Systems vaccinology analysis of a recombinant vaccinia-based vector reveals diverse innate immune signatures at the injection site

Poxvirus systems have been extensively used as vaccine vectors. Herein a systems vaccinology analysis of intramuscular injection sites provides detailed insights into host innate immune responses, as well as expression of vector and recombinant immunogen genes, after vaccination with a new multiplication defective, vaccinia-based vector, Sementis Copenhagen Vector. Chikungunya and Zika virus immunogen mRNA and protein expression was associated with necrosing skeletal muscle cells surrounded by mixed cellular infiltrates. Adjuvant signatures at 12 hours post-vaccination were dominated by TLR3, 4 and 9, STING, MAVS, PKR and the inflammasome. Th1 cytokine signatures were dominated by IFNγ, TNF and IL1β, and chemokine signatures by CCL5 and CXCL12. Multiple signatures associated with dendritic cell stimulation were evident. By day seven, vaccine transcripts were absent, and cell death, neutrophil, macrophage and inflammation annotations had abated. No compelling arthritis signatures were identified. Such innate systems vaccinology approaches should inform refinements in poxvirus-based vector design.


Introduction
). The bars plotting to ≈0% had values ranging from 0 to 3.5x10 -5 %. (B) RNA-Seq reads from each of the five 168 groups aligned to the house-keeping gene, RPL13A, also expressed as a percentage of the number of reads mapping to the mouse genome. (C) IGV 169 visualization of reads aligned to the recombinant structural polyprotein immunogens of ZIKV (prME) and CHIKV (C-E3-E2-6K-E1), which are encoded in 170 the SCV-ZIKA/CHIK vaccine. All reads from all replicates are shown (for details see (S1A Table). As expected, no reads mapped to the non-structural   Table) divided into non-apoptotic signatures (left) and apoptotic signatures (right). KEGG Pathways (purple),

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Go process (black), Reactome Pathways (brown), UniProt Keywords (green).  Table) provided a set of differentially expressed genes (DEGs) (S2B 201   Table; FDR or q <0.01, fold change >2 and sum of all counts across the six samples >6). The up-202 regulated DEGs (n=1390; S2B Table) were analyzed by Cytoscape (S2C Table), with cell death terms 203 suggesting the presence of apoptosis, necroptosis and necrosis (Fig. 1G). Skeletal muscle cells are 204 generally resistant to apoptosis (Schwartz, 2008(Schwartz, , 2018) and VACV's apoptosis inhibitor, B13R (Chea 205 et al., 2019), was also expressed at the injection site (S1B Table). Skeletal muscle cells have recently 206 been shown to be able to undergo necroptosis (Morgan et al., 2018), with skeletal muscle necrosis well The up-regulated DEGs (for MQ vs SCV12hQ; S2B Table) analyzed as above by Cytoscape returned 216 multiple terms associated with innate immune responses (S2C Table). To provide insights into the 217 early innate host immune responses and potential adjuvant signatures induced by SCV-ZIKA/CHIK 218 vaccination, the full DEG list (1608 genes) for MQ vs SCV12hQ (S2B Table) was analyzed by  Table) illustrated a highly significant Toll-like 221 receptor (TLR) signature, dominated by TLR3 and 4, followed by TLR9, 7 and 2 ( Fig. 2A). Although 222 other TLRs (TLR1, 5, 6, 7/8, 8) were also identified, the number of unique DEGs responsible for these 223 annotations was low ( Fig. 2A, numbers in brackets), arguing these were less reliable USRs as they 224 7 arose from subsets of DEGs already used in the annotations for TLR3, 4, 9, 7 and/or 2 (S2D Table, 225 Target molecules in dataset). Given the common signaling pathways used by all TLRs, primarily 226 involving MyD88 and/or TICAM1/TRIF, overlap in genes induced via the different TLRs would be 227 expected.

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The identification herein of TLR4, TLR3, TLR9, TLR7 and TLR2 signatures ( Fig. 2A) is notably 229 consistent with the literature on poxvirus infections in GMO mice (summarized in S2E Table).   Table) of 1608 257 DEGs identified in quadriceps muscles 12 hours post SCV-ZIKA/CHIK vaccination (MQ vs SCV12hQ; S2B Table)). Circle diameters reflect the number 258 of DEGs associated with each IPA USR annotation. Numbers in brackets indicate the number of unique DEGs associated with each annotation over the total 259 number of DEGs associated with the TLR annotation; circles with colored fills contain >3 DEGs uniquely associated with the indicated TLR annotation.

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A46R is expressed in the cytoplasm of infected cells. (B) Cytosolic sensor signatures identified by IPA USR analysis (S2D Table). Sensors divided into 3 261 categories associated with dsRNA (red circles), DNA (blue circles) and inflammasome activation (black circles). Circle diameters reflect the number of 262 DEGs associated with each annotation. VACV genes encoding cytoplasmic inhibitors are shown in blue, with the black cross indicating that the gene/protein 263 is not present or not functional in SCV (or in the Copenhagen strain of VACV), the red cross indicating that the gene was not detected by RNA-Seq of MQ 264 vs SCV12hQ, the green cross indicate that the activity in mice is unknown (see S1B Table).

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Multiple cytoplasmic sensor signatures at 12 hours post vaccination 268 The IPA analysis of DEGs for MQ vs SCV12hQ (S2D Table, Table).

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Poxviruses encode a number of proteins that seek to limit the activity of host immune responses 286 (S1B Table, yellow highlighting), with some of these inhibiting the activities of cytoplasmic sensors 287 (Fig. 2B, blue text). VACV's decapping enzymes (D9 and D10, encoded by D9R and D10R) are 288 expressed at the vaccination site (S1B Table). Both proteins inhibit dsRNA accumulation, and D10 is      Table). These 3 IRFs are also in the top 5 USRs (sorted by p value) when 333 the "direct" only option was used for the IPA USR analysis (S2D Table, Table).

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B19 mRNA is well expressed at the injection site (S1B Table) and is potentially responsible for the     Table, Table, Table; for references see S2F Table). The

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VACV protein A35 inhibits a number of these mediators (see above), as well as inhibiting class II  Table) 444 and Gene Set Enrichment Analyses (GSEAs) were used to determine whether genes from dendritic cell 445 BTMs were significantly represented in the MQ vs SCV12hQ gene list (S2A Table). The GSEAs 446 provided highly significant results (Fig. 4E), illustrating that signatures associated with dendritic cells 447 and their activities can be readily identified at the injection site 12 hours post vaccination. Such 448 signatures likely underpin the immunogenicity of the vector system.  Table), from which a DEG list (n=1413 genes) was 456 generated (S2H Table) by applying the same filters as above (q <0.01, FC >2 and sum of all counts 457 across the six samples >6). Of the 1413 DEGs, 1337 were up-regulated, with 633 (47%) of these also 458 up-regulated DEGs for MQ vs SCV12hQ.

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Cytoscape analyses of up-regulated DEGs from MQ vs SCV12hQ (S2C Table) were compared with 460 MQ vs SCVd7Q (S2I Table). Multiple top signatures (by FDR) associated with T cells and B cells 461 were substantially more significant on day 7 than at 12 hours ( Fig. 5A; Table S2J). For instance, FDR 462 values associated with the GO Process terms "positive regulation of T cell activation" and "T cell  Table). T cell receptor associated KEGG Pathways and GO Component terms were also 465 more significant on day 7 ( Fig. 5A; S2J Table). "T cell receptor complex" was also the top "GO 466 Cellular Component" term by p value for day 7 up-regulated DEGs (S2J Table, Table).  Table) illustrated that the cell death 479 pathway annotations identified at 12 hours (Fig. 1G) were considerably less significant or absent for 480 day 7 (Fig. 5C, S2K Table). Analysis of the 1413 DEGs from MQ vs SCVd7Q (S2H Table) with IPA 481 Diseases and Functions feature (S2L Table), showed a significant reduction in the z-scores of 482 neutrophil-associated annotations on day 7 when compared to 12 hours ( Fig. 5D; S2M Table). (The

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Cytoscape analysis also showed a highly significant reduction in FDR values for neutrophil terms, S2I  (Fig. 1A). A similar IPA Diseases and Functions analysis of macrophage-associated annotations also 488 illustrated a significant reduction by z-scores (Fig. 5D, S2M Table), further indicating that injection site 489 inflammatory responses were abating by day 7 (Soehnlein et al., 2010). 490 The dominant pro-inflammatory cytokine USRs identified at 12 hours post vaccination (Fig. 4A) 491 were substantially lower by day 7 post vaccination with respect to both -log10 p values and z-scores 492 ( Fig. 5E; S2D vs S2N Table). Fold changes in cytokine mRNA expression levels relative to MQ were 493 15 also substantially lower on day 7 (Fig. 5E), with the exception of IFNγ, which had a fold change 494 relative to MQ of 4.17 at 12 h and a fold change of 5.26 relative to MQ on day 7, perhaps due to the 495 emerging Th1 T cell responses (see above). IPA Diseases and Functions also showed reduced 496 significance and z scores on day 7 for Inflammatory response (-log10 p value 110.7 to 59.8, z-score 7.7 497 to 5.5) and Chronic inflammatory disorder (-log10 p value 70.1 to 38.1, z-score -0.23 to -2.2) (S2L 498   Table). These analyses again argue that inflammation at the injection site is abating on day 7, with  Anotations not identified by the IPA analysis were nominally given a -log10 FDR value of zero (y axis). Color coding as for A, but also Green line -UniProt  Table). Numbers in the circles represent the log2 fold change for that cytokine relative to MQ.  Table) and MQ vs SCVd7Q (S2G Table); bars with red outline indicate significant fold change (q<0.05).

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Loss of neutrophils and presence of eosinophils on day 7 post vaccination 519 H&E staining of the injection sites day 7 post vaccination supports the bioinformatics results described 520 in the previous section. When compared with 12 hours (Fig. 1D), necrotic muscle lesions were largely 521 absent, with the cellular infiltrates less disseminated and more focal (Fig. 5F). In addition, in contrast 522 to 12 hours (Fig. 1E), neutrophils (stained with anti-Ly6G) were not observed in the day 7 cellular 523 infiltrates (Fig. 5G), although the occasional neutrophil could be seen in blood vessels, illustrating that 524 the staining had worked (Fig. 5G, insert, arrowhead). In contrast to Fig. 1E, Apoptag staining was also 525 largely negative on day 7 (not shown). Loss of neutrophils is consistent with inflammation resolution 526 (Soehnlein et al., 2010).

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Another feature of the resolving infiltrates on day 7 post vaccination (clearly evident from H&E 528 staining) was the presence of eosinophils (Fig. 5H), despite the retention of a dominant Th1 signature 529 (Fig. 5E). Many of these cells showed the morphological features of immature band eosinophils, as 530 distinct from segmented mature eosinophils (Fig. 5H).

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At 12 hours post vaccination, genes specific to skeletal muscle were generally slightly down-532 regulated ( Fig. 5I; S2A Table), consistent with the SCV infection-associated necrosis or pyroptosis   Table. 549 To determine whether SCV-ZIKA/CHIK vaccination is associated with the induction of an arthritic 550 signature on day 7 post vaccination, feet were collected (by severing at the bottom of the tibias after 551 euthanasia) and analyzed by RNA-Seq (MF vs SCVd7F) (S2P Table). A DEG list was generated after 552 application of two filters q<0.05 and the sum of counts across all 6 samples >6, resulting in only 22 553 genes, of which 8 were up-regulated (S2Q Table). Three of these genes (Daglb, Tgtp2 and Gbp3) were 554 also present in the up-regulated DEGs for day 7 feet of CHIKV infected mice (S2O Table), although the 555 fold change of the latter two were substantially higher after CHIKV infection than after SCV vaccination  with a negative z-score, and cellular infiltrate terms with low z-scores (Table S2Q). Importantly, the z-562 scores and p-values for these annotations were very much lower for SCVd7F than they were for day 7 563 feet from CHIKV infected mice (Fig. 6B) Table), although with only 22 DEGs such a comparison is somewhat underpowered.

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Overall these results suggest that SCV vaccination was not associated with a compelling arthritic 567 signature, even though the injection sites (quadriceps muscles) were in the same legs as the feet that were 568 used to generate the MF vs SCVd7F gene set.  (Table S2Q). Three of these were also up-regulated DEGs for CHIKV arthritis day 7 post infection (Table S2O; q<0.05). Table) and compared with the same annotations identified by 575 IPA analysis of DEGs for CHIKV arthritis (S2O Table).

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Discussion 578 We provide herein a detailed systems vaccinology analysis of a recombinant SCV vaccine in mouse   (Eldi et al., 2017), perhaps explaining the dominant STING signature (Fig. 2B).

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Virulent poxviruses inhibit STING activation via unknown factors, an inhibitory activity not found for 608 MVA (Georgana et al., 2018). This activity is perhaps similarly absent for SCV or is inactive in mice.

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A desirable feature for any vaccine is the avoidance of reactogenicity, a term describing a series of  (McKay et al., 2019). These chemokines featured prominently at the injection site 617 12 hours after SCV vaccination (Fig. 4B), although fold-change had reduced substantially by day 7 618 (log2 4.12 to -0.73, and 8.35 to 3.17, respectively) (S2A and S2G Table). CCL2 is also induced by  Table). All the core enriched genes (S2R Table) were type I IFN  vector genes that were not expressed in vivo post-vaccination (S1B Table) offer other potential 651 20 insertion sites for recombinant immunogens that would ostensibly have minimal impact on vaccine 652 behavior. However, expression of these genes in human muscle might be checked, perhaps via use of 653 human skeletal muscle organoids (Gholobova et al., 2019). The multiple adjuvant pathway stimulated 654 by SCV (Fig. 2) might argue for a certain level of redundancy (Waibler et al., 2007), which might 655 allow certain inhibitors to be reintroduced with the aim of reducing reactogenicity, without 656 compromising immunogenicity. For instance, B13R (also known as SPI-2) is absent in the other pox vectors) as skeletal muscle does not appear readily to undergo apoptosis (Schwartz, 2008, 669 2018), with B13R also well expressed at the injection site (S1B Table). The absence of the chemokine  Table), with anti-IL-5 therapy used for eosinophilic asthma 692 (Walsh, 2020). Transcripts for eosinophil cationic protein (Ear1) and eosinophil peroxidase (Epx) 693 (granule components of inflammatory eosinophils) were also not detected. Eotaxins (CCL11, CCL24 694 and CCL26) were not up-regulated, with IL4, IL13, IL3 and GM-CSF (CSF2) transcripts absent (S2G 695   Table). We were unable to find any eosinophil gene signatures that provided a significant result after 696 GSEAs of the MQ vs SCVd7Q gene set, suggesting that signatures for inflammatory eosinophils are 697 distinct from tissue repair-associated eosinophils, with the signatures for the latter yet to be defined.

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A limitation of using mice to analyze VACV-based vaccines is that several VACV-encoded 699 21 inhibitors are not active in mice (encoded by M2L, A38L) and the activity of others in mice is not 700 known (D9R, H1L, C10L). The activity in mice of B28R and B29R is also not known, but these 701 inhibitors are not active in the Copenhagen strain of VACV. Others were found not to be expressed in 702 mice (C16L, C23L, C22L), although it is unclear whether they are poorly expressed generally or poorly 703 expressed in muscle or poorly expressed in mice. How critical these genes are to the overall 704 interpretations presented herein is difficult to assess, given the presence of multiple overlapping and 705 potentially cross-compensating pathways.  Mouse genome alignments and differential gene expression 744 Mapping to the mouse genome and differential expression analysis was conducted at AGRF under 745 commercial contract using their in-house pipeline. The quality of the raw sequencing reads were 746 22 assessed using FastQC and MultiQC. Adapters were trimmed using the TrimGalore (0.4.4) program 747 and reads with a length <30 bp or quality <10 were removed. Filtered reads were aligned to the Mus 748 musculus reference genome (mm10; GTF file GRCm38.6annotation release 105) using the STAR 749 aligner (v2.5.3a) with default parameters plus a parameter to restrict multi-mapping reads ('-750 outFilterMultimapNmax 2'). Counts per gene were summarized using the featureCounts (v1.4.6-p5) 751 utility in Subread. A counts matrix was generated from the collective samples using in-house scripts 752 and input to R (3.5.0) for differential expression analysis. Differential expression analysis was 753 undertaken using EdgeR (3.22.3) with default settings and no filters, given the importance of key genes 754 with low transcript abundance (Wilson et al., 2017) and the small percentage of cells infected by SCV-755 ZIKA/CHIK in the quadriceps muscles (with whole quadriceps muscles harvested for RNA-Seq).

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Counts were converted to relative counts (CPM) and normalized using the TMM method and modelled 757 using the likelihood ratio test, glmLRT().   799 We thank the animal house staff and Histology and Imaging Services at QIMR B for their assistance.