A single immunization with core-shell structured lipopolyplex mRNA vaccine against rabies induces potent humoral immunity in mice and dogs

The persistence and clinical consequences of rabies virus (RABV) infection have prompted global efforts to develop a safe and effective vaccines against rabies. mRNA vaccines represent a promising option against emerging and re-emerging infectious diseases, gaining particular interest since the outbreak of COVID-19. Herein, we report the development of a highly efficacious rabies mRNA vaccine composed of sequence-modified mRNA encoding RABV glycoprotein (RABV-G) packaged in core-shell structured lipopolyplex (LPP) nanoparticles, named LPP-mRNA-G. The bilayer structure of LPP improves protection and delivery of RABV-G mRNA and allows gradual release of mRNA molecules as the polymer degrades. The unique core-shell structured nanoparticle of LPP-mRNA-G facilitates vaccine uptake and demonstrates a desirable biodistribution pattern with low liver targeting upon intramuscular immunization. Single administration of low-dose LPP-mRNA-G in mice elicited potent humoral immune response and provided complete protection against intracerebral challenge with lethal RABV. Similarly, single immunization of low-dose LPP-mRNA-G induced high levels of virus-neutralizing antibody titers in dogs. Collectively, our data demonstrate the potential of LPP-mRNA-G as a promising next-generation rabies vaccine used in human and companion animals.</p

OVOL expression correlates with the epithelial cell state in multiple human cancer cell lines.

<div><p>(A) qPCR: cDNAs from human primary prostate cancer tissues (n = 40; Origene) was analyzed for the expression of E-cad, OVOL1 and OVOL2. The results were normalized to β-actin and shown relative to the average of all cancer samples for each gene as: .</p> <p>log ((Value of Gene (X) for a Sample) / (Value of Gene (X) for Average Cancer)). The sample values are shown in the dot plots and the correlation of OVOL1 or OVOL2 expression with E-cad was calculated (r). The graph depicts a representative experiment out of two with similar results.</p> <p>(B) Venn diagram: Depicts the RNA-seq results of differentially expressed genes common in the OVOL expressing cells and PC3-Epi, relative to PC3-EMT14. The intersection of A and B represents a common epithelial transcriptional signature of 277 genes. The RNA-seq data was analyzed from at least two biological replicates for each cell line.</p> <p>(C) Dot plot: Expression correlation analyses of the 277 genes identified in the epithelial signature from panel (B). Correlation is depicted between PC3-Epi, PC3-EMT14-OVOL1 or PC3-EMT14-OVOL2.</p> <p>(D) Venn diagram: Compares the results from panel (B) with the genes that correlate with the expression of the OVOLs in 917 human cancer cell lines. From the 129 genes that correlated (r > 0.5) with the OVOLs expression in the 917 cancer cell lines, 67 genes are induced by the expression of both OVOL1 and OVOL2 in PC3-EMT14 (C intersection D).</p> <p>(E) Heat map: ConceptGen analysis of the 67 gene-signature from panel (D) revealed a list of 18 annotated genes with functions related to the epithelial state of the cells.</p> <p>(F) Table: 45 genes negatively correlated with OVOL2 expression (r < -0.5) across 917 cancer cell lines. Among these 45 genes, the 10 shown are also downregulated by OVOL2 expression in PC3-EMT14. Note the TF ZEB1 and the mesenchymal marker vimentin (VIM) are the top genes in this list.</p> <p>See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076773#pone.0076773.s005" target="_blank">Figure S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076773#pone.0076773.s007" target="_blank">Tables S2, S3</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076773#pone.0076773.s009" target="_blank">S4</a>.</p></div

Proposed model of EMT/MET balance in human cancer.

<p>In human cancer cells the mesenchymal and epithelial states are induced and maintained by transcriptional and post-transcriptional (splicing) regulatory programs. These programs are controlled by the feedback regulation between the OVOL and the ZEB1 TFs, critical inducers of MET and EMT respectively. In addition these TFs control the expression of ESRP1, a key-splicing regulator activated in MET and repressed in EMT. Therefore high OVOL and low ZEB1 stabilize the epithelial state decreasing cancer cell invasion and metastasis, and vice versa for the mesenchymal state.</p

Mesenchymal cancer cell populations isolated from co-cultures of epithelial prostate cancer cells and human macrophages.

<div><p>(A) Bright-phase microscopy: morphology changes in the mesenchymal cells PC3-EMT1, PC3-EMT12 and PC3-EMT14 as compared to the epithelial PC3-Epi prostate cancer cells. Scale bars are 200 µm.</p> <p>(B) Immunoblot: Expression of EMT markers in mesenchymal cancer cell lines PC3-EMT1, -EMT2, -EMT12, -EMT14 and -EMT17 compared to epithelial PC3-Epi.</p> <p>(C) Microarray: Gene expression analyses comparing mesenchymal to epithelial cancer cell lines. Venn diagram-I (VD-I) depicts a common EMT-associated signature expressed in the mesenchymal cancer lines PC3-EMT1, -EMT12 and -EMT14 compared to epithelial PC3-Epi. VD-II – Gene signature from VD-I intersected with the signature of ZEB1 silenced cells PC3-EMT1 and -EMT14 using the shRNA-sh4, relative to scramble (Scr) control shRNA.</p> <p>(D) Bright-phase microscopy: PC3-EMT14 cells transfected with ZEB1-shRNAs: sh2, sh4, or Scr. Scale bars are 100 µm.</p> <p>(E) Immunoblot: Expression of EMT markers in PC3-EMT1 and -EMT14 transfected with ZEB1-shRNAs compared to Scr or non-transfected controls.</p> <p>(F) Heat map: 50 genes signature identified in VD-II (panel C). Upregulated genes are in red, downregulated in blue. Fold changes in mesenchymal cancer cells are relative to epithelial PC3-Epi, and in sh4-transfected cells are relative to the Scr control.</p> <p>The immunoblots shown are representative of two independent experiments with similar results. See also Figure S1 and Table S1.</p></div

Mesenchymal cancer cells show increased metastasis while not requiring MET for solid tumor formation.

<div><p>(A) Metastasis: The percentage of ICI-inoculated mice with multiple luciferase signals at 21 and 35 days.</p> <p>(B) Tumor burden: Mice received ICI and were imaged weekly for 35 days. Luciferase expression is represented as regions of interest (ROI-photons/s).</p> <p>(C) IHC: Simultaneous ZEB1 and E-cad staining of metastases detected in the femur and liver of PC3-Epi or PC3-EMT14 injected mice. Note the mesenchymal (ZEB1^high / E-cad^low) (black arrows) and epithelial (ZEB1^low / E-cad^high) (green arrows) cancer cells, depicting the EMT/MET plasticity in subpopulations of PC3-Epi and PC3-EMT14. Scale bars are 100 µm (black) and 20 µm (red).</p> <p>(D) IHC: ZEB1 or E-cad staining of metastasis from the femur of a patient with advanced prostate cancer. Note the mesenchymal-like (ZEB1^high (black arrows)/ E-cad^low (yellow arrows)) and epithelial (ZEB1^low / E-cad^high (green arrows)) cancer cells. Scale bars shown represent 100 µm (black bar) and 50 µm (red bar).</p> <p>(E) Metastasis: The percentage of ICI-inoculated mice with multiple luciferase signals at 21 and 35 days.</p> <p>(F) Tumor burden: Mice received ICI and were imaged weekly for 35 days. Luciferase expression is depicted as regions of interest (ROI-photons/s).</p> <p>(G) IHC: ZEB1 or E-cad staining of metastases in ICI-mice. Note the higher E-cad and lower ZEB1 expression in the metastatic cells expressing OVOL1 or ZEB1-shRNA (sh4). Scale bar represents 100 µm.</p> <p>Graphs show mean +/- sem; p-values were calculated and represented as * p < 0.05; ** p < 0.01. All IHC images are representative of one out of three sections showing similar results. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076773#pone.0076773.s003" target="_blank">Figure S3</a>.</p></div

OVOL1 and OVOL2 induce MET in MDA-MB-231 breast cancer cells.

<div><p>(A) Bright-phase microscopy: morphology changes in the OVO-overexpressing MDA-MB-231 cells towards an epithelial phenotype. Breast cancer cells were transfected with OVOL1, OVOL2 or both TFs. Scales are 100 µm.</p> <p>(B) qPCR: Expression of the epithelial cell markers E-cad and ESRP1, and the EMT-inducing TFs ZEB1, ZEB2, Slug, Snail, and Twist1 in OVOL-overexpressing cells relative to control. Results were normalized to β-actin.</p> <p>(C) Immunoblot: Expression of EMT markers in MDA-MB-231 cells overexpressing OVOL1 and/or OVOL2 (represented by +/- in the table above the blot).</p> <p>(D) Invasion/migration assay: Representative images and graphs of cancer cell invasion using a Boyden chamber assay. Bar graphs depict the lower migratory and invasive potential of cells overexpressing the OVOL TFs. Percent invasion represents the ratio invading/migrating cells. The graph depicts a representative experiment out of three with similar results.</p> <p>(E) qPCR: miRNA expression in MDA-MB-231 cells overexpressing OVOL1, OVOL2, or both, relative to the control. Results were normalized to miR-U6.</p> <p>(F) qPCR: miRNA expression in PC3-Epi, PC3-EMT14-OVOL1 or PC3-EMT14-OVOL2 relative to the control (PC3-EMT14-EV). Results were normalized to miR-16.</p> <p>Graphs show mean +/- sem; p-values were calculated and represented as * p < 0.05; ** p < 0.01; *** p < 0.001. The qPCRs and immunoblots are representative of two independent experiments with similar results.</p></div

OVOL-induced MET reduces the metastatic potential of mesenchymal PC3-EMT14 in vivo.

<div><p>(A) Tumor burden: Luciferase expression is depicted as regions of interest (ROI-photons/s) in mice with orthotopic injections.</p> <p>(B) Metastasis: (Left) the percentage of orthotopically inoculated mice with multiple luciferase signals at 21 and 28 days (Right). depicts the total number of metastases per group divided by the number of mice (n) per group at 28 days.</p> <p>(C) Imaging: Representative images of luciferase expression in mice 28 days after receiving orthotopic injections. Metastases for each group are circled in red in either the ventral or dorsal images and excluding those suspected to correspond to the same tumor.</p> <p>(D) Tumor Weight: The average weights of orthotopic (prostate) tumors resected at 42 days (n = 4).</p> <p>(E) IHC: E-cad, and ZEB1 staining of metastatic tumors in mice that received orthotopic injections from each group inoculated with OVOL expressing cells or control PC3-EMT14. Note the higher E-cad and lower ZEB1 expression in OVOL-expressing cancer cells. Scale bar represents 100 µm. The IHC shows a representative staining of one out of three sections with similar results.</p> <p>Graphs show mean +/- sem; p-values were calculated and represented as ** p < 0.01. See also Figure S4.</p></div

OVOL1 and OVOL2 induce MET in mesenchymal prostate cancer cells.

<div><p>(A) qPCR: mRNA expression in PC3-EMT14-sh4 relative to PC3-EMT14-Scr.</p> <p>(B) Bright-phase microscopy: PC3-EMT14 cells expressing OVOL1 and OVOL2 exhibit an epithelial phenotype as compared to the parental PC3-EMT14. Scale bars are 100 µm.</p> <p>(C) qPCR: Expression of the epithelial cell markers E-cad and ESRP1, and the EMT-inducing TFs ZEB1, ZEB2, Slug, Snail, and Twist1 in PC3-EMT14 cells expressing OVOL1 and OVOL2 relative to the empty-vector (EV) control.</p> <p>(D) Immunoblot: OVOL1 and OVOL2 protein expression in transfected cells was confirmed by using OVOL1 or V5-specific antibodies, respectively.</p> <p>(E) Immunoblot: Expression of epithelial marker E-cad and mesenchymal markers ZEB1 and Vimentin. Changes in Slug expression were minimal and did not correlate with MET. PC3-EMT14 cells expressing IRF6 or ANKRD22, EV and epithelial PC3-Epi are controls.</p> <p>(F) Invasion/migration assay: Representative images and graphs of cancer cell invasion using a Boyden chamber assay. Bar graphs of OVOL1 and OVOL2 expressing PC3-EMT14 cells relative EV. Percent invasion represents the ratio invading/migrating cells. The graph is representative of one out of three independent experiments.</p> <p>(G) qPCR: Expression of PC3-Epi cells transfected with OVOL1-shRNAs (a, b, c) or OVOL2-shRNAs (d, e, f), relative to the non-silencing control PC3-Epi-NS (represented by the dashed line).</p> <p>(H) Immunoblot: PC3-Epi cells transfected with both OVOL1 and OVOL2 shRNAs (sh-OVOL1/2) demonstrate a decrease in E-cad and increase in Vimentin, which suggests a partial EMT transformation.</p> <p>(I) Schematic: The ZEB1 promoter with potential OVOL2 binding sites (orange triangles) according to the general consensus: 5’-A(A/T) (A/T) (C/A) (T/C)GTTA(T/A). Designed TaqMan primer-pairs are shown as black arrows. Numeration is relative to the transcriptional start site (+1). The sequence of a putative OVOL2 binding site that corresponds to the ChIP DNA in the OVOL2 expressing cells is at +320.</p> <p>(J) ChIP qPCR: (Left) shows input chromatin of PC3-EMT14-OVOL2 relative to EV, and demonstrates that similar amounts of DNA were used (Right). depicts the ChIP DNA using OVOL2 antibody. Primers are named after their forward primer (see panel I). Results were normalized to input controls. The graph depicts one representative experiment out of three with similar results.</p> <p>qPCR results were normalized to β –actin. Graphs show mean +/- sem; p-values are represented as * p < 0.05; ** p < 0.01; *** p < 0.001. The qPCRs and immunoblots are representative of two independent experiments with similar results. See also Figure S2.</p></div