NUCLEOTIDE VACCINE

Abstract

The present invention relates to a vaccine comprising a nucleic acid construct such as a DNA construct especially a nucleic acid construct comprising sequences encoding invariant chain operatively linked to antigenic protein or peptide encoding sequences. The vaccine stimulates an immune response, especially an immune response in an MHC-I dependent, but CD4.sup.+ T-cell independent manner.

Claims

1-185. (canceled)

186. An adenoviral vector comprising a nucleotide construct encoding: (a) at least one antigen derived from hepatitis C virus and (b) at least one invariant chain, wherein the invariant chain is directly linked to the at least one antigen by the last amino acid residue of the invariant chain sequence.

187. The adenoviral vector according to claim 186, wherein the at least one invariant chain is of mammalian origin.

188. The adenoviral vector according to claim 187, wherein the at least one invariant chain is of human origin.

189. The adenoviral vector according to claim 186, wherein the encoded at least one invariant chain is at least 85% identical to SEQ ID NO: 2.

190. The adenoviral vector according to claim 189, wherein the encoded at least one invariant chain is identical to SEQ ID NO: 2.

191. The adenoviral vector according to claim 186, wherein the adenoviral vector is derived from a simian source.

192. The adenoviral vector according to claim 186, wherein the adenoviral vector is Modified vaccinia Ankara (MVA).

193. The adenoviral vector according to claim 186, wherein the adenoviral vector is MVA-BN.

194. The adenoviral vector according to claim 186, wherein the adenoviral vector is a replication defective adenovirus or a conditionally replication defective adenovirus.

195. A method for inducing an immune response in an animal, comprising administering to the animal an adenoviral vector according to claim 186.

Description

DETAILED DESCRIPTION OF THE DRAWINGS

[0197] FIG. 1: Schematic drawing of inserts in the adenovirus vector. A) Schematic drawing of Ad-GP expression cassette, B) Schematic drawing of Ad-IiGP expression cassette. Shown is also the situation of various LCMV GP epitopes. Ad-GP: adenoviral-glycoprotein, CMV: Cytomegalovirus promoter, Ii: Invariant chain. LCMV GP 1-498: Glycoprotein from lymfocytic choriomeningitis virus, STOP: Stop codon, PolyA: SV40 polyadenylation signal

[0198] FIG. 2: CD8.sup.+ and CD4.sup.+ T-cell responses to adenovirus encoded epitopes. C57BL/6 mice were vaccinated with 2×10.sup.7 infectious unit (IFU) of Ad-GP or Ad-IiGP in the right hind footpad. On the indicated days post vaccination the number of epitope specific CD8.sup.+ or CD4.sup.+ T cells were determined by intracellular staining for peptide-induced IFN-γ of spleen cells. Bars represent Average (Avg)±standard deviation (SD) of 3-5 animals.

[0199] FIG. 3: CD8.sup.+ and CD4.sup.+ T-cell responses to adenovirus encoded epitopes in F.sub.1 hybrid mice. C57BL/6×BALB/c (H-2.sup.bxd) F.sub.1 mice were vaccinated with 2×10.sup.7 IFU of Ad-GP or Ad-IiGP in the right hind footpad. On day 21 post vaccination the number of epitope specific CD8.sup.+ or CD4.sup.+ T cells were determined by intracellular staining for peptide-induced IFN-γ of spleen cells. Bars represent Avg±SD of 4-5 animals

[0200] FIG. 4: Ad-IiGP exerts CD8.sup.+ T-cell stimulatory effects that are independent of CD4.sup.+ T-cells. MHC-II.sup.−/− or C57BL/6 mice were vaccinated with 2×10.sup.7 IFU of Ad-GP or Ad-IiGP in the right hind footpad. On day 21 or 90 post vaccination the number of epitope specific CD8.sup.+ or CD4.sup.+ T cells were determined by intracellular staining for peptide-induced IFN-γ of spleen cells. Bars represent Avg±SD of 4 animals.

[0201] FIG. 5: Ad-IiGP confers rapid and superior protection against lethal LCMV infection. C57BL/6 mice were vaccinated with 2×10.sup.7 IFU of Ad-GP or Ad-IiGP in the right hind footpad. On the indicated days post vaccination animals were challenged with 20 pfu (plaque forming units) LCMV Arm 53b i.c. (intra cerebral). Mortality was recorded for 14 days. Each group consisted of 5 to 18 animals. ND means no data.

[0202] FIG. 6: Ad-IiGP efficiently protects against high-dose, intravenous LCMV infection. C57BL/6 mice were vaccinated with 2×10.sup.7 IFU of Ad-GP or Ad-IiGP in the right hind footpad. On day 21 post vaccination animals were challenged with 1×10.sup.6 pfu (plaque forming units) LCMV Arm clone13 i.v. (intra venous). 8 days after virus challenge organ virus titer was determined. Points represent individual animals. Dashed line represent detection limit of the assay.

[0203] FIG. 7: Ad-IiGP confers superior protection to lethal LCMV variants with mutations in immunodominant epitopes. C57BL/6 mice were vaccinated with 2×10.sup.7 IFU of Ad-GP, Ad-IiGP or sham infected in the right hind footpad. On day 90 post vaccination animals were challenged with 20 pfu LCMV Arm 53b i.c. carrying mutations in gp33, gp276 or both epitopes. Mortality was recorded for 14 days. For gp33 nil and gp276 nil, each group consisted of 5 animals, with gp33/gp276 double nil, the groups were 10 animals.

[0204] FIG. 8: Frequencies of CD8.sup.+ or CD4.sup.+ T cells reacting to specific LCMV epitopes after Ad-IiGP vaccination and challenge with LCMV variants with mutations in immunodominant epitopes. Surviving animals from the experiment depicted in FIG. 7 were analysed for epitope specific CD8.sup.+ or CD4.sup.+ T cells by intracellular staining for peptide-induced IFN-γ of spleen cells. Bars represent Avg±SD of 3-5 animals.

[0205] FIG. 9: CD8.sup.+ and CD4.sup.+ T cll responses to vaccination with naked DNA-IiGP and DNA-GP. C57BL/6 mice were vaccinated with DNA coated onto 1.6-nm gold particles in a concentration of 2 μg DNA/mg gold, and the DNA-gold complex was coated onto plastic tubes such that 0.5 mg gold was delivered to the mouse per shot (1 μg DNA per shot). Mice were immunized at the abdominal skin using a hand-held gene gun device employing compressed helium (400 psi) as the particle motive force. Mice were inoculated four times with an interval of 1 week and then allowed to rest for 1 week before investigation. The number of epitope specific CD8.sup.+ or CD4.sup.+ T cells was determined by intracellular staining for peptide-induced IFN-γ of spleen cells. Bars represent Avg±SD of 4-5 animals.

[0206] FIG. 10: Prophylactic vaccination with Ad-Ii-GP increases tumor rejection. C57BL/6 mice were vaccinated in the right hind foot-pad with 2×10.sup.7 IFU of adenovirus encoding either full-length glycoprotein of LCMV (Ad-GP) or glycoprotein linked to invariant chain (Ad-Ii-GP). Controls were infected with either adenovirus encoding full-length β-galactosidase or live LCMV (10.sup.3 PFU of LCMV Armstrong 53b). About 3 months later all mice were challenged by subcutaneous injection of 10.sup.6 B16.F10 melanoma cells expressing the LCMV derived GP33 epitope. Initially a tumor was formed in all animals, but the majority of Ad-Ii-GP and LCMV primed mice eventually rejected the tumor. Each group consisted of 7-10 animals.

[0207] FIG. 11: Therapeutic vaccination with Ad-Ii-GP increases average life span in tumor carrying mice. C57BL/6 mice were challenged subcutaneously by injection of 10.sup.6 B16.F10 melanoma cells expressing the LCMV derived GP33 epitope. When tumors were palpable in all mice (day 5 after tumor injection), the animals were vaccinated in the right hind foot-pad using 2×10.sup.7 IFU of adenovirus encoding either full-length glycoprotein of LCMV (Ad-GP) or full-length glycoprotein of LCMV linked to invariant chain (Ad-Ii-GP); controls were vaccinated with either adenovirus encoding full-length β-galactosidase or live LCMV (10.sup.3 PFU of LCMV Armstrong 53b). Mice were sacrificed when the tumor exceeded 12 mm in length or ulceration was observed. The numbers in bold in the center of the figure represents the mean day of death following the tumor challenge. Each group consisted of 7-10 animals.

[0208] FIG. 12: Survival rate following vaccination with either Ad-Ii-VSVGP or Ad-VSVGP. C57BL/6 mice were vaccinated in the right hind foot-pad with 2×10.sup.7 IFU of adenovirus encoding either full-length glycoprotein of vesicular stomatitis virus (Ad-VSVGP) or full-length glycoprotein of vesicular stomatitis virus linked to invariant chain (Ad-Ii-VSVGP). (A) On the indicated days serum samples were collected and in vitro neutralizing antibody titers were determined in a plaque-reduction assay, dots represent individual animals. (B) On the indicated days vaccinated mice were challenged with 10.sup.5 PFU of VSV intranasally, and mortality was registered over the next 14 days. Survival of control (unvaccinated) mice has been included for comparison. Each group consisted of 5-10 animals

[0209] FIG. 13: CD8.sup.+ and CD4.sup.+ T-cell responses to more adenovirus encoded epitopes. C57BL/6 mice (Influenza and OVA) or B6D2 F.sub.1 mice (MHV-68 M2 and M3) were vaccinated with 2×10.sup.7 IFU of the indicated construct in the right hind footpad. On the indicated days the number of epitope specific CD8+ or CD4+ T cells was determined by intracellular staining for peptide-induced IFN-γ of spleen cells. Iso is the isotype control which determined the background. Bars represent Avg±SD of 4-5 animals.

[0210] FIG. 14: Efficiency of Ad-Ii-GP constructs compared to Ad-GP-Lamp-1 constructs measured by CD8.sup.+ T-cell responses to adenovirus encoded epitopes. C57BL/6 mice were vaccinated with 2×10.sup.7 IFU of the indicated construct in the right hind footpad. On day 21 the number of epitope specific CD8+ cells was determined by intracellular staining for peptide-induced IFN-γ of spleen cells. Bars represent Avg t SD of 3 animals.

[0211] FIG. 15: Vectors based on in-frame polylinkers. Ii without stop-codon was amplified by PCR with the sequence for the AsiSI, SwaI, AscI, PmeI, FseI and a stop site included in the 3′ primer and cloned into the pacCMV vector. The resulting vector was numbered 770 (see partial sequence hereof in SEQ ID NO: 7). A corresponding vector termed 768 (see partial sequence hereof in SEQ ID NO: 8) without the Ii sequence incorporated was also constructed.

[0212] FIG. 16: Vectors with IRES2 sites. The vector termed “pacCMV Ii MCS IRES2” (and numbered 1163, see partial sequence hereof in SEQ ID NO: 9) was constructed by cloning the IRES2 from pLP-IRES2-EGFP into the 770 vector by PCR and restriction enzyme digestion. The sequences for I-sceI and SrfI were included in the 3′ primer. The first ATG site after the IRES sequence initiates expression of a second protein. A corresponding vector termed “pacCMV MCS IRES2” and numbered 1165 (see partial sequence hereof in SEQ ID NO: 10) without the Ii sequence incorporated was also constructed

EXAMPLES

[0213] The invention will now be further illustrated with reference to the following examples. It will be appreciated that what follows is by way of example only and that modifications in detail may be made while still falling within the scope of the invention.

Example 1

[0214] CD8.sup.+ and CD4.sup.+ T-cell responses to adenovirus encoded epitopes.

[0215] Mice: C57BL/6 (H-2b), C57BL/6×BALB/c (H-2bxd) F1 hybrids and MHC II−/− mice (B6.129-H2-Ab1.sup.tm1GlmN12 (H-2b)) were obtained from Taconic M&B (Ry, Denmark). All mice used were between 7-10 weeks old and housed in a specific germ free facility. All experimental procedures were performed according to local experimental guidelines.

[0216] Adenovirus vectors: For construction of E1 and E3 deleted adenovirus-expressing, LCMV derived antigen fused to invariant chain we performed 2-step PCR. First we obtained overlapping PCR products containing the full-length mouse invariant chain and full-length LCMV glycoprotein and these were joined by secondary PCR with invariant chain 5′ and glycoprotein 3′ primers. Adenovirus expressing full-length GP was amplified in single step PCR. The obtained fragments were cloned into the pacCMV shuttle vector. The obtained plasmid was co-transfected with pJM17 plasmid into HEK293 cells and viral lysates were obtained. These were cloned by plaque purification before sequencing, large-scale production and purification by CsCl gradient centrifugation as described (Becker et al., 1994. Methods Cell Biol. 43 Pt A:161-189). Infectivity of adenovirus stocks was determined with the Adeno-X Rapid Titer Kit (Clontech). All unmodified virus stocks had particle/IFU ratios between 46 and 201.

[0217] Vaccinations: In all studies, mice to be vaccinated were anaesthetized and injected with 2×10 infectious units in the right hind footpad in a volume of 0.03 ml.

[0218] Virus infection: Mice were infected i.c. with 20 pfu of LCMV Armstrong clone 53b in a volume of 0.03 ml or i.v. with 10.sup.8 pfu of LCMV clone 13 in 0.3 ml. I.c. Infection induces a fatal CD8.sup.+ T cell-mediated meningitis from which immunocompetent mice succumb on days 7 to 10 p.i. (post infection) (Christensen et al., Scandinavian Journal of Immunology 40:373-382).

[0219] Survival study: Mortality was used to evaluate the clinical severity of acute LCMV-induced meningitis. Mice were checked twice daily for a minimum of 2 weeks after i.c. inoculation; deaths occurring less than 3 days after infection were excluded from analysis.

[0220] Organ virus titers: To determine virus titers in organs, these were first homogenized in PBS to yield 10% (v/w) organ suspensions, and serial 10-fold dilutions were prepared. Each dilution was then plated in duplicates on MC57G cells. Forty-eight hours after infection, infected cell clusters were detected using monoclonal rat anti-LCMV (VL-4) antibody, peroxidase-labeled goat anti-rat antibody and o-phenylendiamin (substrate) (Battegay et al., 1991, Journal of Virological Methods 33:191-198). The numbers of pfu were counted, and results expressed as pfu/g tissue.

[0221] Cell preparations: Single cell suspensions of spleen cells were obtained by pressing the organs through a fine steel mesh.

[0222] Abs for flow cytometric: The following monoclonal antibodies (mAbs) were purchased from BD PharMingen (San Diego, Calif.) as rat anti-mouse antibody: Cy-chrome-conjugated anti-CD8, FITC-conjugated anti-CD44, phycoerythrin (PE)-conjugated anti-IFN-γ and PE-conjugated IgG.sub.1 isotype standard.

[0223] Flow cytometric analysis: Staining of cells for flow cytometry was performed according to standard laboratory procedure (Andersson et al., 1994. Journal of Immunology 152:1237-1245; Andreasen et al., 1999, International Immunology 11:1463-1473). For enumeration of LCMV-specific CD8.sup.+ T cells, splenocytes were incubated in vitro for 5 h at 37° C. in 5% CO with relevant peptide (0.1 μg/ml) in the presence of monensin (3 μM Sigma Chemicals co., St. Louis, Mo.) and murine recombinant IL-2 (10 units/well, R&D Systems Europe Ltd, Abingdon, UK). After incubation cells were surface stained, washed, fixed and permeabilized using 0.5% saponin. Cells were then stained with anti-IFN-γ or IgG.sub.1 isotype control for 20 min at 4° C. Samples were analyzed using a Becton Dickinson FACSCalibur, and at least 104 mononuclear cells were gated using a combination of low angle and side scatter to exclude dead cells and debris. Data analysis was conducted using Cell Quest Pro (B&D Biosciences).

[0224] Replication deficient adenovirus vectors expressing lymphocytic choriomeningitis virus full-length glycoprotein (Ad-GP), or lymphocytic choriomeningitis virus full-length glycoprotein N-terminally linked to murine invariant chain ((Ad-IiGP) for schematic representation of the expression cassette see FIG. 1), were generated through standard methods (Becker et al., Methods Cell Biol. 43 Pt A:161-189). C57BL/6 mice were then vaccinated in the right hind paw with 2×10.sup.7 infectious units of Ad-GP or Ad-IiGP and mice were sacrificed 5, 7, 11, 14, 21, 28, 90, 180 or 360 days later. The generation of LCMV glycoprotein specific T-cells were then analysed on splenic cells. Evidently (see FIG. 2), at all time points tested, Ad-IiGP induced numerically superior T-cell responses compared to Ad-GP, and these were accelerated and included both CD4.sup.+ and CD8.sup.+ T-cell responses. Thus peak numbers of T-cells generated after Ad-IiGP vaccination were obtained at 7-14 days after vaccination depending on the epitope, with responses after Ad-GP peaking at day 21 after vaccination.

Example 2

[0225] CD8.sup.+ and CD4.sup.+ T-cell responses to adenovirus encoded epitopes in F.sub.1 hybrid mice: As C57BL/6 mice are homozygous with regard to both MHC class I and MHC class II molecules on all loci, we tested whether Ad-GP and Ad-IiGP could also induce an immune response in C57BL/6×BALB/c F.sub.1 mice that express both the H-2.sup.b and H2.sup.d haplotypes. These mice resemble an out bred population, but with defined haplotypes. The experiments were performed as above, but testing was limited to day 21 after vaccination. As can be seen from FIG. 3, Ad-IiGP efficiently induces CD8.sup.+ T-cell responses towards a multitude of epitopes while Ad-GP seemed to perform worse than in homozygous C57BL/6 mice.

Example 3

[0226] Ad-IiGP exerts CD8.sup.+ T-cell stimulatory effects that are independent of CD4.sup.+ T-cells: As a potential mechanism of Ii function in the enhanced stimulation of CD8.sup.+ T-cells is the ability to traffic to endosomal and lysosomal compartments and stimulate CD4.sup.+ T-cells (Diebold et al., 2001, Gene Ther. 8:487-493) through MHC class II, we performed vaccination of MHC class II deficient mice. To this effect MHC-II or C57BL/6 mice were vaccinated with 2×10.sup.7 IFU of Ad-GP or Ad-IiGP in the right hind footpad. On day 21 or 90 post vaccination the number of epitope specific CD8.sup.+ or CD4.sup.+ T cells were determined by intracellular staining for peptide-induced IFN-γ of spleen cells. As can be seen from FIG. 4, Ad-IiGP efficiently induces CD8.sup.+ T-cell responses directed against several epitopes; in the absence of CD4.sup.+ T cell help however, some responses were lower than what is seen in wild type mice.

Example 4

[0227] Ad-IiGP confers rapid, superior and sustained protection against lethal LCMV infection: As we observed an accelerated response to Ad-IiGP compared to Ad-GP we investigated the ability of vaccination to confer protection both at 21 days post vaccination (peak of Ad-GP) and at 3, 5, 7, 14, 60, 90, 180 and 360 days post vaccination (FIG. 5). Remarkably, we found that Ad-IiGP vaccinated animals vaccinated as little as 3 days previously were protected against intracerebral LCMV infection. Protection conferred by Ad-GP was only partial at 14 and 21 days post infection and no protection were seen at day 60 or later. Furthermore, the Ad-IiGP conferred protection was sustained for 360 days, at which point Ad-GP no longer protected against intracerebral LCMV infection.

Example 5

[0228] Ad-IiGP efficiently protects against high-dose, intravenous LCMV infection: Since we found that Ad-IiGP protected mice against an acute localised infection, we wanted to investigate whether the same held true for a high-dose systemic infection. Accordingly, mice were vaccinated with Ad-GP, Ad-IiGP or sham (PBS), and challenged 21 days later by intravenous injection of 10.sup.6 plaque-forming units of the fast replicating LCMV clone 13 strain. 5 days later animals were sacrificed and infectious titers in the lungs were determined. Although Ad-GP conferred significant protection upon vaccinated animals, Ad-IiGP was superior and reduced titers to a level at or below the detection limit of the assay (FIG. 6).

Example 6

[0229] Comparative analyses of novel constructs with respect to kinetics, magnitude of response, long-term immunity and virus dose needed for immunity.

[0230] All constructs described below are comparatively analysed among each other and to Ad-GP and Ad-IiGP with respect to the kinetics and magnitude of immune response e.g. as described in Examples 1, 2 and 3, long-term immunity e.g. as described in Examples 1, 4 and 5, and virus dose needed for immunity.

[0231] Novel constructs based upon alterations to the Ii fusions. In each construct adenovirus-encoded GP is fused N-terminally to: [0232] 1. the lysosomal targeting sequence of Ii [0233] 2. the CLIP sequence of Ii [0234] 3. the KEY sequence of Ii [0235] 4. the CLIP sequence and the sequence N-terminally adjacent to the CLIP sequence [0236] 5. the CLIP sequence and the sequence C-terminally adjacent to the CLIP sequence [0237] 6. the sequence N-terminally adjacent to the CLIP sequence [0238] 7. the sequence C-terminally adjacent to the CLIP sequence

[0239] Alterations of antigen presentation context. In each construct adenovirus-encoded GP is fused: [0240] 1. C-terminally to LAMP [0241] 2. N-terminally to the N-domain of calreticulin [0242] 3. C-terminally to the N-domain of calreticulin [0243] 4. C-terminally to Hsp70. [0244] 5. N-terminally to Ii and C-terminally to the N-domain of calreticulin (AdIiGPCrt) [0245] 6. N-terminally to Ii and C-terminally to Hsp70 (AdIiGPHsp70)
All of these constructs are furthermore used as the starting point for a series of constructs in which the GP-fusions are followed by an internal ribosomal entry site (IRES) and a gene encoding VP22, HIV tat or Cx43.

[0246] Alterations with regard to intercellular spreading. In each construct adenovirus-encoded GP is fused: [0247] 1. N-terminally to herpes simplex virus encoded VP22 [0248] 2. N-terminally to HIV encoded tat [0249] 3. N-terminally to connexin 43 (Cx43) [0250] 4. N-terminally to other connexins and intercellular gap-junctions constituents.
Furthermore constructs are prepared where adenovirus-encoded GP is followed by an internal ribosomal entry site (IRES) and a gene encoding VP22, HIV tat or Cx43 or other connexins and intercellular gap-junctions constituents.

[0251] All of the above constructs are furthermore altered in any of the following ways: [0252] 1. All of the above constructs followed by an IRES site and a gene encoding an NK-cell (natural killer cell) activation molecule, for example H60. This alteration gives enhanced delivery of co-stimulatory signals and cytokine help. [0253] 2. All of the above constructs involving IRES sites, where the downstream gene is placed under control of a separate promoter. [0254] 3. All of the above constructs involving IRES sites, where the downstream gene is instead encoded on a separate vector. [0255] 4. All of the above constructs placed under inducible promoter systems. [0256] 5. All of the above constructs placed under cell type specific and/or inducible promoter systems. [0257] 6. All of the above constructs where the GP antigenic sequence is replaced with a sequence encoding any of the following antigens, several of which comprise multiple antigens, see examples of specific antigens in FIGS. 12 and 13: VSV-GP, Influenza A NS-1, Influenza A M1, Influenza A NP, LCMV NP, LCMV GP, Ebola GP Ebola NP, murine gammaherpesvirus (MHV-68) M2, M3 (this corresponds to the human EBV and HHV8 viruses) and ORF73, chicken Ovalbumin (OVA), or a helper T-cell epitope. These antigenic sequences will furthermore be combined so at least 2 or more are encoded in the same vector.

[0258] See FIG. 14 for an example of a comparison of the efficiency of Ad-Ii-GP pg33 and gp276 constructs compared to Ad-GP-Lamp-1 pg33 and gp276 constructs as measured by CD8+ T-cell responses. As can be seen, the Ad-Ii-GP constructs are superior to the Ad-GP-Lamp-1 constructs in their capability of evoking a CD8+ T-cell response.

Example 7

[0259] Comparative analyses of novel constructs and alternative administration methods with respect to kinetics, magnitude of response, long-term immunity and virus dose needed for immunity.

[0260] All constructs described in Example 6 are comparatively analysed among each other and to Ad-GP and Ad-IiGP following alternative administration methods. The comparisons are done with respect to the kinetics and magnitude of immune response e.g. as described in Examples 1, 2 and 3, long-term immunity e.g. as described in Examples 1, 4 and 5, and virus dose needed for immunity.

[0261] Alternative administration methods include: [0262] 1. Alterations with regard to enhanced delivery of co-stimulatory signals and cytokine help in which all of the in Example 6 described constructs are co-injected with adenovirus encoded type 1 interferon, for example tetracycline inducible IFN-β. Furthermore, all the in Example 6 described constructs are co-injected with adenovirus encoded cytokine, for example IL-15. [0263] 2. Administration of Ad-IiGP simultaneously with Ad-Tet-onGP at separate sites of the body (Ad-Tet-onGP encodes GP under control of a tetracycline inducible promoter). [0264] 3. Adenovirally delivery of any one of the in Example 6 described inserts/constructs followed by homologous viral vector boosting with the same insert/construct or followed by heterologous viral vector boosting with lentivirus encoded or other adenovirus-encoded delivery of the same insert/construct.

Example 8

[0265] Ad-IiGP confers rapid and superior protection against lethal LCMV infection in absence of major epitopes. Lymphocytic choriomeningitis virus full-length glycoprotein (GP) comprises four CD8.sup.+ specific epitopes of varying antigenicity, measured by the percentile of CD8.sup.+ cells with specificity for the individual epitope against all the CD8.sup.+ cells raised in response to GP vaccination. The predominant population of CD8.sup.+ cells raised against GP is specific for the gp33 epitope, a somewhat smaller population is specific for gp276, and minor populations are specific for gp118 and for gp92. As gp33 and gp276 are the major/immunodominant epitopes, we investigated how nil mutations of either epitope independently or both simultaneously would effect the efficiency of the protection offered by the Ad-IiGP fusion construct.

[0266] C57BL/6 mice were vaccinated with 2×10.sup.7 IFU Ad-GP and Ad-IiGP or sham infected in the right hind footpad. Each group consisted of 5 animals. On day 90 post vaccination, the animals were challenged with 20 pfu LCMV Arm 53b i.c. (intra cerebral) constructs carrying gp33 nil mutations, gp276 nil mutations or gp33/gp276 double nil mutations. Mortality was recorded for 14 days. As can be seen from FIG. 7, Ad-IiGP conferred superior protection against lethal LCMV infection despite the gp33 or gp276 nil mutations. The double nil mutation of gp33/gp276 lead to the survival of 70% of the animals compared to no surviving animals in the Ad-GP and Sham vaccinated groups.

[0267] Surviving animals from the experiment above were analyzed for epitope specific CD8.sup.+ or CD4.sup.+ T cells by intracellular staining for peptide-induced IFN-γ of spleen cells. Expectedly, as can be seen from FIG. 8, in the absence of either gp33 or gp276, the major epitope specificity of the CD8.sup.+ or CD4.sup.+ T cells is gp276 or gp33, respectively. In the absence of both gp33/gp276, the major epitope specificity of the CD8.sup.+ or CD4.sup.+ T cells is gp92.

Example 9

[0268] CD8.sup.+ and CD4.sup.+ T cell responses to vaccination with naked DNA-IiGP and DNA-GP. To investigate an alternative platform for invariant chain fusion vaccines other than adenoviral delivery, we tested the ability of IiGP and GP as naked DNA to raise GP-epitope specific CD8.sup.+ and CD4.sup.+ T cells. The naked DNA comprised the GP and Ii-GP fragments from the Adenoviral vectors illustrated in FIG. 1.

[0269] C57BL/6 mice were vaccinated with DNA coated onto 1.6-nm gold particles in a concentration of 2 μg DNA/mg gold, and the DNA-gold complex was coated onto plastic tubes such that 0.5 mg gold (1 μg DNA per shot) was delivered to the mouse per shot. Mice were immunized at the abdominal skin using a hand-held gene gun device employing compressed helium (400 psi) as the particle motive force. Mice were inoculated four times with an interval of 1 week and then allowed to rest for 1 week before investigation. The number of epitope specific CD8.sup.+ or CD4.sup.+ T cells were determined by intracellular staining for peptide-induced IFN-γ of spleen cells. As can be seen from FIG. 9, DNA-IiGP efficiently induces CD8.sup.+ T-cell responses directed against several epitopes.

Example 10

[0270] Ad-IiGP confers superior protection against challenge with tumor cells expressing the gp33 epitope from LCMV. Under normal circumstances, tumor cells express several different antigens recognized by T cells. To determine the efficiency of this response, B16.F10 melanoma cells expressing gp33 from LCMV were used to challenge vaccinated animals with.

[0271] C57BL/6 mice were vaccinated with 2×10.sup.7 IFU Ad-GP or Ad-IiGP in the right hind footpad. As a control some animals were vaccinated with Ad-β-galactosidase (negative control) or infected with LCMV (positive control). On day 90 after vaccination/infection animals were challenged with 10.sup.6 tumor cells subcutaneously and the tumor growth was followed by measuring the size of the tumor. Initially a tumor will form in all animals, but eventually the immune response directed towards gp33 will eliminate the tumor cells. Each group consisted of 7-10 animals. Prophylactic vaccination with Ad-Ii-GP resulted in tumor free mice in 70% of the cases compared to only 10% in Ad-GP vaccinated animals, as can be seen from FIG. 10.

[0272] In many cases the tumor has already formed when a physician sees the patient. To mimic this situation. C57BL/6 mice were injected with 10.sup.8 B16.F10 tumor cells subcutaneously. After 5 days the tumors were palpably recognizable and could be measured. At this time point animals were vaccinated with 2×10.sup.7 IFU Ad-GP or Ad-IiGP in the right hind footpad. As a control some animals were vaccinated with Ad-β-galactosidase (negative control) or infected with LCMV (positive control). Tumor growth was followed by measuring the size of the tumor, and once the size was greater that 12 mm in any dimension the animals were sacrificed. As can be seen from FIG. 11, in mice given the therapeutic Ad-IiGP vaccine, the speed of tumor development was approximately half of that seen in mice given Ad-GP as therapeutic vaccine, as measured by number of days passing prior to reaching a 12 mm tumor size and animal sacrifice.

Example 11

[0273] To demonstrate that the vaccine construct also can induce protecting antibody response the VSV infection was used.

[0274] Virus and virus quantitation: Vesicular stomatitis virus (VSV) of the Indiana serotype was used throughout this study. Stocks of virus were propagated in L929 cells (ATCC CCL 1) and stored at −70° C. until use. Virus quantitation was performed by plaque assay on monolayers of L929 cells. In brief, serial 10-fold dilutions of virus were prepared in Eagle's minimal essential medium (F11) containing 1% L-glutamine, 1% penicillin/streptomycin, 5% NaHCO.sub.3 and 10% fetal calf serum. One ml of each dilution was added in duplicate to monolayers of L929 cells in petri dishes plated 48 hours earlier. After incubation for 90 min at 37° C. in 5% CO.sub.2, medium containing the virus dilutions was aspirated, and the monolayers were overlaid with a mixture of 2.5 ml 1% agarose and 2.5 ml 2×F11. Monolayers were then incubated for 24 hours at 37° C. in 5% CO.sub.2 before staining with a mixture of 1 ml of 1% agarose and 1 ml of 2×F11 containing 1% neutralred. After further 24 hours of incubation, the numbers of PFU were counted.

[0275] Survival study: Mortality was used as parameter for the severity of VSV infection, based on previous findings that virus titer in CNS correlate strongly with clinical symptoms (Thomsen et al. 1997, Int. Immunol. 9:1757-1766.). Mice were inspected daily for signs of VSV-induced paralysis, and sacrificed when severe paralysis was noted and the animals expected to succumb within the next 24 hours.

[0276] Serum neutralizations test: Serial two-fold dilutions of serum in F11 were mixed with equal volumes of virus diluted to contain approximately 100 PFU/ml. After 1 h of incubation at room temperature, 1 ml of each serum-virus mixture was added in duplicate to monolayers of L929 cells in petri dishes and assayed for the presence of residual virus by plaque assay (see virus and virus quantitation). The highest serum dilution that reduced the number of plaques by at least 50% was taken as the neutralizing titer.

[0277] C57BL/6 mice were vaccinated in the right hind foot-pad with 2×10.sup.7 IFU of adenovirus encoding either full-length glycoprotein of vesicular stomatitis virus (Ad-VSVGP) or glycoprotein linked to invariant chain (Ad-Ii-VSVGP). On day 7, 14, 21 and 110 after vaccination serum samples were collected and in vitro neutralizing antibody titers were determined in a plaque-reduction assay, FIG. 12a. On day 3, 7, 14, 21 and 110 after vaccination, animals were challenged with 10.sup.5 PFU of VSV intranasally, and mortality was registered over the next 14 days, FIG. 12b. As seen in FIG. 12 almost identical responses are seen in the two groups, suggesting that, although not an advantage, the invariant chain allows for antibody production and does not have a detrimental effect on the production.

REFERENCE LIST

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