Poxviral vaccines

Abstract

The present application relates to novel administration regimens for poxviral vectors comprising nucleic acid constructs encoding antigenic proteins and invariant chains. In particular the use of said poxviral vectors for priming or for boosting an immune response is disclosed.

Claims

1. A Modified Vaccinia Ankara virus comprising a nucleic acid construct for use in priming or boosting an immune response, the nucleic acid construct comprising: (i) a nucleic acid sequence encoding at least one antigenic protein or antigenic fragment thereof operatively linked to (ii) a nucleic acid encoding at least one invariant chain.

2. The Modified Vaccinia Ankara virus according to claim 1, wherein the at least one encoded invariant chain is of mammalian origin.

3. The Modified Vaccinia Ankara virus according to claim 1, wherein the encoded at least one invariant chain is characterized by at least one of the following features: (i) the endogenous KEY-region is deleted or substituted by a different sequence; (ii) the methionine in positions 107 and 115 (human invariant chain) or in positions 90 and 98 (murine invariant chain) or the positions corresponding thereto in another invariant chain is substituted by another amino acid; (iii) at least one sorting peptide is added to, removed from or replaces the endogenous sorting peptide of the invariant chain, and/or (iv) at least one CLIP region is added to, removed from or replaces the endogenous CLIP region of the least one invariant chain.

4. The Modified Vaccinia Ankara virus according to claim 1, wherein the encoded at least one invariant chain is a fragment of SEQ ID NO: 1 or SEQ ID NO: 3 of at least 40 consecutive amino acids or has at least 85% sequence identity to the same fragment of SEQ ID NO: 1 or SEQ ID NO:3.

5. The Modified Vaccinia Ankara virus according to claim 1, wherein the at least one antigenic protein is a protein of a pathogenic organism.

6. The Modified Vaccinia Ankara virus according to claim 5, wherein the pathogenic organism is a virus, a bacterium, a protist or a multicellular parasite.

7. The Modified Vaccinia Ankara virus according to claim 1, wherein the invariant chain is selected from p35 or a variant or fragment thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: MVA encoding Invariant chain (li)—NS antigen induces stronger T cell response in mice. Two groups of mice were immunized with 2×10{circumflex over ( )}5 plaque forming units (pfu) of MVA comprising NS-antigen (left panel) and with MVA comprising NS linked to invariant chain (right panel). Total T cell response to NS antigen was measured by IFNγ ELISpot and numbers on y axis represent spot forming cells (SFC)/million splenocytes.

(2) FIG. 2: MVA encoding Invariant chain (li)—NS antigen induces stronger T cell response than the corresponding Adeno in mice. Two groups of mice were immunized with MVA encoding NS (MVA wt) or NS linked to human invariant chain (MVAli). Two additional groups of mice were immunized with a comparable dose of ChAd3 encoding NS (ChAd3 wt) or NS linked to human invariant chain (ChAd3li). Total T cell response to NS antigen was measured by IFNγ ELISpot and numbers on y axis represent spot forming cells (SFC)/million splenocytes.

(3) FIG. 3: Boosting with MVA comprising NS linked to invariant chain augments the generation of HCV-NS specific T cells in macaques. Two groups of 4 macaques were primed with ChAd3IiNS and 50 weeks later boosted with MVA-NS (grey bars) or with MVA-IiNS (black bars). Panel A shows the response by IFNγ ELIspot one week (peak boost) or 3 months post boost (memory). Numbers on y axis represent spot forming cells (SFC)/million PBMC. Panel B shows higher CD8 frequency by IFNγ ICS one week post boost with MVAIiNS (black bars). Numbers on y axis represent % of antigen-specific CD8 T cells producing IFNγ.

(4) FIG. 4: Schematic diagram showing the four isoforms of human invariant chain (p33, p35, p41, p43, isoform c) and the two isoforms of murine invariant chain (p31, p41). In human p35 and p43 an additional 16 residues are present at the N-terminus due to alternative initiation of translation. In human p41 and p43 isoforms and the murine p41 isoform an additional domain is present due to alternative splicing. The human c isoform lacks two exons relative to human p33 and p35 (three exons relative to human p41 and p43) leading to a frame-shift.

EXAMPLES

Example 1

Priming with MVA Comprising NS Linked to Invariant Chain (MVA-hli NS) Augments the Generation of HCV-NS Specific T Cells in Mice

(5) Two groups of Balb/c mice were immunized intramuscularly with 2×10{circumflex over ( )}5 pfu (plaque forming units) of MVA encoding NS or with the same dose of MVA comprising NS linked to human invariant chain. The NS region encompasses about two thirds of the HCV genome and encodes for five different proteins (NS3, NS4A, NS4B, NS5A and NS5B) that result from the proteolytic cleavage of the HCV polyprotein by the encoded NS3 protease. Ten days after immunization, splenocytes were collected and HCV-NS specific T cell response was evaluated by IFNγ ELIspot using pools of peptides spanning NS. The response was evaluated by summing up reactivities against the six individual peptide pools and subtracting background (spots counted in control wells with no peptide). The level of specific T cells targeting NS was higher in mice primed with the li-based MVA vaccine (FIG. 1).

Example 2

Priming with MVA Comprising NS Linked to Invariant Chain (MVA-hli NS) Induces Stronger T Cell Response in Mice than the Corresponding Adenoviral Vector

(6) Two groups of Balb/c mice were immunized intramuscularly with 2×10{circumflex over ( )}5 pfu of MVA encoding NS or with the same dose of MVA comprising NS linked to human invariant chain. Two additional groups of mice were immunized with 2×10{circumflex over ( )}5 iu (infective units) of ChAd3 encoding NS or with the same dose of ChAd3 comprising NS linked to human invariant chain. Peak immune response was evaluated on splenocytes collected 10 and 21 days after immunization with MVA and ChAd3 vectored vaccines, respectively. T cell response was evaluated by IFNγ ELIspot using pools of peptides spanning NS. The results (FIG. 2) show that the li-based MVA vaccine induces higher response than the corresponding li-based ChAd3 vaccine.

Example 3

Boosting with MVA Comprising NS Linked to Invariant Chain (MVA-hli NS) Augments the Generation of HCV-NS Specific T Cells in Macaques

(7) Two groups of 4 macaques were primed with ChAd3IiNS and 50 weeks later boosted with MVA-NS (grey bars) or with MVA-IiNS (black bars). The injected dose was 1×10.sup.10 vp for adenoviral vectors, and 2×10.sup.8 pfu for MVA vectors. Immune response was evaluated on PBMC collected 1 week (peak response) and 3 months (memory response) after priming by IFNγ ELIspot and IFNγ Intracellular staining (ICS) using pools of peptides spanning NS. As shown in FIG. 3, higher ELISpot response was induced in the group receiving MVAIiNS at both time points (black bars). Panel A shows the response by IFNγ ELIspot one week or 3 months post boost. Panel B shows higher frequency of CD8 T cells producing IFNγ by ICS one week post boost with MVAIiNS (black bars).

(8) Materials and Methods

(9) Adenoviral and MVA Vectors

(10) The ChAd3 vector expressing the entire HCV NS3-5B (NS) region from genotype 1b, strain bk, has been described previously (Colloca et al. Sci Transl Med 4(115), 115ra112, 2012). MVA vector expressing the same cassette was derived and prepared as described previously (Cottingham, M. G. et al PLoS ONE 3, e1638, 2008; Di Lullo, G. et al. Virol. Methods 156, 37-43, 2009). The human li (p35, NCBI Reference Sequence: NM_004355) insert was synthetized by GeneArt (Life Technologies, Paisley, UK) and then cloned at the N-terminus of the NS transgene under HCMV and BGHpA control.

(11) Animals and Vaccinations

(12) All experimental procedures were performed in accordance with national and international laws and policies (EEC Council Directive 86/609; Italian Legislative Decree 116/92). The ethical committee of the Italian Ministry of Health approved this research. Animal handling procedures were performed under anesthesia and all efforts were made to minimize suffering and reduce animal numbers. Female 6-week-old Balb/c or C57Bl/6 mice were purchased from Charles River (Como, Italy), and experimental groups of 5 mice each were set. ChAd3 and MVA vectors were administered intramuscularly in the quadriceps by delivering a volume of 50 μl per site (100 μl final volume).

(13) Naïive, female, 11 to 19 years old (weight range 3.2 to 6.5 Kg) Cynomolgus macaques (Macaca fascicularis) from a purpose bred colony housed at the Institute of Cell Biology and Neurobiology (National Research Council of Italy, Rome), were assigned to experimental groups of four animals each. All immunizations were delivered by intramuscular route in the deltoid muscle injecting 0.5 ml of virus diluted in stabilizing buffer. The injected dose was 1×10.sup.10 vp for adenoviral vectors, and 2×10.sup.8 pfu for MVA vectors. During handling, the animals were anesthetized by i.m. injection of 10 mg/kg ketamine hydrochloride.

(14) Peptides

(15) A set of 494 peptides, 15 amino acids in length, overlapping by 11 amino acids and spanning the open reading frame from NS3-NS5B (1985 a.a.) of HCV genotype 1b strain BK were obtained from BEI Resources (Manassas, Va.).

(16) Ex Vivo IFNγ ELISpot with Mouse and Macaque Samples

(17) MSIP S4510 plates (Millipore) were coated with 10 μg/ml of anti-mouse or anti-monkey IFNγ antibody (both from U-CyTech Utrecht, The Netherlands) overnight at 4° C. After washing and blocking, mouse splenocytes or macaque peripheral blood mononuclear cells PBMC) were plated in duplicate at two different densities (2×10.sup.5 and 4×10.sup.5 cells/well) and stimulated overnight with overlapping 15mer peptide pools at a final concentration of 4 μg/ml each single peptide. The peptide diluent DMSO (Sigma-Aldrich, Milan, Italy) and ConA (Sigma-Aldrich, Milan, Italy) were used respectively as negative and positive controls. Plates were developed by subsequent incubations with biotinylated anti-mouse or anti-monkey IFNγ antibody (both from U-CyTech Utrecht, The Netherlands), Streptavidin-Alkaline Phosphatase conjugated (BD Biosciences, NJ) and finally with BCIP/NBT 1-Step solution (Thermo Fisher Scientific, Rockford, Ill.). Plates were acquired and analyzed by an A.EL.VIS automated plate reader. The ELISpot response was considered positive when all of the following conditions were met: IFNγ production present in Con-A stimulated wells; at least 50 specific spots/million splenocytes or PBMC to at least one peptide pool; the number of spots seen in positive wells was three times the number detected in the mock control wells (DMSO); and that responses decreased with cell dilutions. ELISpot data were expressed as IFNγ spot forming cells (SFC) per million splenocytes or PBMC.

(18) Intracellular Cytokine Staining (ICS) and FACS Analysis with Macaque Samples

(19) Briefly, 2×10.sup.6 monkey PBMCs were stimulated at 37° C. in 5% CO.sub.2 for 15-20 hours using peptide pools as antigen at 2 μg/ml each peptide final concentration in presence of anti-human CD28/CD49d costimulatory antibodies (BD Biosciences, NJ) and Brefeldin A (Sigma-Aldrich, Milan, Italy). DMSO (Sigma-Aldrich, Milan, Italy) was used as negative control, and Staphylococcal enterotoxin B (SEB, Sigma-Aldrich, Milan, Italy) was used as positive control. After overnight stimulation, PBMCs where stained with the following surface antibodies: APC anti-monkey CD3, clone SP34-2, PerCp-Cy5.5 anti-monkey CD4, clone L200, PE anti-human CD8, clone RPA-T8 (all from BD Biosciences, NJ). Intracellular staining was performed after treatment with Cytofix/Cytoperm and in the presence of PermWash (BD Biosciences, NJ) using FITC anti-human IFNγ, clone MD-1 (U-CyTech Utrecht, The Netherlands). Stained cells were acquired on a FACS Canto flow cytometer, and analyzed using DIVA software (BD Biosciences, NJ). At least 30,000 CD8+, CD3+ gated events were acquired for each sample.