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, 31 (48), 5713-21

Highly Immunostimulatory RNA Derived From a Sendai Virus Defective Viral Genome

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Highly Immunostimulatory RNA Derived From a Sendai Virus Defective Viral Genome

Xiomara Mercado-López et al. Vaccine.

Abstract

Defective viral genomes (DVGs) are generated during virus replication. DVGs bearing complementary ends are strong inducers of dendritic cell (DC) maturation and of the expression of antiviral and pro-inflammatory cytokines by triggering signaling of the RIG-I family of intracellular pattern recognition receptors. Our data show that DCs stimulated with virus containing DVGs have an enhanced ability to activate human T cells and can induce adaptive immunity in mice. In addition, we describe the generation of a short Sendai virus (SeV)-derived DVG RNA (DVG-324) that maintains strong immunostimulatory activity in vitro and in vivo. DVG-324 induced high levels of Ifnb expression when transfected into cells and triggered fast expression of pro-inflammatory cytokines and mobilization of dendritic cells when injected into the footpad of mice. Importantly, DVG-324 enhanced the production of antibodies to a prototypic vaccine after a single intramuscular immunization in mice. Notably, the pro-inflammatory cytokine profile induced by DVG-324 was different from that induced by poly I:C, the only viral RNA analog currently used as an immunostimulant in vivo, suggesting a distinct mechanism of action. SeV-derived oligonucleotides represent novel alternatives to be harnessed as potent adjuvants for vaccination.

Keywords: Adjuvant; DPs; DVG; Defective genomes; Dendritic cells; IFN; Immunization; MDA5; RIG-I; RIG-I-like receptors; RLRs; RSV; SeV; SeV HD; SeV LD; SeV depleted of defective particles; SeV high content of defective particles; Sendai virus; defective particles; defective virus genome; interferon; melanoma differentiation-associated protein 5; poly I:C; polyinosinic:polycytidylic acid; respiratory syncytial virus: inRSV, formalin-inactivated RSV; retinoic acid-inducible gene 1.

Figures

Figure 1
Figure 1. Activation of human DCs upon SeV Cantell HD infection induces strong CD4+ T cell response
(A) BMDCs were mock-infected or infected with a MOI=1.5 TCID50/cell of SeV Cantell HD or SeV Cantell LD. Infected cells were harvested 6 h post-infection and total RNA was analyzed by PCR to detect copy-back DVGs and standard viral genomic RNA (gSeV). Our DVG PCR is designed to detect most copy-back genomes generated in infected cells. SeV Cantell HD has one predominant copy-back genome that is seen as an amplicons of 278 nt. (B) Human MDDCs were infected with SeV Cantell HD or SeV Cantell LD (MOI=1.5 TCID50/cell). After 6 h, total RNA was extracted and analyzed by RT-qPCR for the expression of viral Np mRNA and cytokines. Data correspond to the average of five independent experiments. Each experiment was performed in triplicates. Bars correspond to SEM. p<0.0001 (Tnf), p=0.0340 (Il-6), p=0.0082 (Ifnb), p<0.0001 (Il-12p35) by one-way ANOVA. Significance after Bonferroni post-hoc test among different conditions is indicated in the graphs as *=p<0.05, ***= p<0.001, and ****= p<0.0001. Bonferroni denominator = 3. (C) Cytokine proteins were measured from the culture supernatants using ELISA. Data correspond to the average of two independent experiments. Each experiment was performed in triplicates. Bars correspond to SEM. p<0.0001 (Tnf), p=0.0027 (Il-6), p=0.0127 (Ifnb) by one-way ANOVA. Significance after Bonferroni post-hoc test among different conditions is indicated in the graphs as *=p<0.05, and ***= p<0.001. Bonferroni denominator = 3. (D) Activated MDDCs were co-cultured with human allogeneic CD4+ T cells (DCs: T cells ratio=1:5). After 5 days, the supernatant was collected and IFNγ was quantified by ELISA. Phytohaemagglutinin (PHA) was used for CD4+ T cell activation as a positive control. Data correspond to the average of three independent experiments. Each experiment was performed in triplicates. Bars correspond to SEM. p<0.0001 by one-way ANOVA. Significance after Bonferroni post-hoc test is indicated in the graphs as ****= p<0.001. Bonferroni denominator = 5. Data are expressed as copy numbers relative to the housekeeping genes Tuba1b and Rps11.
Figure 2
Figure 2. SeV defective viral particles (DPs) enhance the ability of DCs to promote adaptive immune responses in vivo
(A) Representation of the DC immunization protocol. BMDCs pretreated for 24 h with UV-inactivated IAV were incubated with 125 HA Units of pDPs or mock treated. (B) Expression of viral proteins (Np) mRNA and Il-12p40 was determined from a sample of the cells 2 h post-infection by RT-qPCR. Data correspond to the average of three independent experiments. Each experiment was performed in triplicates. Bars correspond to SEM. p=0.0039 (SeV Np), p=0.0130 (Il-6), p=0.0030 (IL-6 protein) by one-way ANOVA. Significance after Bonferroni post-hoc test among different conditions is indicated in the graphs as *=p<0.05, **= p<0.01. Bonferroni denominator = 3. Data are expressed as copy numbers relative to the housekeeping genes Tuba1b and Rps11. (C) Anti-influenza virus IgG, IgG2b, and IgG1 in sera of mice 14 days after immunization. The experiment was repeated independently three times. Data shown is a compilation of all experiments (total number of mice = 9–14/group). p=0.03 (total IgG), p=0.0367 (IgG2b) by Kruskal-Wallis test. Significance after Dunn’s post-hoc test for each treatment is indicated in the graphs as *=p<0.05. Dunn’s denominator = 3. The data was also reanalyzed after eliminating a maximum of one outlier from each group based on the Grubb’s test with a significance level of 0.05. Significance was maintained at p<0.05 for Total IgG. (D) Number of influenza virus specific IFNγ-producing CD8+ T cells in splenocytes from immunized mice. For these experiments splenocytes from 4 mice per each group were pooled for in vitro restimulation. The experiment was independently repeated twice. Bars correspond to SEM of the assay.
Figure 3
Figure 3. Recombinant SeV copy back DVG preserves strong stimulatory activity
(A) Schematic of the construct for the expression of SeV DVG-546. (B) BHK-21 cells expressing the T7 polymerase (BSR-T7) were infected with partially inactivated SeV 52 and transfected with the plasmid encoding DVG-546. Cells and supernatant were collected 48 h later and inoculated into 10-day embryonated hen eggs. (C) LLC-MK2 cells were infected at a MOI of 5 TCID50/cell with virus obtained from three consecutive passages in eggs (PI-P3) of control SeV 52 alone or SeV 52 in the presence of rDPs. DVG-546 in the infected cells was detected 15 h after infection by PCR. DVG from SeV Cantell was used as a positive control (+). (D) Passages PI-P3 of allantoic fluid from eggs containing control SeV 52 alone or in the presence of rDPs were analyzed for their content of DI particles by determining the ratio of infectious particles over total hemagglutinating particles (HA). Data from each individual egg is shown (n=5–8). p=0.0001 (DVG) by Kruskal-Wallis test. Significance after Dunn’s post-hoc test among different conditions is indicated in the graphs as *=p<0.05 and ***= p<0.001. Dunn’s denominator = 3. (E) LLC-MK2 cells infected with SeV 52 alone or containing rDP (P3) were analyzed by RT-qPCR for the expression of the viral protein Np mRNA and (F) Ifnb mRNA. The experiment was independently repeated more than three times. Data shown correspond to the average of three experiments. p=0.0001 by one-way ANOVA with Bonferroni post-hoc test. Significance after Bonferroni post-hoc test among different conditions is indicated in the graphs as ****=p<0.0001. Bonferroni denominator = 3. Data are expressed as copy numbers relative to the housekeeping genes Tuba1b and Rps11
Figure 4
Figure 4. Naked DVG-derived ivtRNA preserves immunostimulatory activity
(A) Representation of deletion mutants of the DVG genome. Deletions (hatched) were performed in the internal sequence (black) without compromising the DVG complementary ends (grey). (B) The electrophoretic analysis for each ivtRNA was performed in an Agilent's 2100 Bioanalyzer. (C) LLC-MK2 cells were transfected with DVG-546, capped DVG-546 (CAP), DVG-546 treated with alkaline phosphatase (AP) or the different mutants and 6 h post-transfection cells were harvested and total cellular RNA was extracted to determine expression of Ifnb mRNA by RT-qPCR. The experiment was independently repeated three times. Each assay was performed in triplicates. Data corresponds to the average of all experiments (total n = 3–8/group). p=0.0001 by one-way ANOVA with Bonferroni post-hoc test. Significance after Bonferroni post-hoc test among different conditions is indicated in the graphs as ****=p<0.0001, **=p<0.01, and **=p<0.05. Bonferroni denominator = 3. Data are expressed as copy numbers relative to the housekeeping genes Actb and Tuba1b.
Figure 5
Figure 5. DVG-derived naked RNA trigger RIG-I signaling
WT and RIG-I KO MEFs were transfected with 1 µg of CpG or with increasing doses of poly I:C or in vitro transcribed DVG-324 as indicated. Total RNA was extracted 8 h later and expression of Ifnb mRNA was determined by RT-qPCR. Data corresponds to the average of three experiments. p=0.0001 by one-way ANOVA with Bonferroni post-hoc test. Significance after Bonferroni post-hoc test among different conditions is indicated in the graphs as ****=p<0.0001. Bonferroni denominator = 12. Data are expressed as copy numbers relative to the housekeeping genes Actb and Tuba1b.
Figure 6
Figure 6. DVG-derived naked RNA shows strong localized pro-inflammatory activity in mice
Mice were injected subcutaneously in the footpad with 50 µg of DVG-324 or poly I:C (high molecular weight). (A–B) Footpad tissue was harvested after 36 h and RNA was extracted for the analysis of cytokine expression. The experiment was independently repeated three times. Each assay was performed in triplicates. Data shown is a compilation of all experiments. p=0.0192 (Ifnb), p=0.0201 (Il6), p=0.006 (Il1b), p=0.009 (Tnf) and p=0.0001 (Cxcl10) by Kruskal-Wallis test. Significance after Dunn’s post-hoc test among different conditions is indicated in the graphs as *=p<0.05, **=p<0.05 and ***= p<0.001. Dunn’s denominator = 3. To control for experimental variation, data was also analyzed by two-way ANOVA for the parameters treatment/experiment. Significance of variation among treatments are: p=0.0158 (Ifnb), p=0.0002 (Il6), p=<0.0001 (Il1b), p=<0.0001 (Tnf), and p<0.0001 (Cxcl10). (B) Analysis of cytokine expression in mice footpads collected at 6 h post infection. Data shown is an average of two independent experiments (n=4–5/group). Each assay was performed in triplicates. Data are expressed as copy numbers relative to the housekeeping gene rps11. p=0.0357 (Ifnb), p=0.0374 (Il1b), p=0.0490 (Tnf), and p=0.0979 (Cxcl10) by Kruskal-Wallis test. Significance after Dunn’s post-hoc test among different conditions is indicated in the graphs as *=p<0.05. Dunn’s denominator = 3. (C–D) Single cell suspensions from draining popliteal lymph nodes were analyzed by flow cytometry. (C) Percentage of CD11c+CD11b+ cells from the live cell gate is shown (n = 2 for PBS and 5 for poly I:C and DVG 324). p=0.387 by one-way ANOVA with Bonferroni post-hoc test. Bonferroni denominator = 3. (D) CD11c+CD11b+ cells were further gated for expression of CD103 and B220 to quantify CD11c+CD11b+CD103 DCs and CD11bloCD11cloB220+ plasmacytoid DCs. Data show a representative plot for poly I:C treatment and two representative plots for DVG-324 treatment. (E) Antibodies in the sera of Balb/c mice three weeks after immunization with a single i.m. dose of 180 µg of inactivated respiratory syncytial virus (inRSV) in the presence of 50 µg poly I:C, 50 µg DVG-324, or PBS. Sera pre-immunization (pre-bleed: PB) was also analyzed. n=4/group. p=0.0013 (total IgG), p=0.0011 (IgG1), p=0.0013 (IgG2b), and p=0.0008 (IgG2a) by Kruskal-Wallis test. Significance after Dunn’s post-hoc test among different conditions is indicated in the graphs as *=p<0.05, **p<0.01, and ***p<0.001. Dunn’s denominator = 4

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