Fig 2: SARS-CoV-2 spike DNA antigens protect from viral replication in vivo(A) Mice were immunized once or twice separated by four weeks with 10ug of pS via electroporation. Serum was collected at day 18 post-final immunization. At 35 days post-final immunization mice were infected intranasally with adeno-associated virus expressing human ACE2 (white). 17 days following AAV6-ACE2 transduction, animals were intranasally infected with 1 × 105 PFU of SARS-CoV-2 VIDO-01 P2. Four days post infection, animals were sacrificed to quantify viral replication. SARS-CoV-2 specific serum IgG endpoint titers (B) and pseudoviral neutralization titers (C) at day 18 post-final immunization. Replication competent virus (D), and viral RNA (E) in the lungs four-days post-infection. Pearson correlations between virus titer and serum IgG endpoints (F) and neutralization titers (G). Pearson correlations between viral RNA copies and serum IgG endpoints (H) and neutralization titers (I). Each point represents the average of duplicate samples from an individual animal, bars represent the mean, lines represent the median, and error bars represent the SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 by student's t-test (A and B), or Kruskall-Wallis ANOVA (D and E). Spearman correlations were used to determine relationships (F-I). Data are representative of one experiment with n = 5 males (squares) and 5 females (circles) per group.

Fig 3: Transcriptome profiling of ACE2-expressing and non-expressing HuH7 cells. (a) Principal component analysis of RNA-seq profiles of parental wild-type cells, singly sorted ACE2-negative and ACE2-positive, and serially sorted ACE2-enriched cells. (b) Volcano plots of differentially expressed genes between ACE2-negative and ACE2-positive cells isolated after a single sort. Genes exhibiting abs(log2FC) > 1 and padj < 0.1 by DESeq2 calculation are colored in red. (c) Volcano plot of differentially expressed genes in parental HuH7 cells and serially ACE2-enriched cells. (d) Venn diagram comparing the number of upregulated genes identified in each of the 2 RNA-seq analyses in (b,c). (e) Correlation of log2 fold change for each gene upregulated in 5B in comparison to its magnitude of upregulation in (c). (f) Gene set enrichment analysis depicting the normalized enrichment scores of the 10 most enriched hallmark pathways and transcription factors target sets in singly sorted ACE2-positive cells relative to ACE2-negative cells. The corresponding normalized enrichment score in serially sorted ACE2-enriched cells relative to wild-type cells is also plotted. Asterisk indicates FDR < 25% by GSEA calculation.

Fig 4: ACE2 heterogeneity in HuH7 cells is mediated at the mRNA level. (a) Immunofluorescence confocal microscopy of semi-permeabilized HuH7 cells with endogenous ACE2 (green), GM130 (red), and DAPI (blue) staining. Scale bar = 10 µm. (b) ACE2-positivity as determined by flow cytometry of intact or semi-permeabilized parental HuH7 cells. Dashed line represents background signal in cells stained only with secondary antibody. (c) Immunoblot of endogenous ACE2 (GeneTex #GTX01160) in lysates from FACS-sorted ACE2-positive and ACE2-negative HuH7 cells. (d) ACE2 transcript levels in FACS-sorted ACE2-positive and ACE2-negative HuH7 cells as determined by qRT-PCR. Asterisk indicates p < 0.05 by two-tailed Student’s t test. Error bars represent standard deviation. All ACE2 immunofluorescence and flow cytometry experiments in this figure were performed with R&D #MAB9332.

Fig 5: ACE2 isoform analysis in HuH7 wild-type and ACE2-enriched cells. Sashimi plot for ACE2 transcripts detected by RNA-seq of HuH7 wild-type cells, ACE2-positive cells isolated after a single sort, or serially ACE2-enriched cells. Histograms of read counts mapping to each individual exon and enumeration of reads containing exon-exon junctions are displayed.