cctattaccg gctgcaactg gtagttaaga gagctgcagc tgccgtcatc cctgagcagg ggggccactt cgttaagcat 7440. gtccctgact cgcatgtttt ccctgacca atccgccaga aggcgctcgc cgcccagcga tagcagttct tgcaaggaag caaagttttt caacggtttg agaccgtccg ccgtaggcat 7560 gcttttgagc gtttgacca gcagttccag gcggtcccac agctcggtca cctgctctac ggcatctcga tccagcatat ctcctcgttt cgcgggttgg ggcggctttc gctgtacggc 7680 agtagtcggt gctcgtccag acgggccagg gtcatgtctt tccacggggcg cagggtcctc 7740 gtcagcgtag tctgggtcac ggtgaagggg tgcgctccgg gctgcgcgct ggccagggtg 7800. cgcttgaggc tggtcctgct ggtgctgaag cgctgccggt cttcgccctg cgcgtcggcc 7860 aggtagcatt tgaccatggt gtcatagtcc agcccctccg cggcgtggcc cttggcgcgc 7920 agcttgccct tggaggaggc gccgcacgag gggcagtgca gacttttgag ggcgtagagc 7980 ttgggcgcga gaaataccga ttccggggag taggcatccg cgccgcaggc cccgcagacg 8040 gtctcgcatt ccacgagcca ggtgagctct ggccgttcgg ggtcaaaaac caggtttccc 8100 ccatgctttt tgatgcgttt cttacctctg gtttccatga gccggtgtcc acgctcggtg 8160 acgaaaaggc tgtccgtgtc cccgtataca gacttgagag gcctgtcctc gagcggtgtt 8220 ccgcggtcct cctcgtatag aaactcggac cactctgaga caaaggctcg cgtccaggcc 8280 agcacgaagg aggctaagtg ggaggggtag cggtcgttgt ccactagggg gtccactcgc 8340 tccagggtgt gaagacacat gtcgccctct tcggcatcaa ggaaggtgat tggtttgtag 8400 gtgtaggcca cgtgaccggg tgttcctgaa ggggggctat aaaagggggt gggggcgcgt 8460 tcgtcctcac tctcttccgc atcgctgtct gcgagggcca gctgttgggg tgagtactcc 8520 ctctgaaaag cgggcatgac ttctgcgcta agattgtcag tttccaaaaa cgaggaggat 8580 ttgatattca cctggcccgc ggtgatgcct ttgagggtgg ccgcatccat ctggtcagaa 8640 aagacaatct ttttgttgtc aagcttggtg gcaaacgacc cgtagagggc gttggacagc 8700 aacttggcga tggagcgcag ggtttggttt ttgtcgcgat cggcgcgctc cttggccgcg 8760 atgtttagct gcacgtattc gcgcgcaacg caccgccatt cgggaaagac ggtggtgcgc 8820 tcgtcgggca ccaggtgcac gcgccaaccg cggttgtgca gggtgacaag gtcaacgctg 8880 gtggctacct ctccgcgtag gcgctcgttg gtccagcaga ggcggccgcc cttgcgcgag 8940 cagaatggcg gtagggggtc tagctgcgtc tcgtccgggg ggtctgcgtc cacggtaaag 9000 accccgggca gcaggcgcgc gtcgaagtag tctatcttgc atccttgcaa gtctagcgcc 9060 tgctgccatg cgcgggcggc aagcgcgcgc tcgtatgggt tgagtggggg accccatggc 9120 atggggtggg tgagcgcgga ggcgtacatg ccgcaaatgt cgtaaacgta gaggggctct 9180 ctgagtattc caagatatgt agggtagcat cttccaccgc ggatgctggc gcgcacgtaa 9240 tcgtatagtt cgtgcgaggg agcgaggagg tcgggaccga ggttgctacg ggcgggctgc 9300 tctgctcgga agactatctg cctgaagatg gcatgtgagt tggatgatat ggttggacgc 9360 tggaagacgt tgaagctggc gtctgtgaga cctaccgcgt cacgcacgaa ggaggcgtag 9420 gagtcgcgca gcttgttgac cagctcggcg gtgacctgca cgtctagggc gcagtagtcc 9480 agggtttcct tgatgatgtc atacttatcc tgtccctttt ttttccacag ctcgcggttg 9540 aggacaaact cttcgcggtc tttccagtac tcttggatcg gaaacccgtc ggcctccgaa 9600 cggtaagagc ctagcatgta gaactggttg acggcctggt aggcgcagca tcccttttct 9660 acgggtagcg cgtatgcctg cgcggccttc cggagcgagg tgtgggtgag cgcaaaggtg 9720 tccctgacca tgactttgag gtactggtat ttgaagtcag tgtcgtcgca tccgccctgc 9780 tcccagagca aaaagtccgt gcgctttttg gaacgcggat ttggcagggc gaaggtgaca 9840 tcgttgaaga gtatctttcc cgcgcgaggc ataaagttgc gtgtgatgcg gaagggtccc 9900 ggcacctcgg aacggttgtt aattacctgg gcggcgagca cgatctcgtc aaagccgttg 9960 atgttgtggc ccacaatgta aagttccaag aagcgcggga tgcccttgat ggaaggcaat 10020 tttttaagtt cctcgtaggt gagctcttca ggggagctga gcccgtgctc tgaaagggcc 10080 cagtctgcaa gatgagggtt ggaagcgacg aatgagctcc acaggtcacg ggccattagc 10140 atttgcaggt ggtcgcgaaa ggtcctaaac tggcgaccta tggccatttt ttctggggtg 10200 atgcagtaga aggtaagcgg gtcttgttcc cagcggtccc atccaaggtt cgcggctagg 10260 tctcgcgcgg cagtcactag aggctcatct ccgccgaact tcatgaccag catgaagggc 10320 acgagctgct tcccaaaggc ccccatccaa gtataggtct ctacatcgta ggtgacaaag 10380 agacgctcgg tgcgaggatg cgagccgatc gggaagaact ggatctcccg ccaccaattg 10440 gaggagtggc tattgatgtg gtgaaagtag aagtccctgc gacgggccga acactcgtgc 10500 tggcttttgt aaaaacgtgc gcagtactgg cagcggtgca cgggctgtac atcctgcacg 10560 aggttgacct gacgaccgcg cacaaggaag cagagtggga atttgagccc ctcgcctggc 10620 gggtttggct ggtggtcttc tacttcggct gcttgtcctt gaccgtctgg ctgctcgagg 10680 ggagttacgg tggatcggac caccacgccg cgcgagccca aagtccagat gtccgcgcgc 10740 ggcggtcgga gcttgatgac aacatcgcgc agatgggagc tgtccatggt ctggagctcc 10800 cgcggcgtca ggtcaggcgg gagctcctgc aggtttacct cgcatagacg ggtcagggcg 10860 cgggctagat ccaggtgata cctaatttcc aggggctggt tggtggcggc gtcgatggct 10920 tgcaagaggc cgcatccccg cggcgcgact acggtaccgc gcggcgggcg gtgggccgcg 10980 ggggtgtcct tggatgatgc atctaaaagc ggtgacgcgg gcgagccccc ggaggtaggg 11040 ggggctccgg acccgccggg agagggggca ggggcacgtc ggcgccgcgc gcgggcagga 11100 gctggtgctg cgcgcgtagg ttgctggcga acgcgacgac gcggcggttg atctcctgaa 11160 tctggcgcct ctgcgtgaag acgacgggcc cggtgagctt gaacctgaaa gagagttcga 11220 cagaatcaat ttcggtgtcg ttgacggcgg cctggcgcaa aa...

Claims

1. The following elements: (a) A first promoter functionally linked to the nucleic acid encoding the first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein; (b) A second promoter functionally linked to a nucleic acid encoding a Toll-like receptor-3 (TLR-3) agonist; and (c) A third promoter functionally linked to the nucleic acid encoding the SARS-CoV-2 N protein. A chimeric adenovirus expression vector containing an expression cassette.

2. The chimeric adenovirus expression vector according to claim 1, wherein the SARS-CoV-2 N protein comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:

2.

3. The chimeric adenovirus expression vector according to claim 1 or 2, wherein element (c) is located between elements (a) and (b) in the expression cassette.

4. A chimeric adenovirus expression vector according to any one of claims 1 to 3, wherein the first SARS-CoV-2 protein comprises a SARS-CoV-2 S protein having a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:1, SEQ ID NO:21, or SEQ ID NO:

22.

5. The chimeric adenovirus expression vector according to any one of claims 1 to 4, wherein the nucleic acid encoding the TLR-3 agonist includes a nucleic acid encoding dsRNA.

6. A chimeric adenovirus expression vector according to any one of claims 1 to 4, wherein the nucleic acid encoding the TLR-3 agonist includes a sequence selected from the group consisting of SEQ ID NO: 13 to 20.

7. A chimeric adenovirus expression vector according to any one of claims 1 to 6, wherein the nucleic acid encoding the first SARS-CoV-2 protein in element (a) comprises a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% identity with the sequence of SEQ ID NO:

3.

8. The chimeric adenovirus expression vector according to any one of claims 1 to 7, wherein the nucleic acid encoding the SARS-CoV-2 N protein includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% identity with the sequence of SEQ ID NO:

4.

9. A chimeric adenovirus expression vector according to any one of claims 1 to 25, wherein the first promoter and the second promoter are the same.

10. The chimeric adenovirus expression vector according to claim 9, wherein both the first promoter and the second promoter are CMV promoters.

11. A chimeric adenovirus expression vector according to any one of claims 1 to 10, wherein the first promoter is a CMV promoter, the second promoter is a CMV promoter, and the third promoter is a β-actin promoter.

12. The chimeric adenovirus expression vector according to claim 1, wherein element (c) is located between elements (a) and (b), and elements (a), (c), and (b) together encode a sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:7, or encodes the sequence of SEQ ID NO:

7.

13. A chimeric adenovirus expression vector according to claim 1, comprising a sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:10, or comprising a sequence of SEQ ID NO:

10.

14. The following elements: (a) A first promoter functionally linked to the nucleic acid encoding a fusion protein including the S1 region of the SARS-CoV-2 S protein, the furin region, and the SARS-CoV-2 N protein; and (b) A second promoter functionally linked to a nucleic acid encoding a Toll-like receptor-3 (TLR-3) agonist. A chimeric adenovirus expression vector containing an expression cassette.

15. The chimeric adenovirus expression vector according to claim 14, wherein the fusion protein comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO:

12.

16. The chimeric adenovirus expression vector according to claim 14 or 15, wherein the nucleic acid encoding the SARS-CoV-2 fusion protein is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:5, or comprises SEQ ID NO:

5.

17. A chimeric adenovirus expression vector according to any one of claims 14 to 16, wherein the first promoter and the second promoter are the same.

18. The chimeric adenovirus expression vector according to claim 17, wherein both the first promoter and the second promoter are CMV promoters.

19. A chimeric adenovirus expression vector according to any one of claims 14 to 18, wherein elements (a) and (b) together encode the sequence of SEQ ID NO:8, or encode the sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:

8.

20. A chimeric adenovirus expression vector according to any one of claims 14 to 19, which is encoded in the sequence of SEQ ID NO:11, or in a sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:

11.

21. The following elements: (a) A first promoter functionally linked to the nucleic acid encoding the SARS-CoV-2 S protein; and (b) A second promoter functionally linked to a nucleic acid encoding a Toll-like receptor-3 (TLR-3) agonist. A chimeric adenovirus expression vector containing an expression cassette.

22. The chimeric adenovirus expression vector according to claim 21, wherein the SARS-CoV-2 S protein is present in at least 95%, 96%, 97%, 98%, or 99% of any one of SEQ ID NO: 1, 21, or 22.

23. The chimeric adenovirus expression vector according to claim 21, wherein the SARS-CoV-2 S protein comprises the sequence of SEQ ID NO:1, SEQ ID NO:21, or SEQ ID NO:

22.

24. The chimeric adenovirus expression vector according to any one of claims 21 to 23, wherein the nucleic acid encoding the SARS-CoV-2 S protein is at least 85%, 90%, or 95% identical to the polynucleotide sequence of SEQ ID NO:3, or comprises the sequence of SEQ ID NO:

3.

25. A chimeric adenovirus expression vector according to any one of claims 21 to 24, wherein the first promoter and the second promoter are the same.

26. The chimeric adenovirus expression vector according to claim 25, wherein both the first promoter and the second promoter are CMV promoters.

27. A chimeric adenovirus expression vector according to any one of claims 21 to 26, wherein elements (a) and (b) together encode the sequence of SEQ ID NO:6, or encode the sequence having at least 85%, at least 90%, or at least 95% identity with respect to SEQ ID NO:

6.

28. A chimeric adenovirus expression vector according to any one of claims 21 to 27, which is encoded by a sequence having at least 85%, at least 90%, or at least 95% identity with SEQ ID NO:9, or is encoded by the sequence of SEQ ID NO:

9.

29. An immunogenic composition comprising a chimeric adenovirus expression vector according to any one of claims 1 to 28 and a pharmaceutically acceptable carrier.

30. A method for inducing an immune response to the SARS-CoV-2 protein in a subject, comprising administering to the subject an amount immunogenically effective for the mammalian subject of a chimeric adenovirus expression vector according to any one of claims 1 to 28 or an immunogenic composition according to claim 29.

31. The method according to claim 30, wherein the route of administration is oral, intranasal, or mucosal.

32. The method according to claim 31, wherein the administration route is oral delivery by swallowing a tablet.

33. The method according to any one of claims 30 to 32, wherein the immune response is induced in the target alveolar cells, absorptive intestinal cells, ciliated cells, goblet cells, club cells, and / or airway basal cells.

34. The method according to any one of claims 30 to 33, wherein the subject is a human.

35. The following elements: (a) A first promoter functionally linked to the nucleic acid encoding the antigen protein; (b) A second promoter functionally linked to a nucleic acid encoding a Toll-like receptor-3 (TLR-3) agonist; and (c) A third promoter functionally linked to the nucleic acid encoding the SARS-CoV-2 N protein. A chimeric polynucleotide containing an expression cassette.

36. The chimeric polynucleotide according to claim 35, wherein the SARS-CoV-2 N protein has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to SEQ ID NO:

2.

37. A chimeric polynucleotide according to claim 35 or 36, which is a chimeric adenovirus expression vector.

38. The chimeric polynucleotide according to any one of claims 35 to 37, wherein the nucleic acid encoding the TLR-3 agonist includes a nucleic acid encoding dsRNA.

39. The chimeric polynucleotide according to any one of claims 35 to 37, wherein the nucleic acid encoding the TLR-3 agonist includes a sequence selected from the group consisting of SEQ ID NO: 13 to 20.

40. The chimeric polynucleotide according to any one of claims 35 to 37, wherein element (c) is located between element (a) and element (b) in the expression cassette.

41. The method according to any one of claims 35 to 40, wherein the antigen protein is derived from bacteria, fungi, viruses, or parasites.

42. The method according to any one of claims 35 to 40, wherein the antigen protein is a cancer antigen.

43. A method for inducing an immune response in a subject, comprising administering a chimeric polynucleotide according to any one of claims 35 to 42 to the subject.

44. The following elements: (a) A first promoter functionally linked to the nucleic acid encoding the first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein; and (b) A second promoter functionally linked to a nucleic acid encoding a Toll-like receptor-3 (TLR-3) agonist. A chimeric adenovirus expression vector containing an expression cassette.

45. The chimeric adenovirus expression vector according to claim 44, wherein the nucleic acid encoding the TLR-3 agonist includes a nucleic acid encoding dsRNA.

46. The chimeric adenovirus expression vector according to claim 44, wherein the nucleic acid encoding the TLR-3 agonist includes a sequence selected from the group consisting of SEQ ID NO: 13 to 20.

47. A chimeric adenovirus expression vector according to any one of claims 44 to 46, further comprising element (c), which is a third promoter functionally linked to a nucleic acid encoding a second SARS-CoV-2 protein.

48. The chimeric adenovirus expression vector according to claim 47, wherein element (c) is positioned between elements (a) and (b) in the expression cassette.

49. A chimeric adenovirus expression vector according to claim 47 or 48, wherein the first SARS-CoV-2 protein in (a) and the second SARS-CoV-2 protein in (c) are different.

50. The chimeric adenovirus expression vector according to claim 47 or 48, wherein the SARS-CoV-2 protein in (a) and the SARS-CoV-2 protein in (c) are the same.

51. A chimeric adenovirus expression vector according to any one of claims 44 to 50, wherein the nucleic acid encoding the first SARS-CoV-2 protein in element (a) and / or the nucleic acid encoding the second SARS-CoV-2 protein in element (c) comprises a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% identity with respect to the sequence of SEQ ID NO:

3.

52. The chimeric adenovirus expression vector according to claim 51, wherein the first and / or second SARS-CoV-2 protein comprises a SARS-CoV-2 S protein having a sequence that is at least 85%, 90%, 95%, 97%, 99%, or 100% identical to the sequence of SEQ ID NO:1 or SEQ ID NO:21 or SEQ ID NO:

22.

53. A chimeric adenovirus expression vector according to any one of claims 44 to 52, wherein the nucleic acid encoding the first SARS-CoV-2 protein in element (a) and / or the nucleic acid encoding the second SARS-CoV-2 protein in element (c) comprises a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% identity with the sequence of SEQ ID NO:

4.

54. The chimeric adenovirus expression vector according to claim 53, wherein the first and / or second SARS-CoV-2 protein comprises a SARS-CoV-2 N protein having a sequence that is at least 85%, 90%, 95%, 97%, 99%, or 100% identical to the sequence of SEQ ID NO:

2.

55. A chimeric adenovirus expression vector according to any one of claims 44 to 54, wherein the nucleic acid encoding the first SARS-CoV-2 protein in element (a) and / or the nucleic acid encoding the second SARS-CoV-2 protein in element (c) comprises a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% identity with respect to the sequence of SEQ ID NO:

5.

56. An immunogenic composition comprising a chimeric adenovirus expression vector according to any one of claims 44 to 55 and a pharmaceutically acceptable carrier.

57. A method for inducing an immune response to the SARS-CoV-2 protein in a subject, comprising administering to the subject an immunogenically effective amount of a chimeric adenovirus expression vector according to any one of claims 44 to 55 or an immunogenic composition according to claim 56.

58. The method according to claim 57, wherein the route of administration is orally, intranasally, or via the mucous membrane.

59. The method according to claim 58, wherein the administration route is oral delivery by swallowing a tablet.

60. The method according to any one of claims 57 to 59, wherein the immune response is induced in the target alveolar cells, absorptive intestinal cells, ciliated cells, goblet cells, club cells, and / or airway basal cells.

61. The method according to any one of claims 57 to 60, wherein the subject is a human.

Images