Yeast virus-derived stimulator of the innate immune system augments the efficacy of virus vector-based immunotherapy

Abstract

To identify novel stimulators of the innate immune system, we constructed a panel of eight HEK293 cell lines double positive for human Toll-like receptors (TLRs) and an NF-κB-inducible reporter gene. Screening of a large variety of compounds and cellular extracts detected a TLR3-activating compound in a microsomal yeast extract. Fractionation of this extract identified an RNA molecule of 4.6 kb, named nucleic acid band 2 (NAB2), that was sufficient to confer the activation of TLR3. Digests with single- and double-strand-specific RNases showed the double-strand nature of this RNA, and its sequence was found to be identical to that of the genome of the double-stranded RNA (dsRNA) L-BC virus of Saccharomyces cerevisiae. A large-scale process of production and purification of this RNA was established on the basis of chemical cell lysis and dsRNA-specific chromatography. NAB2 complexed with the cationic lipid Lipofectin but neither NAB2 nor Lipofectin alone induced the secretion of interleukin-12(p70) [IL-12(p70)], alpha interferon, gamma interferon-induced protein 10, macrophage inflammatory protein 1β, or IL-6 in human monocyte-derived dendritic cells. While NAB2 activated TLR3, Lipofectin-stabilized NAB2 also signaled via the cytoplasmic sensor for RNA recognition MDA-5. A significant increase of RMA-MUC1 tumor rejection and survival was observed in C57BL/6 mice after prophylactic vaccination with MUC1-encoding modified vaccinia virus Ankara (MVA) and NAB2-Lipofectin. This combination of immunotherapies strongly increased at the injection sites the percentage of infiltrating natural killer (NK) cells and plasmacytoid dendritic cells (pDCs), cell types which can modulate innate and adaptive immune responses.

Importance: Virus-based cancer vaccines offer a good alternative to the treatment of cancer but could be improved. Starting from a screening approach, we have identified and characterized an unexplored biological molecule with immunomodulatory characteristics which augments the efficacy of an MVA-based immunotherapeutic agent. The immune modulator consists of the purified dsRNA genome isolated from a commercially used yeast strain, NAB2, mixed with a cationic lipid, Lipofectin. NAB2-Lipofectin stimulates the immune system via TLR3 and MDA-5. When it was injected at the MVA vaccination site, the immune modulator increased survival in a preclinical tumor model. We could demonstrate that NAB2-Lipofectin augments the MVA-induced infiltration of natural killer and plasmacytoid dendritic cells. We suggest indirect mechanisms of activation of these cell types by the influence of NAB2-Lipofectin on innate and adaptive immunity. Detailed analysis of cell migration at the vaccine injection site and the appropriate choice of an immune modulator should be considered to achieve the rational improvement of virus vector-based vaccination by immune modulators.

FIG 1

(A) (Top) Schematic representation of fractionation of yeast strain S. cerevisiae JC7. Cell lysis followed by differential centrifugation generated SN, a suspension of yeast microsomes. Phenol extraction of SN removed lipids and proteins and resulted in a nucleic acid solution (NA). (Bottom) Next, NA was fractionated by electrophoresis in a native 1% agarose gel. The first and second bands were isolated from the gel and digested in the presence of high-dose RNase A (50 ng/μl). NAB1 was RNase A resistant, while NAB2 and the lower-molecular-mass molecules appearing as smears were digested. Arrow, NAB2; asterisk, lower-molecular-mass molecules. (B) NA, NAB1, and NAB2 were treated with RNase A or not and tested on the HEK-hTLR3-luc cell lines. NAB2, like NA, increased the fold induction of luciferase activity. NAB1 had no effect. Treatment with RNase A abolished TLR3 activation by NA and NAB2. The means ± SEMs of two independent experiments are shown.

FIG 2

Stimulation of IGROV cells knocked down for either RIG-1 or MDA-5. Cells were incubated with NAB2-Lipofectin and transfected with the MDA-5 ligand poly(I·C), the RIG-I ligand 3P-RNA (in vitro-transcribed 5′-triphosphate RNA), and the negative-control RNA poly(A) or poly(dA·dT) ligand to assess TLR-independent stimulation by DNA. Secreted IP-10, a surrogate marker for type I IFN induction, was measured in IGROV cells in which RIG-I (A) and MDA-5 (B) were knocked down, and the results were compared with those for the wild type (wt). The activity of NAB2-Lipofectin to induce IP-10 was abolished in MDA-5-knockdown IGROV cells. The means ± SEMs of two independent experiments are shown. w/o, no ligand treatment.

FIG 3

NAB2 configuration. (A) NAB2 was heat denatured in water and renatured in high-concentration MgCl₂ buffer. Then, NAB2 was digested with the dsRNA-specific RNase III and RNase V1 and single-strand RNA-specific RNase T2. Urea and dyes were added, and the samples were run on a 6% polyacrylamide-urea slab gel under denaturing conditions. Lane 1, NAB2; lane 2, NAB2 digested with RNase III; lane 3, NAB2 digested with RNase T2; lane 4, NAB2 digested with RNase V1; lanes L, RNA ladder (numbers on the left right are in bases). NAB2 is sensitive to the two dsRNA-specific RNases. (B) Electron microscopy analysis of NAB2. Platinum-shadowed nucleic acid molecules were observed in a transmission electron microscope. Analyses revealed a linear molecule without bulges or forks.

FIG 4

Enrichment of NAB2 following the improved purification method for NAB2. Agarose electrophoresis analysis of the different pools was performed after each step of the purification. A total of 1.5 to 15 μl of each pool was loaded on a 1% agarose gel. Lysate (204 ml), supernatant after lysis and centrifugation; 1st CF11 (190 ml), pool after the CF11 capture step; 2nd CF11 (125 ml), pool after the polishing CF11 step, DEAE (7 ml), pool after the DEAE step. The double-stranded DNA markers in the second lane are 23, 9.5, 6.6, 4.4, 2.3, 2, 1.3, 1.1, 0.9, and 0.6 kbp. The 6.6- and 4.4-kbp bands are indicated.

FIG 5

Activation of human moDCs. (A) Human monocyte-derived dendritic cells (7.5 × 10⁵) from three healthy donors were seeded once (donor C) or three times (donors A and B) and incubated overnight with 0.3 μg NAB2, 1.5 μg Lipofectin, or 0.3 μg NAB2 plus 1.5 μg Lipofectin. The TLR4 ligand LPS (5 ng), the TLR7/8 ligand R848 (10⁻⁴ M), and the TLR3 ligand poly(I·C) (0.3 μg) were added as controls. The dendritic cell marker CD209 and the maturation markers CD83 and CD86 were quantified by flow cytometry. (B) The secreted cytokines IFN-α, IP-10, IL12(p70), IL-6, and MIP-1β were quantified by FlowCytomix analysis. The means ± SDs for donors A and B are shown. medFI, median fluorescence intensity.

FIG 6

CD107a degranulation assay with human NK cells. Human NK cells (1 × 10⁶) isolated from two healthy donors (donors D and E) were stimulated directly with 0.3 μg NAB2, 1.5 μg Lipofectin, 0.3 μg NAB2 plus 1.5 μg Lipofectin, R848 (10⁻⁴ M), or poly(I·C) (0.3 μg) or indirectly with 50 μl of supernatant from moDCs (donors A and B) treated with the same substances (Fig. 5). The percentages of CD107a^high NK cells in the presence of K562 cells is shown. The percentage of CD107a^high NK cells in the absence of K562 for all conditions was below 2% (data not shown).

FIG 7

(A) Tumor rejection in C57BL/6 mice vaccinated with MVATG9931 and NAB2-Lipofectin (12 mice per group). (B) NAB2-Lipofectin increases the efficacy of MVATG9931 in four independent experiments. The effect was statistically significant, according to hazard ratio analysis.

FIG 8

Skin infiltration assay. (A) Mice were subcutaneously injected on day 1 (D1) and day 8 (D8) with 5 × 10⁵ PFU of MVATG9931. NAB2-Lipofectin (0.3 μg and 1 μg, respectively) was injected on day 2 (D2) and day 9 (D9). Mice were sacrificed on day 10 (D10). Skin around the injection site was cut out and mechanically dissociated. Suspensions of cells from 10 to 14 injection sites were immunofluorescently stained and analyzed by flow cytometry. (B) The percentages of pDCs macrophages, neutrophils, and NK within the total population were calculated, and the fold induction is expressed on the basis of the values obtained with the negative-control group (buffer injection). (C) The percentage of CD86⁺ pDCs in the total cell population was determined. The means ± SEMs of two independent experiments are shown.

FIG 9

Tumor infiltration assay. C57BL/6 mice injected with buffer, MVATG9931 alone, or MVATG9931 followed by NAB2 or NAB2-Lipofectin were sacrificed between days 11 and 14 after tumor cell implantation. Pooled tumors (10 per group) were mechanically dissociated and separated via density gradient centrifugation into PBMCs (A) and the tumor cell fraction (pellet after density gradient) (B). (A) Living PBMCs were stained for CD4, CD8a, and NKp46 (CD335). (B) Percentages of cleaved caspase 3 (casp 3)-positive cells in the tumor cell fraction. The means ± SEMs of three experiments are shown.