MEANS AND METHODS FOR TREATING HERPESVIRUS INFECTION

Abstract

The present invention provides herpesviruses, such as EBV, which lack at least one viral miRNA. Such herpesviruses lacking at least one viral miRNA are advantageously not capable of packaging their genome into the capsid, thereby producing HVLPs, which are substantially free of their herpesvirus genome or the nucleic acid molecule encoding the proteinaceous part of the HVLP and viral miRNA. Such HVLPs may be used as vaccine.

Claims

1. A Herpes virus-like particle (HVLP) comprising Herpes viral proteins which are encoded by at least one nucleic acid molecule which still comprises miRNA coding loci encoding Herpes viral miRNAs, wherein at least one of said miRNA coding loci is genetically modified.

2. The HVLP of claim 1, wherein said genetic modification effects that said at least one Herpes viral miRNA is not expressed or only partially expressed, said at least one Herpes viral miRNA does not bind to its target sequence, said at least one Herpes viral miRNA or its precursor has a wrong 3D structure, the precursor of said at least one Herpes viral miRNA is not further processed, said at least one Herpes viral miRNA or its precursor are degraded by the cell, said at least one Herpes viral miRNA coding loci has a scrambled sequence, said at least one Herpes viral miRNA coding loci is deleted, and/or said at least one Herpes viral miRNA or its precursor comprises mutations, deletions or insertions.

3. The HVLP of claim 1 or 2, wherein said genetic modification leads to an increased immune response when compared to a HVLP that comprises no genetically modified Herpes viral miRNA coding loci, wherein said increase is at least 5% as determined in a quantitative ELISA, comprising measuring the concentration of proinflammatory cytokines in the supernatant of immune cells incubated with the HVLPs of claim 1 or 2 and comparing said cytokine concentration to the cytokine concentration in the supernatant of immune cells incubated with HVLPs that are encoded by a nucleic acid molecule comprising miRNA coding loci identical to the wild type virus.

4. The HVLP of any one of the preceding claims, wherein the at least one nucleic acid molecule encoding said Herpes viral proteins is genetically modified such that it is not packaged in the HVLPs.

5. The HVLP of claim 4, wherein the at least one nucleic acid molecule encoding said Herpes viral proteins lacks a functional cis-acting element required for packaging.

6. The HVLP of claim 4, wherein the at least one nucleic acid molecule encoding said Herpes viral proteins comprises at least one gene encoding a Herpes viral protein required for packaging, which is genetically modified such that said Herpes viral protein is not expressed or non-functional.

7. The HVLP of any one of the preceding claims, wherein said HVLP is substantially free of a Herpes virus genome and/or the at least one nucleic acid molecule.

8. The HVLP of any one of the preceding claims, wherein the at least one nucleic acid molecule encoding said Herpes viral proteins comprises at least one gene encoding a Herpes viral protein required for cellular transformation, which is genetically modified such that said Herpes viral protein is not expressed or non-functional.

9. The HVLP of any one of the preceding claims, wherein the at least one nucleic acid molecule encoding said Herpes viral proteins comprises at least one gene encoding a Herpes viral protein required for inducing virus synthesis, which is genetically modified such that said Herpes viral protein is not expressed or non-functional.

10. The HVLP of any one of the preceding claims, wherein the at least one nucleic acid molecule encoding the Herpes viral proteins comprises a Herpes virus genome, wherein said Herpes virus is selected from the group consisting of Herpes-simplex virus 1, Herpes-simplex virus 2, Varicella-zoster virus, Epstein-Barr virus, Human cytomegalovirus, Kaposi's sarcoma-associated herpesvirus, Human herpesvirus 6, Human herpesvirus 7, Bovine herpesvirus 1, Bovine herpesvirus 2, Bovine herpesvirus 3, Bovine herpesvirus 4, Bovine herpesvirus 5, and Murine gammaherpesvirus 68.

11. The HVLP of any one of the preceding claims, which is an Epstein-Barr VLP (EBVLP), comprising Epstein-Barr virus (EBV) proteins and EBV miRNAs.

12. The EBVLP of claim 11, wherein the at least one modified miRNA coding loci is selected from the group consisting of miR-BHRF1-1, miR-BHRF1-2, miR-BHRF1-3, miR-BART1, miR-BART2, miR-BART3, miR-BART4, miR-BART5, and miR-BART15.

13. The EBVLP of claim 11 or 12, wherein the at least one nucleic acid molecule encoding said EBV proteins comprises at least one gene, encoding an EBV protein required for B-cell transformation, selected from the group consisting of EBNA1, EBNA-LP, EBNA2, LMP1, LMP2, EBNA3A, and EBNA3C, which is genetically modified such that the EBV protein is not expressed or non-functional.

14. The EBVLP of any one of claims 11 to 13, wherein the at least one nucleic acid molecule encoding said EBV proteins comprises at least one gene, encoding an EBV protein required for inducing virus synthesis, selected from the group consisting of BZFL1, BRLF1 and BMLF1, which is genetically modified such that the EBV protein is not expressed or non-functional, wherein said gene is preferably BZLF1.

15. The EBVLP of any one of claims 11 to 14, wherein the at least one nucleic acid molecule encoding said EBV proteins lacks the packaging element TR.

16. The EBVLP of any one of claims 11 to 15, wherein the at least one nucleic acid molecule encoding said EBV proteins comprises at least one gene encoding an EBV protein required for packaging of EBV DNA, selected from the group consisting of BFLF1, BBRF1, BGRF1, BDRF1, BALF3, BFRF1A, and BFRF1, which is genetically modified such that said EBV protein is not expressed or non-functional.

17. The EBVLP of any one of claims 11 to 16, wherein the at least one nucleic acid molecule encoding said EBV proteins comprises an EBV genome.

18. A nucleic acid molecule encoding the Herpes viral proteins of the HVLP of any one of claims 1 to 10 or the EBV proteins of the EBVLP of any one of claims 11 to 17.

19. A vector comprising the nucleic acid molecule of claim 18.

20. A composition of matter comprising at least two nucleic acid molecules encoding the Herpes viral proteins of the HVLP of any one of claims 1 to 10 or the EBV proteins of the EBVLP of any one of claims 11 to 17.

21. The composition of claim 20, wherein said at least two nucleic acid molecules are comprised by at least two vectors.

22. A host cell transfected with the nucleic acid molecule of claim 18, the vector of claim 19 or the composition of claim 20 or 21.

23. A method for generating a HVLP or an EBVLP, the method comprising: (i) culturing the host cell of claim 22 under conditions that allow expression of the Herpes viral proteins or the EBV proteins; and (ii) obtaining said HVLP or EBVLP.

24. The method of claim 23, comprising after step (i) and prior to step (ii) a further step (i′), comprising inducing the replicative phase of the Herpes virus or Epstein-Barr virus, wherein said replicative phase is induced by expressing at least one gene, encoding a Herpes viral protein or an EBV protein that is required for inducing Herpes virus synthesis or EBV synthesis, wherein the at least one gene, comprised by the at least one nucleic acid molecule encoding the Herpes viral proteins of the HVLP or the EBV proteins of the EBVLP, encoding said Herpes viral protein or EBV protein, has been genetically modified, such that said Herpes viral protein or EBV protein is not expressed or non-functional.

25. The method of claim 24, wherein said gene is expressed from a stably transfected vector comprised by said host cell and/or wherein expression of said gene is inducibly regulated.

26. The method of claim 24 or 25, wherein said gene encoding said EBV protein is selected from the group consisting of BZLF1, BRLF1 and BMLF1, wherein BZLF1 is preferred.

27. A composition comprising at least 95% of the HVLP as defined in any one of claims 1 to 10 or the EBVLP as defined in any one of claims 11 to 17.

28. A vaccine composition comprising the HVLP of any one of claims 1 to 10, the EBVLP of any one of claims 11 to 17, or the composition of claim 27.

29. The vaccine composition of claim 28, further comprising an excipient.

30. The vaccine composition of claim 28 or 29, further comprising one or more viral or non-viral polypeptides, one or more viral or non-viral nucleic acid sequences and/or vaccine adjuvants, wherein said one or more viral polypeptides or said one or more viral nucleic acid sequences are not from the same virus as the HVLP or EBVLP in said vaccine composition.

31. Use of the HVLP of any one of claims 1 to 10 or the EBVLP of any one of claims 11 to 17, the composition of claim 27, or the vaccine composition of any one of claims 28 to 30 in the vaccination or treatment of a subject.

32. The use of the nucleic acid molecule of claim 18, the vector of claim 19, the composition of claim 20 or 21, or the host cell of claim 22 in the production of a HVLP or an EBVLP.

33. A kit comprising the HVLP of any one of claims 1 to 10, the EBVLP of any one of claims 11 to 17, the nucleic acid molecule of claim 18, the vector of claim 19, the composition of claim 20 or 21, the host cell of claim 22, the composition of claim 27 and/or the vaccine composition of claims 28 to 30.

Description

FIGURES

[0094] FIG. 1: EBV miRNAs affect major pathways of immunity. (A) Heatmaps of the most strongly regulated genes in wt/B95-8 or ΔmiR EBV-infected B-cells of 6 donors (donor Ad1-Ad6) five days post infection. Differentially expressed gene transcripts with absolute z-scores >1.6 are shown. Blue and red colors indicate down- and up-regulated transcripts, respectively, in wt/B95-8 compared with ΔmiR EBV-infected cells. (B) Regulation of selected genes associated with adaptive immune responses or the p53 signaling pathway. Previously reported targets of EBV miRNAs and common housekeeping genes are shown as well. Blue background shadings indicate genes down-regulated by viral miRNAs. (C) The fractions of EBV miRNAs among all miRNAs. Means of 6 donors is shown.

[0095] FIG. 2: EBV miRNAs inhibit secretion of pro-inflammatory cytokines and expression of molecules involved in antigen processing and presentation. (A) Secretion of various cytokines by B-cells infected with wt/B95-8 or ΔmiR EBV. B-cells, which had been infected 4 or 11 earlier (days post infection, dpi), were cultivated for 4 additional days to determine cytokines levels by ELISA (n=3). CpG DNA was added as indicated. (B) EBV miRNAs regulate IL12B and TAP2. HEK293T cells were co-transfected with miRNA expression vectors and luciferase reporter plasmids carrying a wild type or mutated 3′-UTR (FIG. 7) as indicated (n=3). The luciferase activities were normalized to lysates from cells co-transfected with the wild type 3′-UTR reporter and an empty plasmid. wt: wild type 3′-UTR, mut: mutated 3′-UTR, ø: empty plasmid. P values were calculated by an unpaired two-tailed T test. An asterisk (*) indicates p<0.05 with respect to the luciferase activity of the wild type reporter co-transfected with empty plasmid. (C) Western blot analysis of TAP1 and TAP2 in EBV-infected B-cells. Tubulin (TUBB) and β-Actin (ACTB) serve as housekeeping controls. A positive control is IP07. Representative examples (top) and protein expressions normalized to tubulin (bottom; n=3-5) are shown. (D-E) Cell surface expression of HLA molecules (D) and co-stimulatory and adhesion molecules (E) regulated by EBV miRNAs. Median fluorescence intensity (MFI) was measured after immunostainings for individual surface proteins and ratios (wt/B95-8 divided by ΔmiR EBV-infected B-cells) are shown (n=5-10). Means±SD are shown. n.d.: not detected; wt: wt/B95-8; *: p<0.05, **: p<0.01, ***: p<0.001.

[0096] FIG. 3: EBV miRNAs prevent Th1 differentiation and recognition by EBV-specific CD4.sup.+ T-cells. (A) Schematic overview of co-culture experiments to assess the impact of viral miRNAs on helper T-cell differentiation. (B) Th1 differentiation of naive CD4.sup.+ T-cells upon co-culture with EBV-infected B-cells. Naive CD4.sup.+ T-cells were cultivated for 7 days with autologous, newly infected B-cells and αCD3/αCD28 antibodies at indicated ratios (n=5-6). Proliferating, phorbol 12-myristate 13-acetate (PMA) and ionomycin re-stimulated Th1 cells were quantitated by intracellular IFN-γ staining. Left: representative flow cytometry analyses; right: summary of all experiments. (C) An anti-IL12B antibody (5 μg/ml) suppressed Th1 cell differentiation of naive CD4.sup.+ T-cells co-cultivated with wt/B95-8 or ΔmiR-infected B-cells at B:T-cell ratio of 1:1 (n=8). An irrelevant antibody of the same isotype was used as a control. (D) Schematic overview of co-culture experiments investigating the influence of viral miRNAs on antiviral functions of EBV-specific CD4.sup.+ T-cells. (E) IFN-γ release by polyclonal EBV-specific CD4.sup.+ T-cells co-cultured with autologous (auto), HLA-matched, or mismatched (mis.) B-cells infected with EBV (n=3; FIG. 12). The B:T-cell ratio was 1:1. Matched HLA class II alleles are indicated. ø: only T-cells; n.a.: not applicable. (F) Cytotoxic activity of EBV-specific CD4.sup.+ T-cells. Killing of EBV-infected B-cells was analyzed at various B:T-cell ratios by Calcein release assays. A representative experiment with HLA-matched EBV-infected target B-cells (left; n=3) and the overview of all experiments with HLA-matched B-cells (right) are described. Means±SD are shown. *: p<0.05, **: p<0.01, ***: p<0.001.

[0097] FIG. 4: EBV miRNAs inhibit recognition of EBV-infected B-cells by EBV-specific CD8.sup.+ T-cells. (A) Schematic overview of co-culture experiments investigating the influence of viral miRNAs on antiviral functions of EBV-specific CD8.sup.+ T-cells. (B) IFN-γ release by polyclonal EBV-specific CD8.sup.+ T-cells co-cultured with autologous (auto), HLA-matched, or mismatched (mis.) B-cells infected with EBV (n=3; FIG. 12). The B:T-cell ratio was 1:1. Matched HLA class I alleles are indicated. ø: only T-cells; n.a.: not applicable. (C) Cytotoxic activity of EBV-specific CD8.sup.+ T-cells. Killing of EBV-infected B-cells (wt/B95-8 or ΔmiR EBV) was analyzed at various B:T-cell ratios in calcein release assays. A representative experiment with HLA-matched EBV-infected target B-cells (left; n=3) and the overview of all experiments with HLA-matched B-cells (right) are shown. (D) Reactivity of a CD8.sup.+ T-cell clone directed against a LMP2 epitope IED (HLA-B*40:01-restricted). T-cells were cultivated for 16 hours with HLA-B*40:01-positive B-cells that have been infected for 15 days. IFN-γ secretion levels quantified with ELISA (Left; n=3) and MFI ratios (wt/B95-8 divided by ≢miR EBV-infected B-cells) for HLA-B*40 (Right; n=4) are described. ø: only T-cells; peptide: T-cells loaded with the control peptide. wt: wt/B95-8. Means±SD are shown. *: p<0.05, **: p<0.01.

[0098] FIG. 5: The regulation of functional gene groups by EBV miRNAs KEGG pathway categories were used for categorization of gene functions. Pathways are sorted by statistical significance. The sizes of the orange dots indicate −log10 p-value scores. For each of the six donors, fold change values of differentially expressed transcripts are plotted. As in FIG. 1a, blue or red colors indicate down- or up-regulation by EBV miRNAs, respectively.

[0099] FIG. 6: Quantification of EBV miRNAs after RISC-IP EBV's BHRF and BART miRNAs accumulate in wt/B95-8 EBV-infected B-cells but are barely detectable in ΔmiR EBV-infected B-cells. Means±SD is shown.

[0100] FIG. 7: 3′-UTR reporters and their mutations

[0101] Partial sequences of 3′-UTRs of selected transcripts, which were analyzed in FIG. 2b are shown together with corresponding miRNAs and mutations within the 3′-UTRs in reporter vectors. Complementarities are based on in silico predictions according to the RNAhybrid algorithm and depicted as Watson-Click (‘I’) or G:U (‘:’). Non-matching nucleotide residues are indicated (X). They result from mutated mRNA target sequences in the reporter plasmids.

[0102] FIG. 8: Reactivity of polyclonal EBV-specific CD4.sup.+ T-cells EBV-specific CD4.sup.+ T-cell were co-cultured for 16 hours with autologous B-cells that had been infected five days earlier. IFN-γ secretion levels were then quantified with ELISA.Various B:T-cell ratios were used as indicated.

[0103] FIG. 9: Reactivity of the gp350 specific CD4.sup.+ T-cell clone The gp350-specific CD4.sup.30 T-cell clone, epitope FGQ (HLA-DRB1*1301), was used as effector cells. Autologous B-cells from donor JM (table S2) were used as target cells five and 15 days after infection with the two EBV strains indicated at an B:T-cell ratio of 1:1. After 16 hours of co-culture, IFN-γ and GM-CSF secretion levels were quantified by ELISA. Means±SD are shown.

[0104] FIG. 10: Schematic overview of co-culture experiments investigating the influence of viral miRNAs on antigen presentation to a LMP2-specific CD8.sup.+ T-cells clone

[0105] FIG. 11: Regulation of viral genes by EBV miRNAs (A) Western blot analysis of LMP2A expression in B-cells infected with wt/B95-8 or with ΔmiR EBV at day 15 post infection. A representative example (top) and protein expression normalized to tubulin (bottom n=4) are described. Means±SD are shown. (B) Log.sub.e fold changes of two LMP2 gene variants by viral miRNAs. Analysis was performed as in FIG. 1B but the quantification of expression level was done exon-wise to analyse splicing variants correctly.

[0106] FIG. 12: HLA alleles. List of the donors' HLA alleles (MVZ Martinsried, Germany) identified by deep-sequencing, whose B and T-cells have been used in co-culture experiments in this study. n.a.: not available.

[0107] FIG. 13: Activation of CD4.sup.+ T-cells using Epstein-Barr VLPs A human EBV-immortalized B-cell line (LCL) was incubated with similar numbers of VLPs (1*10{circumflex over ( )}4 particles/cell) with miRNAs or lacking all miRNAs (ΔmiRNAs) for 24 hours and then co-cultivated for another 24 hours with an HLA-matched CD4.sup.+ T-cell clone specific for the EBV tegument protein BNRF1 for another 24h. Activation of T cells was quantified in an IFNγ-ELISA assay. Controls are LCLs or T-cells that have not been co-cultivated with LCLs (T-cells only).

EXAMPLES

[0108] The following Examples illustrate the invention, but are not to be construed as limiting the scope of the invention.

Materials and Methods

[0109] Separation of Human Primary Cells

[0110] Human primary B and T-cells were prepared from adenoidal mononuclear cells (MNC) or peripheral blood mononuclear cells (PBMC) by Ficoll-Hypaque gradient centrifugation. B-cells, CD4.sup.+ T-cells, CD8.sup.+ T-cells, and naive CD4.sup.+ T-cells were separated from adenoidal MNC or PBMC using MACS separator (Miltenyi Biotec) with CD19 MicroBeads, CD4 MicroBeads, CD8 MicroBeads, and Naive CD4.sup.+ T-cell Isolation Kit II, respectively.

[0111] Cell Lines and Cell Culture

[0112] The EBV-positive Burkitt's lymphoma cell line Raji and HEK293-based EBV producer cell lines (Seto et al., PLoS Pathog. 6, e1001063 (2010)), infected human primary B-cells, and isolated T-cells were maintained in RPMI 1640 medium (Life Technologies). HEK293T cells were maintained in DMEM medium. All media were supplemented with 10% FBS (Life Technologies), penicillin (100 U/ml; Life Technologies), and streptomycin (100 mg/ml; Life Technologies). Cells were cultivated at 37° C. in a 5% CO.sub.2 incubator.

[0113] Preparation of Infectious EBV Stocks and Infection of Human Primary B-Cells

[0114] Infectious EBV stocks were prepared as described (Seto, loc. cit.). Briefly, EBV producer cell lines for ΔmiR (4027) and wt/B95-8 (2089) EBV strains were transiently transfected with expression plasmids encoding BZLF1 and BALF4 to induce EBV's lytic phase. Supernatants were collected three days after transfection and debris was cleared by centrifugation at 3000 rpm for 15 minutes. Virus stocks were titered on Raji cells as previously reported (Seto, loc. cit.). For virus infection, primary B-cells were cultivated with each virus stock for 18 hours. After replacement with fresh medium, the infected cells were seeded at an initial density of 5×10.sup.5 cells per ml.

[0115] RNA-Seq and RISC-IP

[0116] At 5 days post infection of human primary B-cells, total RNAs were extracted with Trizol (Life Technologies) and Direct-Zol RNA MiniPrep (Zymo Research) from six different donors (Ad1 to Ad6) (FIG. 1) for RNA-Seq, according to the manufacturers' protocols. In parallel, RISC immunoprecipitation (RISC-IP) was performed as described previously (Kuzembayeva, et al., PLoS ONE. 7, e47409 (2012)). Briefly, lysed cells were incubated with anti-Ago2 antibody (11A9)-conjugated dynabeads (Life Technologies), washed, and co-precipitated RNA was extracted. The cDNA libraries were prepared (vertis Biotechnologie AG, Freising, Germany). For RNA-Seq, total RNAs were depleted of rRNAs by Ribo-Zero rRNA Removal Kit (Illumina), fragmented by ultrasonication, and subjected to first strand synthesis with a randomized primer. For RISC-IP, RNAs were poly (A)-tailed, ligated with an RNA adapter at 5′-phosphates to facilitate Illumina TruSeq sequencing, and subjected to first strand synthesis with a oligo-(dT) primer. The cDNAs were PCR-amplified and sequenced with an Illumina HiSeq2000 instrument at the University of Wisconsin Biotechnology Center DNA Sequencing Facility.

[0117] Analysis of Deep Sequencing

[0118] For RNA-Seq, processing of paired-end reads (poly-A tail filtering, N-filtering, adapter removal) was done using FastQC and R2M (RawReadManipulator). Reads were mapped to the human genome (hg19 ‘core’ chromosome-set) by STAR and feature counts per transcript were determined using featureCounts and GencodeCV19 annotations together with EBV's annotation (GenBank: AJ507799). To screen differentially regulated genes by viral miRNAs, it was used a simple but efficient scoring algorithm based on donor/replicate wise fold changes ranks. For each gene g and replicate k it is calculated the gene specific rank score:

[00001]$r_g=\frac{1}{m}.Math.\underset{k=1}{\overset{n}{.Math.}}.Math.r_{\mathrm{gk}}$

where n is the number of all replicates, m the number of all genes/transcripts, r.sub.gk the rank of gene g in sample k.

[0119] To select highly differentially expressed genes the rank score was transformed into a z-score and selected all transcripts with an absolute z-score >1.6.

[0120] For RISC-IP the mapped reads were normalized using size factors estimated with the R package DEseq2 and filtered for reads mapped to annotated 3′UTR regions using Gencode v19. To identify local quantitative differences in the read enrichments on 3′UTRs between wt EBV compared with ΔmiR EBV-infected B cells, a donor-wise relative enrichment score was calculated. For each genomic position p, the relative expression es.sub.p was calculated as:

[00002]${\mathrm{es}}_p=\frac{e_{\mathrm{tp}}}{e_{\mathrm{tp}}+e_{\mathrm{cp}}}.Math.n_{\mathrm{pu}}$

where e.sub.tp is the expression value at position p in wt EBV-infected cells and e.sub.cp the local expression value in ΔmiR EBV-infected B cells, respectively.

[0121] The normalization factor n.sub.pu=e.sub.tp/max(e.sub.u) was introduced to correct for local maxima in the UTR sequence of interest, where max(e.sub.u) is the maximum expression value in the UTR sequence u. Finally a Gaussian filter was used to minimize local noise. To select 3′-UTRs bound by viral miRNAs, the threshold was set as follows: enrichment score >0.6 for a stretch of >20 nucleotides in the 3′-UTRs in two or more donors.

[0122] KEGG Enrichment Pathway

[0123] Enrichment of specific pathways was estimated by performing a hypergeometric distribution test via the KEGG API Web Service. All calculations were done using Matlab (Mathworks).

[0124] ELISA

[0125] To detect cytokine secretion from infected B-cells, 1×10.sup.6 cells were seeded in 6 well plates at four or 11 days post infection, cultivated for four days with cyclosporine (1 μg/ml; Novartis). Supernatants were harvested and stored at −20° C. Enzyme-linked immunosorbent assays (ELISAs) for interleukin-6 (IL-6), IL-10, IL12B (IL-12p40), IL-12, IL-23, and TNF-α were performed following the manufacturer's protocols (Mabtech). For IL-6, IL-10, and TNF-α, CpG DNA were added as previously described (Iskra, et al., J. Virol. 84, 3612-3623 (2010)) to stimulate infected B-cells. ELISA for IFN-γ levels was performed following the manufacturer's protocol (Mabtech).

[0126] Flow Cytometry and Antibodies

[0127] After immunostainings with fluorophore-conjugated antibodies, single-cell suspensions were measured with LSRFortessa or FACSCanto (BD) flow cytometers and the FACSDiva software (BD Biosciences). Acquired data were analyzed with FlowJo software Ver. 9.8 (FlowJo). The following fluorophore-conjugated antibodies reactive to human antigens were used: anti-human IFN-γ APC (4S.B3, IgG1; Biolegend), anti-CD40 PE (5c3, IgG2b; BioLegend), anti-ICOS-L (B7-H2) PE (2D3, IgG2b; BioLegend), anti-PD-L1 (B7-H1) APC (29E.2A3, IgG2b; BioLegend), anti-CD86 (B7-2) PE (37301, IgG1; R&D Systems), anti-CD54 (ICAM-1) APC (HCD54, IgG1; BioLegend), anti-HLA-ABC APC (W6/32, IgG2a; BioLegend), anti-CD80 PE-Cy5 (L307.4; BD Pharmingen), anti-FAS (CD45) PE (Dx2, IgG1; BioLegend), anti-HLA-DR unlabeled (L234, IgG2a; BioLegend), anti-HLA-DQ unlabeled (SPV-L3, IgG2a ; AbD Serotec), anti-HLA-DP unlabeled (B7/21, IgG3; Abcam), anti-mouse F(ab′)2 APC (polyclonal, IgG; eBioscience), HLA-Bw6 PE (REA143, IgG1; Miltenyi Biotec), isotype IgG1 PE (MOPC-21; BioLegend), isotype IgG2b PE (MPC-11; BioLegend), isotype IgG1 APC (MOPC-21; BD Bioscience), isotype IgG2a APC (MOPC-173; BioLegend), isotype IgG2b APC (MG2b-57; BioLegend).

[0128] Western Blotting

[0129] Cells were lysed with RIPA buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% DOC) and boiled the extracts with Laemmli buffer. Proteins were separated on 10% SDS-PAGE gels (Carl Roth) and transferred to nitrocellulose membranes (GE Healthcare Life Science) using Mini-PROTEAN Tetra Cell (Bio-Rad). Membranes were blocked for 30 minutes with Roti-Block (Carl Roth) followed by antibody incubation. Secondary antibodies conjugated with horseradish peroxidase were used (Cell Signaling) and exposed to CEA films (Agfa HealthCare). Protein levels were quantified with the software ImageJ. The following primary antibodies reactive to human proteins were used: anti-human Tubulin (B-5-1-2; Santa Cruz), anti-human actin (AC-74; Sigma), anti-human IP07 (ab88339; Abcam), anti-human TAP1 (1.28; Acris) and anti-human TAP2 (2.17, Acris). The (TP-14B7) monoclonal antibody reactive to the EBV protein LMP2 was provided by Elisabeth Kremmer.

[0130] Luciferase Reporter Assays

[0131] The 3′-UTRs of IL12B (Ensembl: ENST00000231228) and TAP2 (Ensembl: ENST00000374897) were cloned downstream of firefly luciferase (Fluc) in the expression plasmid psiCHECK-2 (Promega). To construct the viral miRNA expression vectors, TagBFP (Evrogen) was clonedunder the control of the EF1a promoter into pCDH-EF1-MCS (System Biosciences). Single miRNAs of interest were cloned downstream of the TagBFP-encoding gene. Viral miRNAs were obtained by PCR from the p4080 plasmid (Seto, loc. cit.). The psiCHECK-2 reporter and pCDH-EF1 miRNA expressor plasmid DNAs were co-transfected into HEK293T cells by Metafectene Pro (Biontex). After 24 hours of transfection, luciferase activities were measured with the Dual-Luciferase Assay Kit (Promega) and the Orion II Microplate Luminometer (Titertek-Berthold). The activity of Fluc was normalized to the activity of Renilla luciferase (Rluc) encoded in the psiCHECK-2 reporter. It was performed in silico prediction of EBV miRNA binding sites on 3′-UTRs primarily with TargetScan (www.targetscan.org) and employed RNAhybrid (bibiserv.techfak.uni-bielefeld.de/rnahybrid) to screen for 6mer binding sites (Bartel, Cell. 136, 215-233 (2009)). Site-directed mutagenesis were performed with overlapping oligo DNAs and Phusion polymerase (NEB).

[0132] Establishment of EBV-Stimulated Effector T-Cells and T-Cell Clones

[0133] EBV-specific CD8.sup.+ T-cell clones were established from polyclonal T-cell lines that were generated by lymphoblastoid cell lines (LCLs) or mini-LCL stimulation of PBMCs as previously described (Adhikary et al., PLoS ONE. 2, e583 (2007))

[0134] T-Cell Differentiation and Recognition

[0135] Th1 differentiation was assessed by co-culture of sorted naive CD4.sup.+ T-cells and infected B-cells 5 days post infection. 1×10.sup.5 naive CD4.sup.+ T-cells stained with CellTrace Violet (Life Technologies) and 0.5 or 1×10.sup.5 infected B-cells were cultured in 96 well plates with Dynabeads Human T-Activator CD3/CD28 (Life Technologies) and cultivated for 7 days. The neutralizing antibody against IL12B (C8.6; BioLegend) or the corresponding isotype control antibody (MOPC-21; BioLegend) were added for certain experiments at 5 pg/ml. Cells were re-stimulated with PMA and ionomycin (Cell Stimulation Cocktail; eBioscience) for 5 hours and treated with Brefeldin A and Monensin (Biolegend) for 2.5 hours prior to fixation. Th1 population was measured by intracellular IFN-γ staining with FIX & PERM Cell Permeabilization Kit (Life Technologies) and subsequent flow cytometery analysis. The Th1 population was defined as IFN-γ positive T-cells in the fraction of proliferating T-cells identified via CellTrace Violet staining. EBV-specific effector T-cells' activities were measured with ELISA and Calcein release assays. For IFN-γ detection from T-cells, effector and target cells were seeded at 5×10.sup.4 cell per ml (1:1 ratio) each and co-cultured for 16 hours in a 96-well plate (V bottom). IFN-γ were detected with ELISA. IFN-γ concentrations lower than 16 pg/ml were considered as not detected.

[0136] T-Cell Cytotoxicity Assays

[0137] Primary infected B-cells were purified by Ficoll-Hypaque gradient centrifugation, and 5×10.sup.5 target cells were labeled with calcein at 0.5 μg/ml. After three washing steps with PBS, target and effector cells were co-cultured in a 96-well plate (V bottom) with different ratios in RPMI red phenol-free medium to reduce background signals. After four hours of co-culture, fluorescence intensity of the released calcein was measured by the Infinite F200 PRO fluorometer (Tecan). As controls, spontaneous calcein release of target cells cultivated without effector cells and cells lysed with 0.5% Triton-X100 were used to define the levels of no and fully lysed target cells, respectively.

[0138] Statistical Analysis.

[0139] Prism 6.0 software (GraphPad) was used for the statistical analysis and two-tailed ratio T test was applied unless otherwise mentioned.

Example 1

[0140] Targets of EBV's miRNAs using an approach designed to detect cellular mRNAs the virus targets to foster its efficient infection were searched. Two stocks of EBV, a laboratory strain (wt/B95-8) that expresses 13 miRNAs and its deleted derivative (ΔmiR) that expresses none, were used to infect freshly isolated B-cells from six donors. RNAs were isolated on day 5 following infection and sequenced. Genes that were differentially expressed were identified with those having a z-score >1.6 shown in FIG. 1A. These genes included the viral miRNA targets LY75/DEC205 (Skalsky et al., PLoS Pathog. 8, e1002484 (2012)) and IPO7 (Dölken et al., Cell Host Microbe. 7, 324-334 (2010)). The identified, regulated genes were grouped according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway categories (FIG. 5) based on consistently down-regulated genes in wt/B95-8 EBV infected cells. This grouping was enriched in the pathways linked to apoptosis, cell cycle regulation, and p53 signaling (Seto et al., PLoS Pathog. 6, e1001063 (2010)). This grouping also strikingly revealed that in newly infected cells, EBV's miRNAs regulate a wide array of immune functions encompassing antigen processing, HLAs and co-stimulatory molecules, and cytokine-cytokine receptor interaction (FIG. 1 B, FIG. 5). RNA-induced silencing complex (RISC-IP) was immunoprecipitated and found 14.5% (±2.4% SD) of all miRNAs were of viral origin in wt/B95-8 EBV-infected cells (FIG. 1C and FIG. 6). It was also found that different mRNAs were detected in the RISC differently among the cell samples as has been found in PAR-CLIP experiments (Skalsky, loc. cit.) (GEO: GSE41437). Therefore, the analyses were focused primarily on candidate mRNAs identified by their differential expression in all samples (FIG. 1A) and used RISC-IP results to confirm them.

Example 2

[0141] It was confirmed that EBV's miRNAs regulate cytokines central to immune functions. The supernatants from B-cells infected with the two strains of EBV were assayed for the levels of interleukin-6 (IL-6), IL-10, TNF-α, IL12B (IL-12p40), IL-12 (p35/p40), and IL-23 (p19/p40). CpG DNA was added, which stimulates TLR9, for the detection of IL-6 and TNF-α secreted from EBV-infected cells (Iskra, et al., J. Virol. 84, 3612-3623 (2010)). The wt/B95-8 EBV-infected B-cells secreted less IL-6, TNF-α, and IL-12p40 than B-cells infected with ΔmiR EBV. In contrast, release of the anti-inflammatory cytokine IL-10 appeared to be unaffected by viral miRNAs (FIG. 2A) consistent with the transcriptome analysis (FIG. 1B). Secretion of IL-12 (p35/p40) and IL-23 (p19/p40), both of which contain the IL-12p40 subunit (Szabo et al., Annu. Rev. Immunol. 21, 713-758 (2003)), was significantly reduced in wt/B95-8 EBV-infected cells compared with ΔmiR EBV-infected cells (FIG. 2A).

Example 3

[0142] It was found that EBV miRNAs directly regulate a cytokine-encoding gene IL12B, which encodes IL-12p40. The finding was verified with luciferase reporter assays. EBV's miR-BHRF1-2, miR-BART1, or miR-BART2 repressed the luciferase activity of the IL12B reporter (FIG. 2B). The predicted binding sites of miR-BART1 or miR-BART2 were mutated, which abrogated their ability to inhibit the IL12B reporter (FIG. 2B and FIG. 7) confirming the direct controls of viral miRNAs on this gene transcript. MiR-BART10 and miR-BART22 were analysed, which are present in field strains of EBV but not in wt/B95-8 EBV, similarly (FIG. 2B and FIG. 7). Mutations of their predicted target sites only partially relieved the inhibition by both miRNAs, suggesting the presence of additional binding sites for these miRNAs in the IL12B transcript. In summary, it was confirmed that cytokines are regulated by EBV miRNAs, and validated IL12B as a direct target of multiple viral miRNAs.

Example 4

[0143] Additionally, levels of proteins pivotal to antigen processing and presentation, including TAP1 and TAP2, whose transcript levels were reduced in wt/B95-8 compared with ΔmiR EBV-imfected cells were quantified (FIG. 1B). Both TAP1 and TAP2 were decreased by EBV's miRNAs (FIG. 2C). They form a heterodimer, which mediates the cytoplasmic transport of antigenic peptides into the ER lumen, where they are loaded onto MHC class I molecules stabilizing them (Horst et al., J. lmmunol. 182, 2313-2324 (2009)). MHC class I molecules and all three subclasses of MHC class II molecules (HLA-DR, HLA-DQ and HLA-DP) were reduced as were co-stimulatory and adhesion molecules by 15 days post infection (FIG. 2, D and E).

Example 5

[0144] RISC-IP and in silico algorithms indicated that the 3′-UTR of TAP2 is targeted by EBV miRNAs. In luciferase reporter assays miR-BHRF1-3 repressed the TAP2 reporter (FIG. 2B). Mutations of the target motif abrogated repression of luciferase, indicating that TAP2 is a direct target of miR-BHRF1-3 (FIG. 2B and FIG. 7). Similarly, miR-BART17, which is encoded by field strains of EBV, directly targeted the 3′-UTR of the TAP2 transcript (FIG. 2B and FIG. 7). Therefore, EBV miRNAs down-regulate genes with pivotal functions in peptide antigen processing, transport and presentation early after infection.

Example 6

[0145] Viral miRNAs inhibit the secretion of IL-12 early after infection (FIGS. 1B and 2A). This inhibition blocked differentiation of type 1 helper T (Th1) cells, a process for which IL-12 is critical (Szabo, loc. cit.). Naive CD4.sup.+ T-cells were co-cultured with autologous EBV-infected B-cells (FIG. 3A). The wt/B95-8 EBV-infected B-cells repressed Th1 differentiation compared with ΔmiR EBV-infected cells (FIG. 3B). An antibody that neutralizes the functions of IL12B, but not an isotype control antibody, suppressed Th1 differentiation when the cells were co-cultured with ΔmiR EBV-infected cells (FIG. 3C), indicating that IL-12 secreted from EBV-infected or activated B-cells per se drives the generation of Th1 cells. Thus, EBV miRNAs suppress the release of IL-12 from infected cells, a function that can abrogate antiviral control by virus-specific Th1 cells.

Example 7

[0146] Further, inhibition of MHC class II, co-stimulatory, and adhesion molecules by EBV miRNAs (FIGS. 1B and 2D, E) impaired MHC class II-mediated recognition of infected cells by CD4.sup.+ T-cells. CD4.sup.+ T-cells were expanded ex vivo by repeated stimulation with an irradiated wt/B95-8 EBV-infected autologous lymphoblastoid cell line (LCL). The EBV-specific CD4.sup.+ T-cells were then co-cultured with autologous B-cells that had been infected with the two EBV strains 5 days earlier (FIG. 3D). Release of IFN-γby EBV-specific CD4.sup.+ T-cells was substantial when co-cultured with ΔmiR EBV-infected cells as targets but was consistently reduced when co-cultured with wt/B95-8 EBV-infected B-cells at all cell ratios tested (FIG. 8).

[0147] This effect was observed in autologous and HLA-matched but not in HLA-mismatched situations (FIG. 3E and FIG. 12) indicating that the observed activation of CD4.sup.+ T-cells was HLA class II-restricted. An EBV antigen-specific CD4.sup.+ T-cell clone was tested directed against the FGQ, an epitope from an EBV glycoprotein gp350 (Adhikary, J. Exp. Med. 203, 995-1006 (2006)) and observed reduced T-cell activities with target B-cells infected with wt/B95-8 EBV compared with ΔmiR EBVs five days after infection (FIG. 9). T-cell activity against B-cells was barely detected at 15 days post infection when the viral antigen gp350 was no longer present because it is a component of the virus particle and presented immediately after B-cell infection (Adhikary, loc.cit.) but is not synthesized during latency (Kalla et al. Proc. Natl. Acad. Sci. U.S.A. 107,850-855 (2010)).

[0148] EBV-specific CD4.sup.+ T-cells have cytolytic activity (Adhikary, loc. cit.). In allogeneic HLA-matched conditions, EBV-specific CD4.sup.+ T-cells consistently showed stronger cytolysis of target B-cells infected with ΔmiR EBV than cells infected wt/B95-8 EBV (FIG. 3F). EBV miRNAs clearly inhibited the recognition of infected B-cells by HLA class II-restricted CD4.sup.+ T-cells early after infection.

[0149] It was found also that EBV miRNAs impair recognition of infected B-cells by MHC class I-restricted, EBV-specific CD8.sup.+ T-cells in addition to CD4.sup.+ T-cells. These tests used co-culture assays with EBV-infected B-cells and polyclonal EBV-specific CD8.sup.+ T-cells as well as CD8.sup.+ T-cell clones specific for certain EBV antigens. IFN-γ secretion by the CD8.sup.+ T-cells was measured upon overnight cultivation with primary B-cells that had been infected with the two different EBV strains 15 days earlier (FIG. 4A). In accordance with their HLA restriction (and only in autologous and matched settings), CD8.sup.+ T-cells released IFN-γ after co-culture with ΔmiR EBV-infected B-cells but less so when co-cultured with wt/B95-8 EBV-infected B-cells (FIG. 4B and FIG. 12). Similarly, B-cells infected with ΔmiR EBV were significantly killed by EBV-specific CD8.sup.+ T-cells relative to B-cells infected with wt/B95-8 EBV expressing miRNAs (FIG. 4C). Finally, IFN-γ release of the CD8.sup.+ T-cell clone specific for the IED epitope of viral protein LMP2 presented by HLA-B*40 (FIG. 10) (Lautscham et al., J. Exp. Med. 194,1053-1068 (2001)) was strongly and consistently reduced when co-cultured with wt/B95-8 EBV-infected B-cells compared with ΔmiR EBV-infected B-cells (FIG. 4D). HLA-B*40 but not LMP2 expression was affected by EBV miRNAs (FIG. 4D and FIG. 11). These results suggest that EBV miRNAs control antigen processing and presentation to protect infected B-cells from the recognition by EBV-specific CD8.sup.+ T-cells.

Example 8

[0150] EBV eventually resides in most people in non-proliferating B-cells largely invisible to a host's immune response (Thorley-Lawson, J. Allergy Clin. Immunol. 116, 251-261 (2005)). However, it induces proliferation of the B-cells it initially infects and fosters their survival. It was found that EBV encodes miRNAs that regulate multiple facets of a host's adaptive immune response in newly infected B-cells. EBV-infected B-cells lacking viral miRNAs are deficient both in affecting these responses and in other miRNA-dependent functions including an inhibition of apoptosis (Seto, loc. cit.). These latter defects have precluded comparisons of B-cells newly infected with wt/B95-8 or ΔmiR in humanized mouse models because of the defects in survival of the latter cells (C. Münz, personal communication). Functional assays in culture show compellingly that EBV's miRNAs inhibit the secretion of cytokines, inhibit antigen processing and presentation, inhibit the differentiation of CD4.sup.+ T-cells and their recognition of infected B-cells, and inhibit the recognition of those cells by EBV-specific CD8.sup.+ T-cells. The breadth of EBV's use of its miRNAs to inhibit adaptive and innate immune responses (Nachmani et al., Cell Host Microbe. 5, 376-385 (2009)) is unprecedented and would foster its efficient establishment of a life-long infection.

Example 9

[0151] VLP production was induced by transfection of producer cells as described in Hettich et al. (Gene Therapy, 2006, vol. 13, pages 844-856). The supernatant was filtered through a 1.2 μm filter and concentrated by ultracentrifugation at 100,000×g for 2 hours. Finally, the pellet was resuspended in 1.5 mL PBS.

[0152] A human EBV-immortalized B-cell line (LCL) was plated into a 96-well plate (5*10{circumflex over ( )}4 cells/well) and incubated with VLPs (1*10{circumflex over ( )}4 particles/cell) with miRNAs or lacking all miRNAs (ΔmiRNAs) in a total volume of 200 μl/well. After 24 h of incubation, 100 μl of the culture medium was removed and the cells were washed by adding 100 μl of RPMI without supplements and centrifugation for 5 minutes at 300×g. Again, 100 μl of the medium were removed and LCLs were mixed with an HLA-matched CD4+ T-cell clone (100 μl cell culture medium containing 5*10{circumflex over ( )}4 cells) specific for the EBV tegument protein BNRF1 (ratio LCLs:T cells=1:1) and then co-cultivated for another 24 hours. Activation of T cells was quantified in a IFNγ-ELISA assay according to the manufacturer's protocol (human IFNγ-ELISA development kit (ALP), Mabtech). The assay was performed with 5 technical replicates. Results of the assay are shown in FIG. 13.

[0153] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein the terms “about” and “approximately” means within 10 to 15%, preferably within 5 to 10%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0154] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0155] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0156] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0157] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0158] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.

[0159] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.