ALPHA-TOCOPHEROL-BASED ADJUVANTED SOLVENT FOR DNA VACCINES

Abstract

The present invention discloses a delivery system for nucleic acid vaccines comprising an emulsion of tocol and esters hereof. Vaccines and new ways of administration of DNA vaccines are disclosed.

Claims

1. A delivery system for nucleic acid vaccines comprising an emulsion of tocol and esters hereof.

2. The delivery system according to claim 1 comprising α-tocopherol and esters hereof.

3. The delivery system according to claim 2, wherein the emulsion is α-tocopherol acetate or the racemat dl-α-tocopherol acetate.

4. The delivery system according to claim 1, further comprising an emulsifier chosen from the group of non-ionic surfactants, cationic surfactants and amphoteric surfactants.

5. The delivery system according to claim 4, further comprising at least one excipient.

6. The delivery system according to claim 5, comprising disodiumphosphate/sodiumdihydrogenphosphate.

7. The delivery system according to claim 5, comprising NaCl.

8. The delivery system according to claim 5, comprising polydimethylsiloxan (PDMS).

9. The delivery system according to claim 1 additionally comprising an immunomodulator or immunostimulator.

10. (canceled)

11. A nucleic acid vaccine for immunizing an animal, comprising the delivery system according to claim 1.

12. The nucleic acid vaccine according to claim 11, comprising the influenza genes NP and M from H1N1 1918, HA and NA from H1N1 2009, and HA and NA from H2N3 1968.

13. A method for immunizing an animal against an infectious disease comprising administering to the animal the nucleic acid vaccine according to claim 11.

14. The method according to claim 13, wherein said administration is by the intramuscular or intracutaneous or subcutaneous injection route.

15. The method according to claim 13, wherein said administration is done by injection to the skin with a syringe and needle or with a needle free jet injector device.

16. The delivery system according to claim 4, wherein said non-ionic surfactant is polyoxyethylene sorbitan mono-oleate (polysorbate), polyoxyethylene monolaurate, a polyoxyethylene fatty acid ester, a polyoxyalkyl ether, a polyoxyethylene castor oil derivative, polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lethicin or gelatin.

17. The delivery system according to claim 16, wherein said non-ionic surfactant is polyoxyethylene stearate or polyoxyethylene cetyl ether

18. The delivery system according to claim 4, wherein said anionic surfactant is a salt of an alkyl ester.

19. The delivery system according to claim 18, wherein said anionic surfactant is sodium lauryl sulfate.

20. The nucleic acid vaccine according to claim 11, wherein said animal is a human being.

21. The method according to claim 13, wherein said animal is a human being.

Description

FIGURE LEGENDS

[0038] FIG. 1. (a) H1N1pdm09 HA-specific IgG and (b) H3N2 1968 HA-specific IgG in rabbit sera post vaccination were measured by ELISA. Arrows indicate vaccination time points. Group A was immunized by i.d.+EP with influenza genes inserted into pSSI standard expression vector. Group B was immunized by IDAL with influenza genes inserted into pSSI vector, premixed with Diluvac®Forte. Thus, similar high antibody response was obtained in rabbits whether immunized by i.d: injection of DNA in PBS followed by EP (Gr.A) or by needle-free delivery of DNA in Diluvac®Forte (Gr.B). Group C was immunized by IDAL with influenza genes inserted into NTC8385-VA1 vector backbone and premixed with Diluvac®Forte. Group D was immunized by IDAL with influenza genes inserted into the NTC9385R vector, and premixed with Diluvac®Forte. Thus, similar IgG antibody response was obtained from three different plasmid backbones when the DNA was diluted in Diluvac®Forte and delivered with needle-free IDAL (Gr.B, C and D).

[0039] FIG. 2. DNA vaccine induced antibodies were measured in an HI assay and the HI titers are given as the geometric mean titer. The ability of the DNA vaccine-induced antibodies to react against H1N1pdm09 are seen in (a). The ability to cross-react with the antigentically different swine H1N1 (H1N1-2007) were also measured (b). Arrows indicate vaccination time points. Group A is immunized by i.d.+EP with influenza genes inserted into pSSI vector. Group B is immunized by IDAL with influenza genes inserted into pSSI vector, premixed with Diluvac®Forte. Group C is immunized by IDAL with influenza genes inserted into NTC8385-VA1 vector, premixed with Diluvac®Forte. Group D is immunized by IDAL with influenza genes inserted into NTC9385R vector, premixed with Diluvac®Forte. Thus, the DNA vaccine groups induced similar functional HAI titers whether the DNA was diluted in PBS and delivered by i.d. needle followed by EP (Gr.A), or if the DNA was diluted in Diluvac®Forte and delivered by needle-free IDAL (Gr.B). Similar HAI titers were induced when the influenza genes were inserted into three different plasmid backbones, diluted in Diluvac®Forte and delivered with needle-free IDAL (Gr. B, C and D).

[0040] FIG. 3. Dose-titration response in influenza DNA vaccinated pigs. IgG responses measured by ELISA against recombinant HA and NA protein, homologous to the DNA vaccine (a-c). In addition, the three vaccinated groups also developed serum IgG responses against vaccine non-homologous proteins; rec. HA, rec. NP and the M2e ectodomain peptide (d-f).

[0041] FIG. 4. Hemagglutination inhibition (HI) antibody response in DNA vaccinated pigs. HI antibody responses were measured against the human isolates H1N1v A/California/07/09 and H3N2 A/Aichi/02/68, homologous to the vaccine, (a and b). HI antibody responses were also measured against two isolates from swine, H1N2 A/swine/DK/10525/2008 and H1N1v A/swine/DK/10409/2013 (c and d).

[0042] FIG. 5. Influenza neutralizing activity in DNA vaccinated pigs. Neutralizing activity against the human isolates H1N1v A/California/07/09, homologous to the vaccine, were observed in animals from vaccinated group 3-5, at 2 weeks after 2nd vaccination, day 35 (a). The kinetics of the neutralizing response was tested for two pigs, receiving different doses of vaccine (b).

EXAMPLE 1

[0043] Construction of Expression Vectors

[0044] Influenza DNA vaccine genes were designed from nucleotide sequences published in GenBank (1918 NP: A/Brevig Mission/1/18(H1N1) AY744935, 1918 M: A/Brevig Mission/1/18(H1N1) AY130766, 2009 HA: A/California/04/2009(H1N1)pdm09 ACP41105, 2009 NA: A/California/04/2009(H1N1)pdm09 ACP41107, 1968 HA: A/Aichi/2/1968(H3N2) AB295605, 1968 NA: A/Aichi/2/1968(H3N2) AB295606). The genes were made synthetically and designed to include the appropriate restriction enzymes and Kozak sequence (GCCACC), −1 base upstream from the start codon, for efficient cloning and transcription in the pSSI (Statens Serum Institut, DK), NTC8385-VA1 (Nature Technologies Corporation, Lincoln, Neb., U.S.) [1] and NTC9385R (Nature Technologies Corporation, Lincoln, Neb., U.S.) [1] expression vectors. The genes were synthesised using only codons from highly expressed human genes (codon optimised). By this, the nucleotide codons are altered (humanised), but the encoded amino acids are identical to those encoded by the viral RNA. The genes were further cloned individually into the pSSI, NTC8385-VA1 and NTC9385R expression vectors. The pSSI expression vector backbone contains kanamycin resistance gene, cytomegalovirus immediate-early promotor, intron A and polyadenylation signal. A tissue plasminogen activator (tPA) signal sequence is not included in the vector, since the influenza HA and NA genes carry their own signal sequences for secretory expression. The influenza NP and M genes have no need for secretory signals since they express internal proteins located inside the virus and the infected cells. The pSSI vaccine construct was produced in the E. coli strain DH5α, using kanamycin as selection antibiotic. Endotoxin free DNA purification of the vaccine clones were prepared by EndoFree Plasmid Giga Kit (QIAGEN). All inserts and vaccine clones were control sequenced. Both the NTC8385-VA1 and the NTC9385R vaccine constructs use antibiotic-free selection systems and have been produced in the HyperGRO™ fermentation process [2].

Vaccine Delivery Mode

[0045] The vaccine constructs were delivered in two different modes to the animals. 1) Intradermal (i.d.) needle injection of naked DNA in PBS, at two sites in shaved abdominal skin, followed by electroporation using OncoVet™ system (CytoPulse Sciencies/Cellectis, Romainville, France) over each injected area. 2) Needle-free i.d. injection using IntraDermal Application of Liquids (IDAL) immunization technique (MSD Animal Health, Summit, N.J., U.S.) distributed at two abdominal injection sites. Before IDAL injection, the vaccine constructs are premixed at 1:1 volume ratio with the Diluvac®Forte Adjuvant (Intervet/MSD), an aqueous oil-in-water emulsion based on dl-α-tocopherol (Vitamin E). Diluvac®Forte also contains DL-α-tocopheryl acetat, Polysorbate, Sodium chloride, Disodium phosphate/Potassium dihydrogen phosphate, Simithicone and water.

[0046] Immunizations

[0047] Ten-week-old female nulliparous New Zealand white rabbits were housed at Statens Serum Institute Animal Facility (Copenhagen, Denmark). Animal experiments were performed by certified animal handlers and according to the Animal Experimentation Act of Denmark and European Convention ETS 123. Acclimatization was at least 10 days prior to any experimental procedures. The rabbits were divided into four groups with four to five rabbits in each group and they were all vaccinated at week 0 and 3 of the experiment. Both vaccinations contained an identical mix formulation of 10 pmole of each influenza gene plasmid, see Table 1. Blood serum was collected before first vaccination (week 0), week 2, before second vaccination (week 3) and week 5. Thus the amount of plasmids delivered was adjusted to equal molar amounts because they had slightly different sizes.

[0048] Serum Antibody Determination by ELISA

[0049] ELISA plates (96 wells) were coated with 100 μl of recombinant influenza haemagglutinin protein antigen A/California/04/09(H1N1pdm09) or A/Aichi/2/1968(H3N2) (Sino Biological, Beijing, China), 2 μg/ml in 50 mM carbonate buffer, overnight at 4° C. Wells were blocked with 2% skim milk powder in PBS buffer to (1% BSA, 10% FCS and 1% Triton X-100) for one hour at room temperature. Plates were washed with 1% triton/PBS. Rabbit sera, diluted in 2% skim milk powder blocking buffer, were added and incubated for one hour at room temperature. The plates were again washed and incubated with horseradish peroxidase-conjugated anti-rabbit-IgG antibody (Sigma A1949) for one hour. Following washing, color was developed with TMB (Kem-En-Tec, Denmark) for 30 minutes and the reaction was stopped by adding 0.2M H2504. Absorbance was read at OD450 nm.

TABLE-US-00002 TABLE 1 Overview of rabbit vaccinations (week 0 and 3) Numbers Delivery Plasmid-encoded Expression Dose/ Group of rabbits mode.sup.a immunogens vector vaccination Diluent A 4 i.d. needle + HA(H1N1)pdm09 pSSI 10 pmole PBS EP NA(H1N1)pdm09 NP(H1N1)1918 M(H1N1)1918 HA(H3N2)1968 NA(H3N2)1968 B 5 IDAL Same as Group A pSSI 10 pmole Diluvac ®Forte C 5 IDAL Same as Group A NTC8385- 10 pmole Diluvac ®Forte VA1 D 5 IDAL Same as Group A NTC9385R 10 pmole Diluvac ®Forte .sup.aEP: electroporation, IDAL: IntraDermal Application of Liquids

[0050] Haemagglutination Inhibitory Assay

[0051] Rabbit sera were treated with receptor destroying enzyme (RDE(II), Seiken, Japan) as described by the manufacturer. Viruses were titrated using a haemagglutination assay according to the protocols of the WHO [3] with 0.75% guinea pig red blood cells in U-bottom plates (U96 MicroWell Plates, Nunc) and incubated for one hour. Virus was standardized to 100% haemagglutination endpoint titer of 8 haemagglutination units (HAU). The haemagglutination inhibition (HI) assay was performed according to the protocols of the WHO [3] with 0.75% guinea pig red blood cells in U-bottom plates (U96 MicroWell Plates, Nunc) and the HI titers were read as the reciprocal of the last dilution of sera that completely inhibited haemagglutination.

[0052] Results From Rabbits Vaccinated With Influenza DNA Vaccine

[0053] ELISA assay demonstrated high IgG specific serum antibodies against the vaccine components A/California/04/09(H1N1) and A/Aichi/2/1968(H3N2). Specific IgG were observed two weeks post 1st vaccination in all vaccinated rabbit groups, to similar titers (FIG. 1). After 2nd vaccination at week three, the specific IgG titers increased further. It is possible that the antibody titers could have increased further at later time points, but the experiment was terminated after five weeks for practical reasons. Haemagglutination inhibition assay measures how well sera from vaccinated rabbits inactivate virus binding to red blood cells. HI antibody titers >40 corresponds to seroprotection rate after vaccination [4], and this was achieved in rabbit sera of all groups against homologous vaccine virus A/California/04/09(H1N1) after 2nd vaccination (FIG. 2a). To determine the cross reactivity obtained by using pandemic genes as DNA vaccine, we also tested HI titers against heterologous swine virus strain H1N1-2007 (FIG. 2b). All vaccinated rabbit groups did develop cross-reactive IgG against the antigentically different swine H1N1 after vaccination, but only to low HI titers. Heterologous human influenza strain H3N2 (A/Perth/16/2009) was also tested in the HI assay (FIG. 2c), however no vaccine-induced inhibition was observed for this strain in any rabbit group.

[0054] Conclusion

[0055] The data show that DNA immunizations with pandemic genes from three different virus strains (H1N1 2009, H1N1 1918, H3N2 1968) induce strong specific antibody response as detected in ELISA. The haemagglutination inhibition assay demonstrates that the vaccine also induce functional antibodies against both homologous virus strain to the vaccine and against heterologous swine influenza strain. The different mode of delivery gave similar immunological response. Diluting the plasmid DNA in Diluvac®Forte and deliver needle-free into the skin resulted in equal immune response as the present state of the art methodology with electroporation, but is much more user- and animal-friendly. The mix of DNA vaccine with the adjuvant Diluvac®Forte was possible with several different plasmid backbones and resulted in similar immunological response. The mixing to of naked DNA with Diluvac®Forte resulted in a homogenous solution suitable for the needle-free delivery without any precipitation of DNA.

EXAMPLE 2

[0056] Pigs Vaccinated With Influenza DNA Vaccine

[0057] Three groups (group 3-5) of 5-6 pigs were immunized twice (see arrows), three weeks apart, with different doses of the same DNA based influenza vaccine consisting of HA+NA genes from H1N1pdm09, HA+NA genes from 1968 H3N2 and NP+M genes from 1918 H1N1 viruses. Two groups were used as control groups; group 1 received nothing and group 2 received only the α-tocopherol-based aqueous solution (Diluvac Forte®, MSD Animal Health) which the vaccine constructs were premixed with for practical use of the IDAL® delivery device. All three vaccinated groups (group 3-5) developed detectable serum IgG responses measured by ELISA against recombinant HA and NA protein, homologous to the DNA vaccine (FIG. 3a-c). In addition, the three vaccinated groups also developed serum IgG responses against vaccine non-homologous proteins; rec. HA, rec. NP and the M2e ectodomain peptide (FIG. 3d-f). HI antibody responses were measured against the human isolates H1N1v A/California/07/09 and H3N2 A/Aichi/02/68, homologous to the vaccine, (FIGS. 4a and b). HI antibody responses were also measured against two isolates from swine, H1N2 A/swine/DK/10525/2008 and H1N1v A/swine/DK/10409/2013 (FIGS. 4c and d). Neutralizing activity against the human isolates H1N1v A/California/07/09, homologous to the vaccine, were observed in animals from vaccinated group 3-5, at 2 weeks after 2nd vaccination, day 35 (FIG. 5a). The kinetics of the neutralizing response was tested for two pigs, receiving different doses of vaccine (FIG. 5b). The neutralizing response seem to follow the development of specific IgG detected in ELISA (FIG. 3a).

[0058] Maternal IgG seem to exist at day 0 at the 1st vaccination time point. These vain over time, but a 2nd vaccination at day 21 can boost the neutralizing response.

[0059] In summary, the influenza DNA vaccine has induce a serum IgG response against both influenza proteins homologous to the vaccine and influenza proteins not expressed by io the vaccine, as measured by ELISA (see group 3-5). The antibody titers seem to correlate with the doses of the vaccine given to the different groups. The control groups, which did not receive the DNA vaccine (group 1 and 2), did not develop any influenza specific IgG response. Influenza DNA vaccinated animals develop a functional antibody response against virus isolates both homologous to the vaccine and non-homologous. Higher HI titer seem to correlate with animals receiving higher doses of the vaccine. The control groups (group 1 and 2) has in general no or low response.

REFERENCES

[0060] 1. Williams, J. A., Vector Design for Improved DNA Vaccine Efficacy, Safety and Production. Vaccines, 2013. 1(3): p. 225-249. [0061] 2. Carnes, A. E. W., J. A., Process for plasmid DNA fermentation. U.S. Pat. No. 7,943,377, 2011. [0062] 3. WHO Recommendations for Influenza Vaccine Composition [http://www.who.int/influenza/vaccines/virus/en/]. [0063] 4. Coudeville, L., et al., Relationship between haemagglutination-inhibiting antibody titres and clinical protection against influenza: development and application of a bayesian random-effects model. BMC Med Res Methodol, 2010. 10: p. 18.