NOVEL ADENOVIRAL VECTOR SYSTEM FOR GENE DELIVERY

STATEMENT OF SEQUENCE LISTING

A computer-readable form (CRF) document of the Sequence Listing is submitted with this application. The document, entitled 68142-03_Seq_Listing_ST25_txt, is generated on Mar. 8, 2021. Applicant states that the content of the computer-readable form is the same and the information recorded in computer readable form is identical to the written sequence listing.

FIELD OF INVENTION

This disclosure relates to a novel adenoviral vector's construction and its uses thereof. Particularly, it relates to a unique cell line system to generate a novel bovine adenovirus vector that provides more gene insertion capabilities and better immunogenicity for inserted antigens. The unique cell line is used for generating and growing of the new adenovirus vectors for gene delivery or recombinant vaccine production.

BACKGROUND

Adenovirus (AdV) vector-based vaccines have been shown to elicit a balanced humoral and cell mediated immune (CMI) responses by activating innate immunity through both Toll-like receptor (TLR)-dependent as well as TLR-independent pathways. Due to the high prevalence of AdV in humans, the development of AdV-specific neutralizing antibodies, known as ‘pre-existing vector immunity’, is one of the potential concerns for several human AdV (HAdV) vector-based vaccine delivery systems. To address this concern, a number of less prevalent HAdVs or nonhuman AdVs have been developed as vaccine delivery vectors. These nonhuman AdV vectors are based on bovine AdV (BAdV), simian AdV (SAdV), porcine AdV (PAdV), ovine AdV (OAdV), Canine AdV (CAdV), avian AdV (AAdV) and murine AdV (MAdV). It has been demonstrated that there were no reductions in humoral and CMI responses against the vaccine immunogen and the resultant protection efficacy of a BAdV type 3 (BAdV-3) vector based H5N1 influenza vaccine even in the presence of exceptionally high levels of pre-existing HAdV vector immunity. In addition, pre-existing HAdV-neutralizing antibodies in humans did not cross-neutralize BAdV-3, and HAdV-specific CMI responses did not cross-react with BAdV-3. Bio-distribution, pathogenesis, transduction, and persistence studies in animal models and cell lines have suggested that the safety aspects of BAdV vectors are similar to that of HAdV vectors. It has been illustrated that the cell internalization of BAdV-3 is independent of the HAdV type C5 (HAdV-C5) receptors [Coxackievirus-AdV receptor (CAR) and αvβ3 or αvβ5 integrin], but it is indicated that the α(2,3)-linked and α(2,6)-linked sialic acid receptors serve as major receptors for BAdV-3 internalization. It appears that BAdV-based vectors may serve as excellent vaccine vectors for humans without any concerns of pre-existing HAdV vector immunity.

However, in the current version of BAdV vector system, only about 0.6 kb deletion in the E1 region (only E1A) of BAdV genome can be made in a bovine cell line expressing human adenoviral E1 gene products. In addition, 1.2 Kb E3 deletion can easily be made in BAdV genome since E3 proteins are not essential for BAdV replication. Repeated efforts to expand the E1 deletion to both E1A and E1B regions were unsuccessful since HAdV E1B proteins do not complement BAdV E1B proteins' functions. Therefore, there is a need to generate a new cell line that could support the replication of BAdV vector having full E1 deletion (E1A and E1B) with or without E3 deletion. BAdV vectors having full E1 and E3 deletions can accommodate larger size foreign gene insert to provide more flexibility of gene expression.

SUMMARY OF THE INVENTION

In the current version of BAdV vector system, only 0.6 kb deletion in the E1 region of BAdV vector can be made in a bovine cell line that expresses HAdV E1 gene products. This disclosure provides a unique cell line that efficiently supports the replication of BAdV vector having the full E1 region deletion with or without E3 deletion, thereby extending the foreign gene cassette insertion capacity by at least 2 Kb. Such deletion would allow insertions up to 5.5 Kb foreign gene cassette containing one or more genes. This next generation of BAdV vectors and the unique cell line that expresses appropriate E1 gene products enhances the utility as well as acceptability of this vector system. This vector system can be used in both recombinant vaccine development and gene therapy applications.

A BAdV-3 with all of its E1 region and E3 region deleted is provided in this disclosure.

A BAdV-3 with all of its E1 region and E3 region deleted and replaced by at least one foreign gene up to 5.5 kb for expression can be created under this disclosure.

This disclosure provides a gene delivery system or a vaccine production system. The system comprises a bovine or equivalent cell line transfected with HAdV E1 region and BAdV EB1L gene (or E1BS and E1BL genes), and a BAdV-3 with E1A, E1BS, E1BL and E3 regions deletion and one or more targeted genes or antigenic domains for gene delivery.

In some preferred embodiment the aforementioned vaccine production system expresses hemagglutinin (HA) gene of an influenza virus.

In some preferred embodiment the aforementioned bovine cell line is a BHH3 that supports replication of E1, E3-deleted BAdV-3 vectors.

In some preferred embodiment the aforementioned bovine cell line is FBRT-HE1-BE1B /Full (FBRT-HE1 fetal bovine retinal cells that expresses HAdV-5 E1A, E1B-S and E1BL proteins and BAdV-3 E1B-L protein), FBRT-HE1-BE1BL, BHH3-E1BL, or BHH3-BE1B/Full.

In some preferred embodiment the aforementioned vaccine production system expresses and processes VP2 protein of Foot and Mouse Disease virus (FMDV) from the P1 gene product.

The disclosure also provides a method of generating immunogenicity against H5NA influenza virus HA antigen in a subject. The method comprising administering to the subject an effective amount of BAdV3 vaccine vector expressing H5HA. In some preferred embodiment, the dose of such BAdV3 vaccine vector is at least 30-fold less than a HAdV counterpart expressing the same H5HA.

These and other features, aspects and advantages of the present invention will become better understood with reference to the following figures, associated descriptions and claims.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 Bovine adenovirus type 3 with E1 region deletion;

SEQ ID NO:2 Bovine adenovirus type 3 with E3 region deletion;

SEQ ID NO:3 amino acid sequence of Influenza A virus (A/Hong Kong.156/97 (H5N1) hemagglutinin subtype H5 (H5) gene;

SEQ ID NO:4 Foot-and-mouth disease virus P1 protein sequence;

SEQ ID NO:5 Modified Foot-and-mouth disease virus P1 protein sequence with Furin cleavage sites;

SEQ ID NO:6 Furin cleavage site;

SEQ ID NO:7 DNA sequence of Influenza A virus (A/Hong Kong.156/97 (H5N1) hemagglutinin subtype H5 (H5) gene;

SEQ ID NO:8 DNA sequence of Foot-and-mouth disease virus P1 gene with furin cleavage site inserted and bovine codon optimized; and

SEQ ID NO:9 protein sequence of Foot-and-mouth disease virus P1 with furin cleavage site.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. HA-specific serum IgG antibody responses in mice immunized once with BAd-H5HA or HAd-H5HA. Mice were immunized intramuscularly (i.m.) or intranasally (i.n.) once with 1×10⁶(A), 3×10⁶(B), 1×10⁷(C), 3×10⁷(D) or 1×10⁸(E) PFU of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated i.m. or i.n. with PBS or 1×10⁸PFU of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, serum samples were collected, diluted to 1:500, and the development of HA-specific IgG antibody responses were monitored by ELISA. Data are represented as the mean ±standard deviation (SD) of the optical density (OD) readings. Statistically significant responses are shown as compared to PBS group (+) or i.n. versus i.m. route of inoculation in the same group (*). * or +, significant at p<0.05; ** or ++, significant at p<0.01; *** or +++, significant at p<0.001; and **** or ++++; significant at p<0.0001. The statistical analysis was done by Bonferroni post-test and two-way ANOVA using Graph Pad Prim 6. BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-H5HA, human adenoviral vector expressing HA of a A/Hong Kong/156/97(H5N1) influenza virus; BAd-ΔE1E3 (BAd empty vector); HAd-ΔE1E3, (HAd empty vector); PBS, phosphate-buffered saline; and ns, no significance at p>0.05.

FIG. 2. HA-specific serum IgG1 antibody responses in mice immunized once with BAd-H5HA or HAd-H5HA. Mice were immunized intramuscularly (i.m.) or intranasally (i.n.) once with 1×10⁶(A), 3×10⁶(B), 1×10⁷(C), 3×10⁷(D) or 1×10⁸(E) PFU of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated i.m. or i.n. with PBS or 1×10⁸PFU of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, serum samples were collected, diluted to 1:500, and the development of HA-specific IgG antibody responses were monitored by ELISA. Data are represented as the mean ±standard deviation (SD) of the optical density (OD) readings. Statistically significant responses are shown as compared to PBS group (+) or i.n. versus i.m. route of inoculation in the same group (*). * or +, significant at p<0.05; ** or ++, significant at p<0.01; *** or +++, significant at p<0.001; and **** or ++++; significant at p<0.0001. The statistical analysis was done by Bonferroni post-test and two-way ANOVA using Graph Pad Prim 6. BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-H5HA, human adenoviral vector expressing HA of a A/Hong Kong/156/97(H5N1) influenza virus; BAd-ΔE1E3 (BAd empty vector); HAd-ΔE1E3, (HAd empty vector); PBS, phosphate-buffered saline; and ns, no significance at p>0.05.

FIG. 3. HA-specific serum IgG2a antibody responses in mice immunized once with BAd-H5HA or HAd-H5HA. Mice were immunized intramuscularly (i.m.) or intranasally (i.n.) once with 1×10⁶(A), 3×10⁶(B), 1×10⁷(C), 3×10⁷(D) or 1×10⁸(E) PFU of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated i.m. or i.n. with PBS or 1×10⁸PFU of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, serum samples were collected, diluted to 1:500, and the development of HA-specific IgG antibody responses were monitored by ELISA. Data are represented as the mean ±standard deviation (SD) of the optical density (OD) readings. Statistically significant responses are shown as compared to PBS group (+) or i.n. versus i.m. route of inoculation in the same group (*). * or +, significant at p<0.05; ** or ++, significant at p<0.01; *** or +++, significant at p<0.001; and **** or ++++; significant at p<0.0001. The statistical analysis was done by Bonferroni post-test and two-way ANOVA using Graph Pad Prim 6. BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-H5HA, human adenoviral vector expressing HA of a A/Hong Kong/156/97(H5N1) influenza virus; BAd-ΔE1E3 (BAd empty vector); HAd-ΔE1E3, (HAd empty vector); PBS, phosphate-buffered saline; and ns, no significance at p>0.05.

FIG. 4. HA-specific serum IgG2b antibody responses in mice immunized once with BAd-H5HA or HAd-H5HA. Mice were immunized intramuscularly (i.m.) or intranasally (i.n.) once with 1×10⁶(A), 3×10⁶(B), 1×10⁷(C), 3×10⁷(D) or 1×10⁸(E) PFU of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated i.m. or i.n. with PBS or 1×10⁸PFU of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, serum samples were collected, diluted to 1:500, and the development of HA-specific IgG antibody responses were monitored by ELISA. Data are represented as the mean ±standard deviation (SD) of the optical density (OD) readings. Statistically significant responses are shown as compared to PBS group (+) or i.n. versus i.m. route of inoculation in the same group (*). * or +, significant at p<0.05; ** or ++, significant at p<0.01; *** or +++, significant at p<0.001; and **** or ++++; significant at p<0.0001. The statistical analysis was done by Bonferroni post-test and two-way ANOVA using Graph Pad Prim 6. BAd-HSHA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-HSHA, human adenoviral vector expressing HA of a A/Hong Kong/156/97(H5N1) influenza virus; BAd-ΔE1E3 (BAd empty vector); HAd-ΔE1E3, (HAd empty vector); PBS, phosphate-buffered saline; and ns, no significance at p>0.05.

FIG. 5. HA-specific IgA antibody responses in nasal washes of mice immunized once with BAd-H5HA or HAd-H5HA. Mice were immunized intramuscularly (i.m.) or intranasally (i.n.) once with 1×10⁶(A), 3×10⁶(B), 1×10⁷(C), 3×10⁷(D) or 1×10⁸(E) PFU of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated i.m. or i.n. with PBS or 1×10⁸PFU of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, nasal wash samples were collected, diluted to 1:5, and the development of HA-specific IgG antibody responses were monitored by ELISA. Data are represented as the mean ±standard deviation (SD) of the optical density (OD) readings. Statistically significant responses are shown as compared to PBS group (+) or i.n. versus i.m. route of inoculation in the same group (*). * or +, significant at p<0.05; ** or ++, significant at p<0.01; *** or +++, significant at p<0.001; and **** or ++++; significant at p<0.0001. The statistical analysis was done by Bonferroni post-test and two-way ANOVA using Graph Pad Prim 6. BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-H5HA, human adenoviral vector expressing HA of a A/Hong Kong/156/97(H5N1) influenza virus; BAd-ΔE1E3 (BAd empty vector); HAd-ΔE1E3, (HAd empty vector); PBS, phosphate-buffered saline; and ns, no significance at p>0.05.

FIG. 6. HA-specific IgA antibody responses in lung washes of mice immunized once with BAd-H5HA or HAd-H5HA. Mice were immunized intramuscularly (i.m.) or intranasally (i.n.) once with 1×10⁶(A), 3×10⁶(B), 1×10⁷(C), 3×10⁷(D) or 1×10⁸(E) PFU of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated i.m. or i.n. with PBS or 1×10⁸PFU of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, lung wash samples were collected, diluted to 1:10, and the development of HA-specific IgG antibody responses were monitored by ELISA. Data are represented as the mean ±standard deviation (SD) of the optical density (OD) readings. Statistically significant responses are shown as compared to PBS group (+) or i.n. versus i.m. route of inoculation in the same group (*). * or +, significant at p<0.05; ** or ++, significant at p<0.01; *** or +++, significant at p<0.001; and **** or ++++; significant at p<0.0001. The statistical analysis was done by Bonferroni post-test and two-way ANOVA using Graph Pad Prim 6. BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-H5HA, human adenoviral vector expressing HA of a A/Hong Kong/156/97(H5N1) influenza virus; BAd-ΔE1E3 (BAd empty vector); HAd-ΔE1E3, (HAd empty vector); PBS, phosphate-buffered saline; and ns, no significance at p>0.05.

FIG. 7. HA518 epitope-specific IFNγ secreting CD8+T cells in the spleens of mice immunized once with BAd-H5HA or HAd-H5HA. Mice were immunized intramuscularly (i.m.) or intranasally (i.n.) once with 1×10⁶(A), 3×10⁶(B), 1×10⁷(C), 3×10⁷(D) or 1×10⁸(E) PFU of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated i.m. or i.n. with PBS or 1×10⁸PFU of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, the spleens were collected, and the splenocytes were evaluated for HA-specific cell-mediated immune responses using IFNγ-ELISpot assay. The data represent mean ±standard deviation (SD) of the number of spot-forming units (SFU). Statistically significant responses are shown as compared to PBS group (+) or i.n. versus i.m. route of inoculation in the same group (*). * or +, significant at p<0.05; ** or ++, significant at p<0.01; *** or +++, significant at p<0.001; and **** or ++++; significant at p<0.0001. The statistical analysis was done by Bonferroni post-test and two-way ANOVA using Graph Pad Prim 6. BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-H5HA, human adenoviral vector expressing HA of a A/Hong Kong/156/97(H5N1) influenza virus; BAd-ΔE1E3 (BAd empty vector); HAd-ΔE1E3, (HAd empty vector); PBS, phosphate-buffered saline; and ns, no significance at p>0.05.

FIG. 8. HA518 epitope-specific IFNγ secreting CD8+T cells in the respiratory lymph nodes (RLN) of mice immunized once with BAd-H5HA or HAd-H5HA. Mice were immunized intramuscularly (i.m.) or intranasally (i.n.) once with 1×10⁶(A), 3×10⁶(B), 1×10⁷(C), 3×10⁷(D) or 1×10⁸(E) PFU of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated i.m. or i.n. with PBS or 1×10⁸PFU of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, the RLN were collected, and the pooled RLN cells were evaluated for HA-specific cell-mediated immune responses using IFNγ-ELISpot assay. The data represent mean number of spot-forming units (SFU) from pooled samples. BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of a A/Hong Kong/156/97(H5N1) influenza virus; HAd-H5HA, human adenoviral vector expressing HA of a A/Hong Kong/156/97(H5N1) influenza virus; BAd-ΔE1E3 (BAd empty vector); HAd-ΔE1E3, (HAd empty vector); PBS, phosphate-buffered saline.

FIG. 9. HA518 epitope-specific IFNγ secreting CD8+T cells in the inguinal lymph nodes (ILN) of mice immunized once with BAd-H5HA or HAd-H5HA. Mice were immunized intramuscularly (i.m.) or intranasally (i.n.) once with 1×10⁶(A), 3×10⁶(B), 1×10⁷(C), 3×10⁷(D) or 1×10⁸(E) PFU of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated i.m. or i.n. with PBS or 1×10⁸PFU of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, the ILN were collected, and the pooled ILN cells were evaluated for HA-specific cell-mediated immune responses using IFNγ-ELISpot assay. The data represent mean number of spot-forming units (SFU) from pooled samples. BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of a A/Hong Kong/156/97(H5N1) influenza virus; HAd-H5HA, human adenoviral vector expressing HA of a A/Hong Kong/156/97(H5N1) influenza virus; BAd-ΔE1E3 (BAd empty vector); HAd-ΔE1E3, (HAd empty vector); PBS, phosphate-buffered saline.

FIG. 10. HA518 epitope-specific IFNγ secreting CD8+T cells in the lungs of mice immunized once with BAd-H5HA or HAd-H5HA. Mice were immunized intramuscularly (i.m.) or intranasally (i.n.) once with 1×10⁶(A), 3×10⁶(B), 1×10⁷(C), 3×10⁷(D) or 1×10⁸(E) PFU of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated i.m. or i.n. with PBS or 1×10⁸PFU of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, the lungs were collected, and the pooled lung lymphocytes were evaluated for HA-specific cell-mediated immune responses using IFNγ-ELISpot assay. The data represent mean the number of spot-forming units (SFU) from pooled samples. BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of a A/Hong Kong/156/97(H5N1) influenza virus; HAd-H5HA, human adenoviral vector expressing HA of a A/Hong Kong/156/97(H5N1) influenza virus; BAd-ΔE1E3 (BAd empty vector); HAd-ΔE1E3, (HAd empty vector); PBS, phosphate-buffered saline.

FIG. 11. Lung influenza virus titers in mice immunized once with BAd-H5HA or HAd-H5HA. Mice were immunized intramuscularly (i.m.) or intranasally (i.n.) once with 1×10⁶(A), 3×10⁶(B), 1×10⁷(C), 3×10⁷(D) or 1×10⁸(E) PFU of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated i.m. or i.n. with PBS or 1×10⁸PFU of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after immunization, mice were challenged with 100 MID₅₀of A/Vietnam/1203/2004(H5N1)-PR8/CDC-RG influenza virus, and three days after the challenged, mice were euthanized, and the lungs were collected to determine lung virus titers. The data are shown as mean Logo) TCID₅₀±SD and the detection limit was 0.5 Log₁₀TCID₅₀/ml. Statistically significant responses are shown as compared to PBS group (+) or i.n. versus i.m. route of inoculation in the same group (*). * or +, significant at p<0.05; ** or ++, significant at p<0.01; *** or +++, significant at p<0.001; and **** or ++++, significant at p<0.0001. The statistical analyses were done by Bonferroni post-test and two-way ANOVA using Graph Pad Prim 6. BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of a A/Hong Kong/156/97(H5N1) influenza virus; HAd-H5HA, human adenoviral vector expressing HA of a A/Hong Kong/156/97(H5N1) influenza virus; BAd-ΔE1E3 (BAd empty vector); HAd-ΔE1E3, (HAd empty vector); PBS, phosphate-buffered saline; ns, no significance at p>0.05; NA, not applicable.

FIG. 12. Genomic map of BAdV3 wild type and ΔE1E3 deletions.

FIG. 13. Expression and processing of FMDV P1 gene construct in BAd-P1-F infected cells. BHH3-F5 [bovine human hybrid clone 3 expressing human adenovirus type 5 (HAdV-5) E1, E1B-S, E1B-L and bovine adenovirus type 3 (BAdV-3) E1B-L proteins] cells were mock (PBS) [lane: BHH3-F5] infected or infected with BAdAE1FE3 (BAd empty vector having full E1deletion as well as E3 deletion) or BAd-P1-F [BAdAE1FE3 expressing foot-and-mouth disease virus (FMDV) P1 containing the furin cleavage domains, SEQ ID NO:5]. The mock or infected cells were harvested 48 h post-infection and processed for total cell protein extraction. 20, 40 or 80 μg of protein was processed for Western blot analysis. 293 cells similarly infected with HAd-ΔE1E3 (HAd empty vector having full E1 deletion as well as E3 deletion) or HAd-P1-F (HAd-ΔE1E3 expressing FMDV P1 containing the furin cleavage domains) were harvested 48 h post-infection and processed for total cell protein extraction. 10 or 100 μg of extract protein were processed for Western blot analysis. HAd-P1-F infected cell extract served as a positive control. An anti-VP2 FMDV mAb antibody (1C8B8) was used to confirm the expression and processing of P1-furin protein into VP2 and other viral proteins (VP1, VP3 and VP4). The molecular weight markers (M) are shown on the right. The dominant band at approximately 60 kDa represents the major product of P1-furin containing VP2.

FIG. 14. SEQ ID NO:4: FMDV structural proteins VP4, VP2, VP3, VP1 separated by natural sequences. The amino acids highlighted in red can be replaced by consensus furin cleavage site (RRHRR, SEQ ID NO:6).

FIG. 15. SEQ ID NO:5: Amino acid sequences of FMDV structural proteins VP4, VP2, VP3, VP1 separated by the furin cleavage site (RRHRR SEQ ID NO:6)

FIG. 16. Immunoblot demonstrating expression of 47kDa BAdV-3 E1B large protein in BHH3 cell clones stably transfected with the BAdV-3 E1B large gene cassette. BHH3 cells were stably transfected with the BAdV-3 E1B large gene cassette and a number of independently generated cell clones were isolated following selection. These clones were grown as independent cell clones. Cells from a few clones (5, 1, 14, 6, and 4) were harvested and processed for western blot. Mock or wild type (WT) BAdV-3-infected cell extracts served as negative and positive control, respectively. Immunoblotting was done with a BAdV-3 E1B large protein-specific antibody raised in a rabbit. The arrow on the right side indicates the 47 kDa BAdV-3 E1B large protein band. The locations of selected molecular weight markers are indicated on the left.

FIG. 17. Replication kinetics of (A) wild type bovine adenovirus type 3 (WT BAdV-3), (B) BAdV-3 with EIA and E3 deletions (BAdV-ΔE1AE3), and (C) BAdV-3 with full EIA, EIB and E3 deletions (BAdV-ΔE1FE3) in MDBK, FBRT-HE1, BHH3 or BHH3-E1B/L5 (BHH3 expressing BAdV-3 EIB large protein). Cells were infected with a multiplicity of infection (m.o.i.) of 5 plaque-forming units (p.f.u.) per cell with purified WT BAdV-3, BAdV-ΔE1AE3 or BAdV-ΔE1AE3 and harvested at 6, 24, 36, 48 and 60 h post infection. Virus-infected cells along with the medium were collected and virus titration were performed by tissue culture infectious dose 50 (TCID₅₀) assays.

DETAILED DESCRIPTION

While the concepts of the present disclosure are illustrated and described in detail in the figures and the description herein, results in the figures and their description are to be considered as exemplary and not restrictive in character; it being understood that only the illustrative embodiments are shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.

Unless defined otherwise, the scientific and technology nomenclatures have the same meaning as commonly understood by a person in the ordinary skill in the art pertaining to this disclosure.

Adenovirus (AdV) vector-based vaccines have been shown to elicit balanced humoral and cell-mediated immune (CMI) responses by activating innate immunity through both Toll-like receptor (TLR)-dependent as well as TLR-independent pathways. Due to the high prevalence of AdV in humans, the development of AdV-specific neutralizing antibodies, known as ‘pre-existing vector immunity’, is one of the potential concerns for several human AdV (HAdV) vector-based vaccine delivery systems. To address this concern, a number of less prevalent HAdVs or nonhuman AdVs have been developed as vaccine delivery vectors. These nonhuman AdV vectors are based on bovine AdV (BAdV), simian AdV (SAdV), porcine AdV (PAdV), ovine AdV (OAdV), Canine AdV (CAdV), avian AdV (AAdV) and murine AdV (MAdV).

It has been demonstrated that there were no reductions in humoral and CMI responses against the vaccine immunogen and the resultant protection efficacy of a BAdV type 3 (BAdV-3) vector-based H5N1 influenza vaccine even in the presence of exceptionally high levels of pre-existing HAdV vector immunity. In addition, pre-existing HAdV-neutralizing antibodies in humans did not cross-neutralize BAdV-3, and HAdV-specific CMI responses did not cross-react with BAdV-3. Bio-distribution, pathogenesis, transduction, and persistence studies in animal models and cell lines have suggested that the safety aspects of BAdV vectors are similar to that of HAdV vectors. It has been illustrated that the cell internalization of BAdV-3 is independent of the HAdV type C5 (HAdV-C5) receptors [Coxackievirus-AdV receptor (CAR) and αvβ3 or αvβ5 integrin], but it is indicated that the α(2,3)-linked and α(2,6)-linked sialic acid receptors serve as major receptors for BAdV-3 internalization. It appears that BAdV-based vectors may serve as excellent vaccine vectors for humans without any concerns of pre-existing HAdV vector immunity.

Vaccine formulation features that are important for developing effective pre-pandemic influenza vaccine strategies include the development of a balanced humoral and CMI responses that could offer cross-protection, safety and efficacy with a single dose, dose sparing for vaccine delivery to a large number of individuals, and the capacity to produce a large number of vaccines at short notice. In our previous studies we have demonstrated that AdV vector-based vaccines could elicit potent humoral and CMI responses in mice conferring cross-protection depending on the immunogen/s of choice. Since AdV vectors have been evaluated for their efficacy as gene delivery vehicles in many clinical trials in humans, it is well understood how to produce a clinical grade of purified AdV vector lots in exceptionally large quantity under GLP conditions in certified cell lines in a short time span.

Several studies using AdV vector-based influenza vaccines have been conducted both in animal models and as clinical trials in humans to explore the potential of this vector system. Previously we have shown that Ad vector-based vaccines can provide complete protection against challenge with homologous and heterologous (antigenically distinct) influenza virus strains. Moreover, Ad vector vaccines containing multiple HA from different HA subtypes, or expressing nucleoprotein (NP) of H5N1 influenza, conferred either complete protection or significant reduction in lung virus titers, respectively, following challenge with pandemic H5, H7 or H9 influenza viruses.

The purpose of this study was to determine some of the parameters that are important for a pre-pandemic influenza vaccine including route of immunization, dose sparing, protection from a single dose, and protection against a heterologous influenza virus strain. Without being bound to any theory, the hypothesis behind this study is that a combination of HA-specific CMI responses and cross-reactive (though not necessarily cross-neutralizing) humoral immune responses will provide heterologous protection against an antigenically distinct H5N1 influenza virus. In addition, this study also focused on determining the vector type and route of immunization for enhanced immunogenicity conferring efficient protection.

In this study, we have compared the immunogenicity and efficacy of BAdV-3 vector (BAd-H5HA) expressing hemagglutinin (HA, SEQ ID NO:3) of a H5N1 influenza virus with that of HAdV-C5 vector (HAd-H5HA) expressing HA of a H5N1 influenza virus in a mouse model. The vaccine doses [1×10⁶, 3×10⁶, 1×10⁷, 3×10⁷, or 1×10⁸plague-forming units (PFU)] and the routes of immunization [intranasal (i.n.) or intramuscular (i.m.)] were evaluated to determine whether the BAdV vector system will serve as a better vaccine vector compared to the HAdV vector. Overall, significantly higher levels of humoral and CMI responses, and higher vaccine efficacy, in mice were observed with the BAd-H5HA vector compared to that of HAd-H5HA. The best protection efficacy, with a significantly lower vaccine dose, was observed in the mouse group inoculated i.n. with BAd-H5HA. These results suggest that the BAdV-3-based vector system is a better vaccine delivery vehicle for developing pre-pandemic influenza vaccines.

Overall, there were no significant differences in the serum anti-HA IgG, IgG1, IgG2a, and IgG2b or mucosal IgA responses in mouse groups inoculated i.m. either with BAd-H5HA or HAd-H5HA. However, mice inoculated with BAd-H5HA had higher increases in the numbers of IFNγ-secreting HA518-specific CD8 T cells in the spleen, lung and RLN than did those inoculated with HAd-H5HA. In contrast, there were no significant differences in the numbers of IFNγ-secreting HA518-specific CD8 T cells in the ILN of the mouse groups inoculated i.m. either with BAd-H5HA or HAd-H5HA. An intravenous bio-distribution study in mice has demonstrated that the BAd vector genome persists longer and with higher copy numbers in the spleen and lungs than that of the HAd vector Similar bio-distribution following the i.m. route of immunization may be responsible for the higher level of CMI responses with the BAd-H5HA vector. Additional studies will be required to determine the mechanism/s of these observations.

The HK/156 HA expressed in the AdV vectors and the HA in the challenged virus VN/1203/RG are antigenically different, so it was not surprising that the serum samples from either BAd-H5HA- or HAd-H5HA-inoculated animal groups did not have detectable levels of HI or VN titers against the challenge virus. We purposely opted for antigenically distinct HAs as an immunogen and a challenge virus to test our hypothesis that a combination of CMI responses and non-neutralizing antibodies will provide heterologous protection. The mouse group inoculated i.m. with 3×10⁷PFU of BAd-H5HA were completely protected from the challenge with VN/1203/RG, whereas detectable levels (0.84±0.5 log) of lung virus titers were observed in the mouse group i.m.-inoculated with 3×10⁸PFU of HAd-H5HA. These observations suggest that significantly higher levels of CMI responses and non-neutralizing antibodies elicited with BAd-H5HA may be responsible for enhanced protection at a significantly lower dose. In mouse groups inoculated i.n. with BAd-H5HA, levels of serum anti-HA IgG, IgG1, IgG2a, IgG2b, mucosal anti-HA IgA, and the numbers of IFNγ-secreting HA518-specific CD8 T cells in the spleen, lung and RLN, were significantly higher than in the groups inoculated i.m. with BAd-H5HA or inoculated i.m. or i.n. with HAd-H5HA. Additional experiments are required to determine whether the high levels of humoral (systemic and mucosal) and CMI responses are due to better transduction of and/or the levels and duration of persistence of the BAd vector genomes following immunization with BAd-H5HA. Since BAd-3 utilizes the α(2,3)-linked and α(2,6)-linked sialic acid receptors as the major receptors for virus internalization, while HAd-C5 uses CAR for virus entry in susceptible cells, we expect that BAd-H5HA will better transduce the respiratory tract following i.n. inoculation compared to HAd-H5HA. Moreover, the levels and duration of persistence of the BAd vector genome copy numbers in the lungs were found to be significantly higher than that of the HAd vector in an intravenous bio-distribution study. There is a possibility that a similar situation may occur following the i.n. inoculation.

Complete protection from challenge with an antigenically distinct H5N1 influenza virus VN/1203/RG was observed even in the mouse group inoculated i.n. with 1×10⁶PFU of BAd-H5HA, whereas similar level of protection with HAd-H5HA was only obtained with a much higher vector dose of 1×10⁸PFU. These observations suggest that BAd vector-based i.n. vaccine delivery system has considerable promise for dose sparing, because an approximately 30-fold lower vaccine dose of BAd-H5HA compared to HAd-H5HA conferred complete protection. Dose sparing not only lowers vaccine costs, but also increases the capacity to produce a large number of doses especially in an event similar to the influenza pandemic. Of course, additional studies will be needed to determine the long-term efficacy of cross-protective humoral and CMI responses induced by BAdV-3-based vectors.

The results described in this manuscript suggest that the BAdV-3-based vector system has many advantages over HAd systems as a vaccine delivery vehicle for developing pre-pandemic influenza vaccines. Further studies in another animal model of influenza, such as ferrets, will be essential to fully explore the potential of this vaccine delivery system. Additional studies will be required to determine the best combinational antigens or immunogenic domains that could elicit broadly cross-protective immune responses when delivered through the BAd vector system for developing a universal influenza vaccine for pandemic preparedness and to offer a better vaccine option against seasonal influenza viruses.

Material and Methods
Cell Lines, AdV Vectors, and Influenza Viruses

BHH3 (bovine-human hybrid clone 3) , BHH2C (bovine-human hybrid clone 2C), 293 (human embryonic kidney cells expressing HAdV-C5 E1 proteins), and MDCK (Madin-Darby canine kidney) cell lines were propagated as monolayer cultures in minimum essential medium (MEM) (Life Technologies, Gaithersburg, Md.) containing either 10% reconstituted fetal bovine serum or fetal calf serum (Hyclone, Logan, Utah) and gentamycin (50 μg/ml).

The construction and characterization of BAd-H5HA [BAdV-3 E1 and E3 deleted vector expressing HA of A/Hong Kong/156/97(H5N1) (HK/156)], BAd-ΔE1E3 (BAdV-3 E1 and E3 deleted empty vector), HAd-H5HA [HAdV-Cc5 E1 and E3 deleted vector expressing HA of HK/156], HAd-ΔE1E3 (HAdV-C5 E1 and E3 deleted empty vector), are described previously. For example, BHH3 cell lines that support the replication of BAdV vectors having only E1A deletion with or without E3 deletion are described in van Olphen A L, and Mittal S K: “Development and characterization of bovine x human hybrid cell lines that efficiently support the replication of both wild-type bovine and human adenoviruses and those with E1 deleted.” J Virol 2002. 76:5882-92. FBRT-HE1 cell line (fetal bovine retinal cells which express HAdV-5 E1A, E1B-S and E1B-L proteins) is described in van Olphen A L, Tikoo S K, and Mittal S K: “Characterization of bovine adenovirus type 3 E1 proteins and isolation of E1-expressing cell lines.” Virology 2002. 295:108-18.

BAd-H5HA and BAd-ΔE1E3 were grown and titrated in BHH3 (bovine-human hybrid clone 3 that expresses HAdV-5 E1A, E1B-S and E1B-L proteins) cells, and HAd-H5HA and HAd-ΔE1E3 were grown in 293 cells and titrated in BHH2C cells. Both BAd-H5HA and HAd-H5HA expressed HA to similar levels in vector-infected cells as observed by immunoblotting (data not shown). These vectors were purified by cesium chloride density gradient ultracentrifugation as described previously.

A/Vietnam/1203/2004(H5N1)-PR8/CDC-RG [VN/1203/RG] that was created by reverse genetics (RG) in the A/PR/8/1934(H1N1) [PR8] background was grown in embryonated hen eggs and titrated in the eggs and/or MDCK. The HA gene in the vaccine vectors was from HK/156, which is antigenically distinct from the challenge virus VN/1203/RG.

Immunogenicity and Protection Studies in Mice

All immunization and protection studies in mice were performed in a USDA-approved BSL-2+facility with the approvals of the Institutional Animal Care and Use Committee

(IACUC), and the Institutional Biosafety Committee (IBC). Six-to-eight-week-old BALB/c mice (Harlan Sprague Dawley Inc., Indianapolis, Ind.) served as the subjects for immunization and protection studies following approved guidelines.

The mouse groups (10 animals/group) were mock-inoculated (with phosphate-buffered saline (PBS), pH 7.2) or inoculated i.n. or i.m. with 1×10⁶, 3×10⁶, 1×10⁷, 3×10⁷, or 1×10⁸PFU of BAd-H5HA or HAd-H5HA. The mouse groups inoculated i.n. or i.m. with 1×10⁸PFU of BAd-ΔE1E3 or HAd-ΔE1E3 served as vector controls. Four weeks post-inoculation, 5 animals/group were anesthetized with ketamine-xylazine (90 mg/kg ketamine and 10 mg/kg xylazine) by intraperitoneal injections, the blood samples were collected via retro-orbital puncture, nasal washes were collected by washing the nasal passage with 0.5 mL of PBS, and the lung washes were prepared after homogenizing one lung from each animal in 1 mL of PBS as described previously. The serum samples, nasal washes and lung washes were used to evaluate the humoral immune responses. The second lung was processed to collect CD3+ T cells from the lung cells using MagniSort® Mouse CD3 Positive Selection Kit following the manufacturer's instructions (Affymetrix eBioscience San Diego, Calif.), and used to monitor CMI responses. The spleens, respiratory area lymph nodes (RLN) and inguinal lymph nodes (ILN) were also collected to evaluate CMI responses. The remaining five animals per group were challenged i.n. with 100 mouse infectious dose 50 (MID50) of VN/1203/RG. Three days post-challenge, the animals were euthanized under ketamine-xylazine anesthesia as described above, and the lungs were collected for determination of the lung virus titers as described previously.

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA was performed as described earlier. 96-well ELISA plates (eBioscience, San Diego, Calif) were coated with purified HA protein (0.5 μg/mL) of HK/156 (MyBioSource, Inc., San Diego, Calif., USA) and incubated overnight at 4° C. Following blocking with 1% bovine serum albumin (BSA) in PBS, diluted serum samples (1:500 dilution for IgG & IgG1 and 1:50 for IgG2a and IgG2b), 1:5 diluted nasal washes, or 1:10 diluted lung washes were added and incubated at room temperature for 2 h. During the standardization process, various dilutions of each type of sample were tested to establish the best dilution. The horseradish peroxidase-conjugated goat anti-mouse IgG, IgG1, IgG2a, IgG2b, or IgA antibodies, (Invitrogenl Fisher Scientific Corp.) at a recommended dilution for each antibody was added and incubated at room temperature for 2 h. The color development was obtained with a BD OptEIATM ELISA sets TMB substrate (Fisher Scientific Corp.). The reaction was stopped with 2N sulfuric acid solution and the optical density readings were obtained at 450 nm using a SpectraMax® i3x microplate reader (Molecular Devices, Sunnyvale, Calif.).

ELISpot Assays

The ELISpot assays were performed as described previously. The splenocytes, lung lymphocytes, RLN, and ILN were used for interferon-gamma (IFNγ) ELISpot assays after stimulating the cells with HA518 (IYSTVASSL) peptide (H-2K^d-restricted CTL epitope for HA). The number of spot-forming units (SFU) were counted using a dissection microscope.

Statistical Analyses

Two-way ANOVA with Bonferroni post-test were performed to determine statistical significance. The statistical significance was set at p<0.05.

EXAMPLES
Example 1. Comparison of Wild Type BAdV3 Vector Sequence Versus BAdV3ΔE1E3 Vector Sequence

A map of wild type BAdV3 vector genome outline compared to BAdV3ΔE1E3 is shown in FIG. 12. As it shows, the entire E1 region of BAdV3 comprising 2574 nucleotides (SEQ ID NO:1) are deleted, as well as 1245 nucleotides of E3 region (SEQ ID NO:2) are also deleted. Such E1 and E3 region deleted BAdV vector increased the foreign gene cassette insertion capacity by at least 2 Kb compared to previously conventional adenovirus vector, which was not able to delete entire E1 region and still viable to produce virus in its host cell lines.

Example 2. Unique Cell Line for Producing BAdV3ΔE1E3 Viruses with or without Foreign Gene Expression

The cell line used for production of empty BAd3ΔE1E3 virus or BAd3VΔE1E3 vector with insert of HA gene is BHH3 that is also transfected with human adenovirus E1 region and BAdV-3 EB1L gene. This cell line will support BAd3ΔE1E3 virus replication despite the deletion of complete E1 and E3 regions in the BAdV3 vector.

In this disclosure cell lines that support the replication of BAdV vectors having E1 full deletion (including E1A, E1B-S and E1B-L) with or without E3 deletion are following:

- BHH3-BE1BL (BHH3 that expresses HAdV-5 E1A, E1B-S and E1B-L proteins and BAdV-3 E1B-L protein) cell line.
- BHH3-BE1B/Full (BHH3 that expresses HAdV-5 E1A, E1B-S and E1B-L proteins and BAdV-3 E1B-S and E1B-L proteins) cell line.
- FBRT-HE1-BE1BL (FBRT-HE1 that expresses HAdV-5 E1A, E1B-S and E1B-L proteins and BAdV-3 E1B-L protein) cell line.
- FBRT-HE1-BE1B/Full (FBRT-HE1 that expresses HAdV-5 E1A, E1B-S and E1B-L proteins and BAdV-3 E1B-S and E1B-L proteins) cell line.

In previous versions of the vectors only E1A and E3 region were deleted in BAdV-3 genome, the foreign gene cassette insertion capacity was approximately 2 Kb less compared to the E1A, E1BS and E1BL, as well as E3 region deleted vectors. However, when E1A, E1BS, E1BL of the BAdV-3 genome are deleted, the bovine cell line expressing human E1A, E1BS and E1BL cannot rescue the BAdV-3 vector, rendering foreign gene expression impossible. In the current disclosure, we transfected at least one BAdV-3 E1BL gene into a bovine cell line, together with the HAdV E1A, HAdV E1BS, and HAdV E1BL genes.

Example 3
Induction of Enhanced Humoral Immune Responses with BAd-H5HA Compared to HAd-H5HA

The mouse groups inoculated i.n. or i.m. once with 1×10⁶(FIG. 1A), 3×10⁶(FIG. 1B), 1 ×10⁷(FIG. 1C), 3×10⁷(FIG. 1D) or 1×10⁸(FIG. 1E) PFU of HAd-H5HA or BAd-H5HA elicited dose-dependent increases in anti-HA IgG antibody levels. These levels in the i.m.-inoculated HAd-H5HA groups were similar to or slightly better than those observed in the similarly inoculated BAd-H5HA groups. In the i.n.-inoculated BAd-H5HA groups, however, anti-HA IgG antibody levels were significantly higher than those in the similarly or i.m.-inoculated HAd-H5HA groups or the i.m.-inoculated BAd-H5HA groups. The control groups inoculated i.n. or i.m. with PBS, HAd-ΔE1E3, or BAd-ΔE1E3 did not yield anti-HA IgG antibody levels above background (FIG. 1).

We have shown previously that animals immunized with Ad vectors expressing HA produce high levels of hemagglutination inhibition (HI) and virus-neutralizing (VN) antibody titers against homologous influenza virus strains, therefore, here we only tried to determine HI and VN titers against an antigenically distinct influenza virus strain (VN/1203/RG), and noticed that none of the vaccinated groups developed any detectable HI and VN titers above the empty vector controls.

To determine whether the route of immunization or the vector type will influence the levels of IgG subclasses, the serum samples collected from the mouse groups inoculated i.n. or i.m. once with 1×10⁶(FIGS. 2A, 3A & 4A), 3×10⁶(FIGS. 2B, 3B & 4B), 1×10⁷(FIGS. 2C, 3C & 4C), 3×10⁷(FIGS. 2D, 3D & 4D) or 1×10⁸(FIGS. 2E, 3E & 4E) PFU of HAd-H5HA or BAd-H5HA were analyzed for anti-HA IgG1 (FIG. 2), IgG2a (FIG. 3) and IgG2b (FIG. 4) levels by ELISA, respectively. As expected, there were dose-dependent increases in anti-HA IgG1, IgG2a and IgG2b antibody levels. These levels in the i.m.-inoculated HAd-H5HA groups were similar to or slightly better or lower than those observed in the i.m.-inoculated BAd-H5HA groups. However, in the i.n.-inoculated BAd-H5HA groups, anti-HA IgG1, IgG2a and IgG2b antibody levels were significantly higher than those in the i.n.- or i.m.-inoculated HAd-H5HA groups or the i.m.- inoculated BAd-H5HA groups (FIGS. 2, 3 & 4). Control groups inoculated i.n. or i.m. with PBS, HAd-ΔE1E3, or BAd-ΔE1E3 did not yield anti-HA IgG1 (FIG. 2), IgG2a (FIG. 3) or IgG2b (FIG. 4) antibody levels above background.

Furthermore, to ascertain if the route of immunization or the vector type will influence the development of HA-specific IgA responses at the mucosal level, lung washes (FIG. 5) as well as nasal washes (FIG. 6) from the mouse groups inoculated i.n. or i.m. once with 1×10⁶(FIGS. 5A & 6A), 3×10⁶(FIGS. 5B & 6B), 1×10⁷(FIGS. 5C & 6C), 3×10⁷(FIGS. 5D & 6D) or 1×10⁸(FIGS. 5E & 6E) PFU of HAd-H5HA or BAd-H5HA were analyzed for anti-HA IgA levels by ELISA. As expected, there were dose-dependent increases in anti-HA IgA antibody levels in the lung washes as well as nasal washes. IgA antibody levels in the lung washes and nasal washes in the i.m.-inoculated BAd-H5HA or HAd-H5HA groups were detected only with doses of 1×10⁷PFU and onwards (FIGS. 5 & 6). IgA antibody levels in the lung washes and nasal washes of the i.m.-inoculated BAd-H5HA groups were similar to or slightly higher than those in the i.m.-inoculated HAd-H5HA, whereas IgA antibody levels in the lung washes and nasal washes in the i.n.-inoculated BAd-H5HA or HAd-H5HA groups were detected with the lowest dose of 1×10⁶PFU and onwards. In the i.n.-inoculated BAd-H5HA groups, anti-HA IgA antibody levels in the lung washes (FIG. 5) and nasal washes (FIG. 6) were significantly higher than those in the i.n.- or i.m.-inoculated HAd-H5HA groups or the i.m.-inoculated BAd-H5HA groups. The control groups inoculated i.n. or i.m. with PBS, HAd-ΔE1E3, or BAd-ΔE1E3 did not yield anti-HA IgA antibody levels above background (FIGS. 5 & 6).

Example 4

Induction of enhanced CMI responses with BAd-H5HA compared to HAd-H5HA CMI responses against influenza viruses are important for virus clearance following infection and play an important role in heterologous as well as heterosubtypic protection against influenza viruses. To verify whether the route of immunization or the vector type will impact the development of the CMI responses against influenza in vaccinated mice, we analyzed splenocytes (FIG. 7), pooled RLN cells (FIG. 8), pooled ILN cells (FIG. 9), and pooled lung lymphocytes (FIG. 10) from AdV vector-inoculated groups for HA-specific CMI responses following in vitro stimulation with HA518 using an IFNγ-specific ELISpot assay. The numbers of IFNγ-secreting HA518-specific CD8 T cells from the mouse groups inoculated i.n. or i.m. once with 1×10⁶(FIGS. 7A, 8A, 9A & 10A), 3×10⁶(FIGS. 7B, 8B, 9B & 10B), 1×10⁷(FIGS. 7C, 8C, 9C & 10C), 3×10⁷(FIGS. 7D, 8D, 9D & 10D) or 1×10⁸(FIGS. 7E, 8E, 9E & 10E) PFU of HAd-H5HA or BAd-H5HA are shown. There were dose-dependent increases in the numbers of IFNγ-secreting HA518-specific CD8 T cells in splenocytes (FIG. 7), pooled RLN cells (FIG. 8), pooled SLN cells (FIG. 9), and pooled lung lymphocytes (FIG. 10) of the vaccinated groups compared with the empty vector (Ad-ΔE1E3) or PBS control groups. There were significantly higher numbers of IFNγ-secreting HA518-specific CD8 T cells in splenocytes of the i.m.- or i.n.-inoculated BAd-H5HA groups compared to the i.m.- or i.n.-inoculated HAd-H5HA groups (FIG. 7), and the numbers were consistently higher in the i.m.-inoculated vaccine groups compared to that of the i.n. inoculated vaccine groups.

There were substantially higher numbers of IFNγ-secreting HA518-specific CD8 T cells in the RLN of the i.m.- or i.n.-inoculated BAd-H5HA groups compared to those of the i.m.- or i.n.-inoculated HAd-H5HA groups (FIG. 8), and the numbers were consistently higher in the i.n.-inoculated vaccine groups compared to those of the i.m.-inoculated vaccine groups. Whereas in ILN of the i.m.-inoculated vaccine groups, higher numbers of IFNγ-secreting HA518-specific CD8 T cells were detected compared to those of the empty vector (Ad-ΔE1E3) or PBS control groups or the i.n.-inoculated vaccine groups (FIG. 9), and the numbers were consistently higher in the i.m.-inoculated vaccine groups compared to those of the i.n.-inoculated vaccine groups. Furthermore, considerably elevated numbers of IFNγ-secreting HA518-specific CD8 T cells in the lung lymphocytes of the i.m.- or i.n. inoculated BAd-H5HA groups compared to those of the i.m.- or i.n.-inoculated HAd-H5HA groups, respectively, were visualized (FIG. 10), and the numbers were consistently higher in the i.n.-inoculated vaccine groups compared to those of the i.m.-inoculated vaccine groups.

Example 5
Development of Enhanced Protection in Mice Immunized with BAd-H5HA Compared to HAd-H5HA

Since the influenza virus VN/1203/RG, which was used as a challenge virus to evaluate the efficacy of protection, does not cause significant morbidity or mortality in mice, significant reductions in lung viral titers in vaccinated animals following challenge is a useful measure of the virus clearance and vaccine protective efficacy. The mouse groups were immunized i.n. or i.m. once with 1×10⁶(FIG. 11A), 3×10⁶(FIG. 11B), 1×10⁷(FIG. 11C), 3×10⁷(FIG. 11D) or 1 ×10⁸(FIG. 11E) PFU of HAd-H5HA or BAd-H5HA, and subsequently challenged with 100 MID₅₀of VN/1203/RG. For the i.n. route of inoculation, the lowest vaccine dose of 1×10⁶PFU of BAd-H5HA conferred complete protection following challenge (FIG. 11A). The lowest vaccine dose for i.n.-inoculated HAd-H5HA that yielded complete protection following challenge was 3×10⁷PFU (FIG. 11E). For the i.m. route of inoculation, the lowest vaccine dose for BAd-H5HA that conferred complete protection following challenge was 3×10⁷PFU (FIG. 11D). For the i.m.-inoculated HAd-H5HA animal group, the log lung virus titer with a dose of 1 ×10⁸PFU was 1.84±1.14, compared to 4.90±0.42 and 4.6±0.46 log lung virus titers in the empty vector (HAd-ΔE1E3) or PBS control groups, respectively (FIG. 11E). Even at a challenge dose of 3×10⁸PFU per animal for i.m.-inoculated HAd-H5HA animal group, log lung virus titers of 0.84±0.5 was observed.

Example 6. Expression and Processing of FMDV P1 Gene Construct in BAd-P1-F Infected Cells

This is another example of a BAdV-3 vector with full E1 region and E3 region deletion with a foreign gene cassette in the E1 deleted region to express a target antigen in a bovine cell line that complement E1 gene functions. In this example, foot and mouse disease virus (FMDV) P1 gene is inserted to BAdV-3ΔE1E3, and the resulted vector construct is transfected into bovine human hybrid clone 3 expressing human adenovirus type 5 (HAdV-5) E1, E1B-S, E1B-L and bovine adenovirus type 3 (BAdV-3) E1B-L proteins. Successful detection of the P1 gene and its processed structural proteins are shown in FIG. 13. The cells were mock (PBS) [lane: BHH3-F5] infected or infected with BAdΔE1FE3 (BAd empty vector having full E1 deletion as well as E3 deletion) or BAd-P1-F [BAdΔE1FE3 expressing foot-and-mouth disease virus (FMDV) P1 containing the furin cleavage domains]. The mock or infected cells were harvested 48 h post-infection and processed for total cell protein extraction. 20, 40 or 80 μg of protein was processed for Western blot analysis. 293 cells similarly infected with HAd-ΔE1E3 (HAd empty vector having full E1 deletion as well as E3 deletion) or HAd-P1-F (HAd-ΔE1E3 expressing FMDV P1 containing the furin cleavage domains) were harvested 48 h post-infection and processed for total cell protein extraction. 10 or 100 μg of extract protein were processed for Western blot analysis. HAd-P1-F infected cell extract served as a positive control. An anti-VP2 FMDV mAb antibody (1C8B8) was used to confirm the expression and processing of P1-furin protein into VP2 and other viral proteins (VP1, VP3 and VP4). The molecular weight markers (M) are shown on the right. The dominant band at approximately 60 kDa represents the major product of P1-furin containing VP2.

Example 7. Generation of a Cell Line Expressing E1 Proteins (E1A, E1B small and E1B large) of human AdV type C5 (HAdV-C5) and the E1B large protein of BAdV-3

Our repeated efforts to generate BAdV-3 vectors with a full deletion in E1 region using cell lines expressing HAdV-C5 E1 proteins (BHH3 or FBRT-HE1) were unsuccessful, whereas we could generate BAdV vectors having deletion of E1A in these cell lines. These observations suggested that either both E1B proteins (small and large) or at least E1B large protein function/s is/are not complemented for BAdV-3 E1B protein/s. Using BHH3 or FBRT-HE1 cell line, we generated stable clones expressing BAdV-3 E1B proteins (small and large) or expressing BAdV-3 E1B large. BHH3 cell clones expressing 47 kDa BAdV-3 E1B large protein as demonstrated by immunoblotting is shown (FIG. 16). On the basis of cell growth and expression characteristics, BHH3-BE1B/L5 was selected for further use.

Example 8. Growth Kinetics of WT BAdV-3, BAdV-ΔE1AE3 (BAdV-3 with E1A and E3 deletions), and BAdV-ΔE1FE3 (BAdV-3 with full E1A, E1B and E3 deletions)

Growth kinetics of WT BAdV-3, BAdV-ΔE1AE3, and BAdV-ΔE1FE3 were performed in MDBK, FBRT-HE1 (expressing HAdV-C5 E1 proteins), BHH3 (expressing HAdV-C5 E1 proteins) or BHH3-E1B/L5 (BHH3 expressing BAdV-3 E1B large protein). The purpose was to determine whether expression of BAdV-3 E1B large protein is essential for the replication of BAdV-ΔE1FE3 (having 2574 bps E1 deletion and 1245 bps E3 deletion). WT BAdV-3 replicated equally well in all cell lines (FIG. 17A) since its replication does not require any complementation of E1 functions. Since BAdV-ΔE1AE3 requires the complementation of E1A function, it replicated to similar titers in E1A complementing cell lines (BHH3, FBRT-HE1 and BHH3-E1B/L5) (FIG. 17B). Whereas, BAdV-ΔE1FE3 replicated only in BHH3-E1B/L5 cells (FIG. 17C) indicating that BAdV-3 vectors with full E1 deletion requires the complementation of F1B large function from the BAdV-3 E1B large protein.

Those skilled in the art will recognize that numerous modifications can be made to the specific implementations described above. The implementations should not be limited to the particular limitations described. Other implementations may be possible.

It is intended that that the scope of the present methods and compositions be defined by the following claims. However, it must be understood that this disclosure may be practiced otherwise than is specifically explained and illustrated without departing from its spirit or scope.

It should be understood by those skilled in the art that various alternatives to the embodiments described herein may be employed in practicing the claims without departing from the spirit and scope as defined in the following claims.

NOVEL ADENOVIRAL VECTOR SYSTEM FOR GENE DELIVERY

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