The progeroid phenotype of CS was further supported by epigenomic hallmarks of ageing: the prediction of DNAm of repetitive elements suggested an hypomethylation of Alu sequences in CS, and the epigenetic clock returned a marked increase in CS biological age respect to healthy and UVSS cells. The epigenomic remodelling of accelerated ageing in CS displayed both commonalities and differences with other progeroid diseases and regular ageing. CS shared DNAm changes with normal ageing more than other progeroid diseases do, and included genes functionally validated for regular ageing. Collectively, our results support the existence of an epigenomic basis of accelerated ageing in CS and unveil new genes and pathways that are potentially associated with the progeroid/degenerative phenotype.Cockayne syndrome (CS) and UV‐sensitive syndrome (UVSS) are rare genetic disorders caused by mutation of the DNA repair and multifunctional CSA or CSB protein, but only CS patients display a progeroid and neurodegenerative phenotype, providing a unique conceptual and experimental paradigm. As DNA methylation (DNAm) remodelling is a major ageing marker, we performed genome‐wide analysis of DNAm of fibroblasts from healthy, UVSS and CS individuals. Differential analysis highlighted a CS‐specific epigenomic signature  enriched in three categories: developmental transcription factors, ion/neurotransmitter membrane transporters and synaptic neuro‐developmental genes. A large fraction of CS‐specific DNAm changes were associated with expression changes in CS samples, including in previously reported  We studied DNA methylation (DNAm) in a unique human model where impairment of the same protein results or not in a progeroid disease. Functional analyses of DNAm of accelerated ageing reveal enrichment in developmental transcription factors, ion/neurotransmitter transporters, and synaptic neurodevelopmental genes. A large fraction of DNAm changes has been associated with expression changes in multiple patient samples. Progeroid Cockayne syndrome is a pertinent model also to study ageing. CSCockayne syndromeCSACockayne syndrome group A proteinCSBCockayne syndrome group B proteinDMPsdifferentially methylated positionsDMRsdifferentially methylated regionsDNAmDNA methylationGOGene OntologyTFstranscription factorsUVultravioletUVSSUV‐sensitive syndrome1LMNA gene, the Down syndrome (DS) is due to trisomy of chromosome 21. The Werner syndrome (WS) and Cockayne syndrome (CS) result from mutation in multifunctional proteins best known for their role in DNA repair   that is characterized by severe photosensitivity but no sign of premature ageing , driving genomic instability  have been identified . In order to focus on the DNAm alterations that are associated with the age‐accelerated phenotype of CS, we took advantage of the availability of fibroblasts from two very rare UVSS patients, that are mutated but non‐progeroid. We thus included UVSS in the group of ‘Non‐Progeroid’ controls, together with WT cells, and compared them with CS cells. This analysis identified CpG sites mapping in genes enriched in three major functional categories, and, importantly, the most significant of them display a methylation/transcription correlation according to CS transcriptomic datasets. We also demonstrated a dramatic increase of the epigenetic age of CS fibroblasts, and comparison with available datasets revealed a CS epigenetic signature closer to physiological ageing than HGPS, WS or DS, underscoring CS as a relevant model of accelerated ageing. In support of this finding, we report that a majority of top differentially methylated genes have been functionally validated in age‐related processes.22.1CSA) and three healthy subjects (WT), with no siblings, and at similar and early passage number (PN 14). These fibroblasts are available because of single biopsies obtained for diagnostic purposes of the DNA repair defect, and are not repeatable longitudinally. An overview of the study design is reported in Figure ComBat algorithm to adjust for the platform effect, no differences between the 450 k and EPIC samples were observed  and performed again the PCA analysis, which returned a result substantially overlapping the previous one  and, for the seven replicated samples, averaging methylation values from the 450 k and EPIC platforms. PCA and boxplots calculated on this merged dataset are reported in Figure PCA results reported in the previous paragraph suggested the existence of large genome‐wide DNA methylation differences between mutated and WT cells. Among the CpG sites showing DNAm changes, we aimed focusing on those specifically related to the progeroid phenotype that characterizes CS samples (independently from the subtype) and distinguishes them not only from WT, but also from UVSS samples, and thereby identify CS‐specific DNA methylation changes. For this, we compared CpG DNAm between CS‐derived fibroblasts (‘Progeroid’ group) versus WT and UVSS cells, merged in the same ‘Non‐Progeroid’ group, thus prioritizing CS‐specific epigenetic changes (progeroid‐related) and understating those associated with the impaired response to UV, common to CS and UVSS Figure .p‐value < 0.05; absolute difference in mean DNAm values >10%; 2.6% of total probes), 9872 of which were hypomethylated and 1725 hypermethylated in the progeroid group  in Islands . Hypomethylated DMPs were strongly enriched in Open Sea (isolated CpGs in the genome) and Shelves (regions located within 2–4 kbp from CpG islands), whereas hypermethylated DMPs were enriched in Shores (regions located within 0–2 kbp from CpG islands), Shelves and Open Sea regions  between the non‐progeroid and progeroid samples Figure . Out of s Figure . Unsupers Figure . Furtherp‐value < 0.05; Table To strengthen the analysis of genomic regions undergoing DNA methylation changes specifically in CS, we searched for differentially methylated regions (DMRs). We focused on probes located on CpG Islands and surrounding regions (Shores and Shelves) of gene‐associated regions, and applied a pipeline based on a Multivariate Analysis of Variance (MANOVA) of adjacent probes, which has been validated for relevant age‐associated changes  in ageing. Similarly, 43% (9/21) of the top hypermethylated genes have a functional role in ageing, some with a higher number of reports such as RPTOR, HDAC4 and ATP132A , we removed the dispensable terms using REVIGO  on the genes associated with methylated positions (MPs) and methylated regions (MRs) that resulted from the progeroid vs non‐progeroid comparison, using the Genome Ontology (GO) database. GSEA is a method that determines whether an a priori defined set of genes shows statistically significant concordant differences between two biological states (here progeroid versus non‐progeroid). Since both MPs and MRs results displayed high semantic redundancy of significantly enriched GO terms , with GO terms clustered by their semantic proximity  other progeroid syndromes: HGPS (fibroblasts from the GSE196148 dataset), WS . When we considered the entire set of microarray probes . More than 70% of the DMPs common to CS and MSC datasets showed the same direction of epigenetic changes (hyper‐ or hypomethylation compared to the respective controls) while this percentage was lower for HGPS and DS datasets . Forty‐eight DMRs were common to all datasets , 17% (302 DMRs) and 33% (605 DMRs) of CS‐specific DMRs were also significant in HGPS, DS and normal ageing, respectively , and direct correlation for 3 genes  that include genes common to multiple progeroid diseases and normal ageing.Transcriptomic data were not available from our samples; we therefore assessed by RT‐qPCR the expression of several key genes identified as differentially methylated in the progeroid vs non‐progeroid comparison. We selected DMRs according to one or more of the following criteria: the lowest post‐mortem cerebella, and their respective healthy controls. For each dataset, Fisher's exact tests resulted in observed odds ratios greater than 1, revealing a global strong methylation/transcription association in CS ‐derived neurons and mesenchymal stem cells, as well as two datasets from S Figure . Interesa Figure , 60% of a Figure . Importaa Figure . Moreovea Figure .PDE4B), and one, ZIC4, in three datasets  versus WT + UVSS cells (non‐progeroid conditions), and no changes for PDE4B  were differentially expressed in at least one transcriptome dataset Figure . Severals Figure . AlthougB Figure , indicat3The cause of premature ageing features in Cockayne syndrome remains largely unknown, and increasing evidences suggest that the DNA repair defect alone does not explain the progeroid phenotype , which collectively provide robustness to CS‐specific epigenomic signatures  vs ‘Non‐Progeroid’ (WT + UVSS) samples in a differential analysis. We identified 11,597 DMPs in CS, 85% of which were hypomethylated, consistent with a study performed during regular ageing in cultured cells . Using this pipeline and thanks to the pivotal role of UVSS samples (mutated but non‐progeroid), we identified a CS‐specific epigenetic signature highlighting a restricted list of 222 DMRs (64% hypomethylated). Successive steps of selection  obviously restricted the number of hits but, importantly, the main gene categories remained highly represented along the successive analyses (see below). Indeed, the top 20 hypo‐ and hypermethylated associated genes in the 222 StringentDMRs that constitute the CS epigenetic signature, in large part encode transcription factors implicated in developmental processes and membrane transporters  that is, two categories also found in DMRs analyses of single genes as well as pathways (see below). Importantly, the relevance of this concentric strategy to identify ageing‐related differentially methylated gene in CS was confirmed by the observation that half of the top hits correspond to genes functionally validated by experimental and genetic approaches in humans or animal models for playing a role in ageing or age‐related processes. Consequently, the other half of genes constitutes a pool of novel genes potentially involved in ageing or specific to accelerated ageing, revealed by our approach, and which will be necessary to validate in further studies.We do not exclude that our analysis, in particular for StringentDMRs, has been too stringent and relevant genes have been excluded, and/or these findings could be complemented if novel available UVSS fibroblasts at a compatible passage number become available or additional significant CS cases are studied. However, we consider that the major common DNAm differences result from samples derived from patients with different genetic and clinical conditions.i.HOXA9 , TFs of the T‐box  and Fox  families, Wnt transcription regulators , and GATA, SOX, and PAX family members , LHX , NKX  and PITX  families, to our knowledge have not been associated with ageing, possibly being novel developmental regulators factors that play a role in CS as well as in regular ageing. HOXA11, ZIC1, and to some extent HOXA9 demonstrated a strong correlation between DNAm and expression levels in patient fibroblasts, suggesting a functional impact of these CS‐specific methylations changes.Gene enrichment in embryogenesis/developmental transcription factors (dTFs) prevails over all other GO terms, suggesting a major implication in CS‐specific defects, and to a larger extent than observed in other precocious ageing diseases  in the following biological processes/functions: (i) developmental transcription factors and regulators, (ii) ion/neurotransmitter transporters and (iii) synaptic neuro‐developmental genes, in remarkable agreement with single gene analyses above.These findings are consistent with the quasi‐programmed theory of ageing that claims detrimental over‐activity of developmental factors during ageing  involves re‐activation of developmental programmes , which are implicated in regulating the membrane potential in excitable cells like neuronal or muscle cells  that carry a wide variety of substrates including ions, nutrients and/or neurotransmitters through plasma/organelle membranes, also in fibroblasts , tight junctions  and gap junctions . The expression of CLDN1 strongly correlated with DNAm levels in our samples. Cell adhesion factors play an important role in maintaining tissue architecture as well as tissue homeostasis due to their involvement in the formation of epithelial barriers that regulate exchanges, and is therefore not surprising that they are possibly involved in ageing . Among them, the expression of 3.4A general trend in ageing‐related DNAm studies is that changes (hypo‐ or hypermethylation) are not necessarily correlated with changes in gene expression, even in regions with a highly reproducible epigenetic remodelling during ageing that define the ‘methylation clock’ Horvath, . We firspost‐mortem cerebella , underscoring the robustness of our findings. We are aware that other DNAm changes may not be associated with transcription alterations, and further genome‐wide studies will determine case by case when this occurs. Interestingly, this analysis identified expression changes mainly in genes enriched in transmembrane ion channels of the nervous system, in agreement with neurodegeneration observed in CS patients and also during regular ageing  and the European Commission ‘Geneskin: Genetics of human genodermatosis’  approved this study. UVSTA24VI  were a kind gift of Dr. Graciela Spivak, Stanford University, USA. HGPS fibroblasts were from the BioLaM biobank at IGM‐CNR, Bologna, Italy .Patient fibroblasts were derived from skin biopsies excised from unexposed body sites by dermatologists. Patients  provided informed consent to receive diagnosis and genetic analysis. The French Agency of Biomedicine  , as well as the application of a 10% delta methylation cut‐off, was used to identify differentially methylated positions (DMPs). To identify differentially methylated regions (DMRs), we applied multivariate analysis of variance (MANOVA) on sliding windows of three chromosomally adjacent probes, as described in Bacalini et al. . More de5.3MethylGSA  as implemented in 5.4corrplot R package was used to calculate the correlation matrix between DNAm changes in the four datasets. DNAm changes were calculated as the difference between mean DNAm values in diseased samples and DNAm values in controls , or as the difference between mean DNAm values in old samples and DNAm values in young samples (for MSC dataset).Publicly available Infinium450k  and InfiniumEPIC (GSE100825) datasets were downloaded from the GEO database. GSE52114 includes DNAm profiling of mesenchymal stem cells obtained from 22 young individuals (age range 2–29 years) and 12 old individuals (age range 61–92 years). GSE52588 includes DNAm profiling of whole blood DNA from 29 subjects affected by DS (age range 10–43 years), their unaffected siblings (age range 9–52 years) and their mothers (age range 41–83 years). GSE196148 includes DNAm profiling of fibroblasts from one HGPS patient  and two healthy controls . GSE100825 includes DNAm profiling of three subjects affected by WS (age range 44–53 years) and three age‐ and sex‐ matched healthy controls. In each dataset, DNAm differences between progeroid patients and healthy controls were assessed using the same analytical pipeline  used for CS samples. The 5.5p‐value < 0.05 and fold change > 1) where directly extracted from publications. Otherwise, datasets were downloaded from the GEO database. Differential expression analyses were performed on the GSE144557 dataset , analysed results and wrote the manuscript. CCr performed bench experiments and supervised the genome‐wide DNAm experiments. PG and CF provided advice for the biostatistics analysis and, together with AS discussed results and contributed to the manuscript. GL provided unpublished data on DNAm of HGPS fibroblasts, and SH provided the unpublished methylation clock for fibroblasts; both contributed to the manuscript. MR planned the experiments together with CCr, analysed results and wrote the manuscript.This work was supported by Agence Nationale de la Recherche , Institut Pasteur DARRI  and Institut Pasteur PTR (PTR111‐2017).The authors declare that they have no conflict of interests.Data S1:Click here for additional data file.


However, the mechanism through which TNF-α regulates lymphatic metastasis in cervical cancer remains unclear.  Lymphatic metastasis is a common phenomenon of cervical cancer. Tumor necrosis factor-α for 48 h, and then the corresponding conditional medium (CM-TNF-α or CM) was collected. The level of vascular endothelial growth factor (VEGFC) in the corresponding CM was then detected using an enzyme-linked immunosorbent assay (ELISA). Next, human lymphatic endothelial cells (HLECs) were cultured in CM-TNF-α or CM for 48 h. Cell viability was measured using the cell counting kit-8 (CCK-8) assay, and angiogenesis was detected using a tube formation assay. Subsequently, the expressions of AKT, p-AKT, ERK, and p-ERK in HLECs were detected using western blotting. In addition, to further investigate the effect of TNF-α on the progression of cervical cancer, a C33A subcutaneous xenograft model was established in vivo.  In this study, cervical cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with or without TNF-α significantly stimulated cervical cancer cells to secrete VEGFC. Additionally, the CM collected from the TNF-α-treated cervical cancer cells notably promoted the proliferation, migration, and angiogenesis of HLECs; however, these changes were reversed by MAZ51, a VEGFR3 inhibitor. Moreover, TNF-α obviously elevated D2-40 and VEGFC protein expressions in tumor tissues, promoting lymphangiogenesis and lymphatic metastasis in vivo. Meanwhile, TNF-α markedly upregulated p-AKT and p-ERK expressions in tumor tissues, whereas these changes were reversed by MAZ51.  We found that TNF-α could promote tumorigenesis, lymphangiogenesis, and lymphatic metastasis in vitro and in vivo in cervical cancer via activating VEGFC-mediated AKT and ERK pathways. These results may provide new directions for the treatment of cervical cancer. Collectively, TNF- Cervical cancer remains one of the main cancer in women, particularly in developing countries –4. At prTumor microenvironment (TME) refers to the environment around a tumor, including the surrounding blood vessels, immune cells and son on . A promiα (TNF-α) [α can promote tumor development and metastasis [α was able to facilitate the occurrence and development of gastric cancer [α, lymphatic microvessel formation, and tumorigenesis in cervical cancer remains unclear. The aim of the present study was therefore to investigate the mechanism through which TNF-α regulates lymphatic microvessel formation in cervical cancer.The extensively studied pro-inflammatory cytokine, tumor necrosis factor- (TNF-α) , can be  (TNF-α) , 23. Reptastasis  by inductastasis . In addic cancer . Neverth2 at 37°C. HLECs were obtained from Procell and cultured in a medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% endothelial cell growth supplement  [HeLa and C33A human cervical cancer cell lines were purchased from the American Type Culture Collection and cultured in DMEM  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin  with 5% CORESARCH) .α  for 48 h at 37°C, and the corresponding CM (CM-TNF-α) was collected. Meanwhile, in the control group, cervical cancer cell lines were cultured in DMEM for 48 h at 37°C, and the corresponding CM was collected [HeLa and C33A cells were stimulated with different concentrations of TNF-ollected . The CM The level of VEGFC in the CM was detected using a VEGFC assay kit  . The VEGα for 48 h, followed by incubation with 10 μl CCK-8 reagent  for 2 h, and then the absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific) [Cell viability was measured using the CCK-8 assay by culturing HLECs in either CM or CM-TNF-entific) .α for 48 h. Next, HLECs were incubated with 100 μl of 50 μm EdU for 1 h, washed with PBS, and incubated with 1 mg/ml DAPI for 10 min. Next, the EdU-positive HLECs were measured using a fluorescence microscope  [Cell proliferation was measured using an EdU staining assay. The EdU detection kit was purchased from Guangzhou RiboBio (cat. no. C10310-1). Firstly, HLECs were cultured in CM or CM-TNF-® (8 μm pore size) was purchased from Corning, Inc. HLECs were cultured in CM or CM-TNF-α for 48 h. Next, HLECs (1 × 105 cells) were placed in the Matrigel®-coated well at 37°C. Next, the number of tube node of HLECs was observed using a microscope  [The number of tube node formed in the HLECs was observed using a tube formation assay. Matrigel-coated 24-well TranswellOlympus) .5/cell) in a 6-well plate overnight, creating a wound in the monolayer using a 200 μl pipette tip, washing the cells with PBS, and then observing the scratch widths using a microscope  after 0 and 24 h [Cell migration was determined using the wound-healing assay by culturing HLECs  and observed using a microscope  .μg per lane of protein was separated by 10% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). The membranes were then incubated with primary antibodies: anti-AKT , anti-p-AKT , anti-ERK , anti-p-ERK , and anti-GAPDH  overnight at 4°C. GAPDH was used as the internal reference. Next, the membranes were incubated with corresponding HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, an enhanced chemiluminescent substrate kit  was used to observe the protein bands [The total protein from cells and tumor tissues was extracted, the protein concentration was quantified using a BCA Protein Assay Kit  and 40 in bands . Anti-AK7 cells were subcutaneously injected into the left flank of nude mice. Next, when the tumor volume reached ~200 mm3, mice were randomly divided into three groups: control, TNF-α, and TNF-α+MAZ251 groups. TNF-α was intraperitoneally injected into mice in TNF-α and TNF-α+MAZ251 groups three times a week at 54 μg/kg for 3 weeks. In addition, MAZ51 was intraperitoneally injected into mice in the TNF-α+MAZ251 group once a day at 8 mg/kg for 15 days. Meanwhile, normal saline was intraperitoneally injected into mice in the control group. The tumor volume was measured weekly according to the following formula: volume = length × width2/2 [2 (40% volume/min). And the tumors were photographed and weighted. Meanwhile, plasma samples were collected using anticoagulation tubes and then centrifuged for 10 min at 2,000 × g at 4°C.BALB/c nude mice (4-6 weeks old) were provided by Charles River Laboratories, Inc. All animals were maintained following the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. In addition, the experiments of animal study were approved by the Ethics Committee of HY cell biotechnology (No. HY2021-33). C33A cells at the density of 1 × 10width2/2 . In 3 weμl blocking solution was dropped onto the slices at room temperature for 1 h. The section was then incubated with a primary anti-D2-40 or PDPN antibody overnight at 4°C. Next, the section was washed with PBS for 3 times. Subsequently, the section was incubated with secondary antibodies for 50 min at room temperature. DAB was then used for chromogenic reactions. In addition, the slices were placed in a hematoxylin solution for redyeing. Finally, the coverslip was placed over the section, and the staining results were observed using a microscope  [A tumor tissue section was dewaxed with xylene. Antigens from the tumor tissue section were extracted with 0.01 M heated citrate buffers (pH 6.0). Next, 200 oration) . ImageJ ΔΔCt- method [The TRIpure Total RNA Extraction Reagent  was used to isolate the RNA from cells. Then, the EnTurbo™ SYBR Green PCR SuperMix kit  was used to perform RT-PCR. The cycling conditions for qPCR were as follows: 95°C for 3 min, followed by 95°C for 10 s, 58°C for 30 s, and 72°C for 30 s for 40 cycles. The information of primers: GAPDH forward, 5′-CATCATCCCTGCCTCTACTGG-3′ and reverse, 5′-GTGGGTGTCGCTGTTGAAGTC-3′; VEGFC forward, 5′-ACGAGCTACCTCAGCAAGACG-3′ and reverse, 5′-CTCCAGCATCCGAGGAAAAC-3′; D2-40 forward, 5′-CTATAAGTCTGGCTTGACAACTCT-3′ and reverse, 5′-CATCTTTCTCAACTGTTGTCTGTG-3′; VEGFR forward, 5′-GGGCATGTACTGACGATTATGG-3′ and reverse, 5′-GGAGGAATGGCATAGACCGTA-3′. The relative level of VEGFC, D2-40, and VEGFR was calculated using the 2t method , 37.P < 0.05 indicated a statistically significant difference [The statistical analysis was performed using GraphPad Prism software version 7.0 . Data are presented as the mean ± standard deviation and analyzed using a one-way analysis of variance and Tukey's post hoc test. fference , 38.α was found to stimulate cell secretion of VEGF [α obviously upregulated the level of VEGFC in the CM of HeLa and C33A cells in a dose-dependent manner (Figures α promoted the production of VEGFC in cervical cancer cells.It has been reported that VEGFC is the most representative and important factor promoting the formation of tumor lymphangiogenesis , 40. In  of VEGF . Our res Figures . These rα on the formation of lymphangiogenesis in cervical cancer, HLECs were cultured in CM-TNF-α. As indicated in Figures α markedly promoted the viability and proliferation of HLECs. However, these phenomena were reversed in the presence of VEGFR3 inhibitor MAZ51 (Figures α significantly increased the number of tube node formed in HLECs, and that effect was notably inhibited by MAZ51 (Figures α could promote HLEC proliferation and angiogenesis by upregulating VEGFC.With the aim of investigating the effect of TNF- Figures . Meanwhi Figures . All theα on HLEC migration, wound healing, and transwell assays were conducted. The results showed that CM-TNF-α significantly promoted HLEC migration, but MAZ51 clearly inhibited this promotion (Figures α could increase HLEC migration by upregulating VEGFC.In order to study the role of TNF- Figures . These rα regulates the lymphangiogenesis of HLECs, the expressions of p-AKT and p-ERK were evaluated by western blotting. The results indicated that CM-TNF-α markedly increased the levels of p-AKT and p-ERK in HLECs, and these increases were markedly suppressed by MAZ51 (Figures α could promote the expressions of p-AKT and p-ERK in HLECs by upregulating VEGFC.The AKT and ERK signaling pathways have been reported to play an important role in cervical cancer progression , 43. In  Figures . Collectα on cervical cancer tumorigenesis and lymph node metastasis, a C33A subcutaneous xenograft model was established in vivo. As shown in Figures α remarkably promoted tumor volume and weight in C33A subcutaneous xenografts; however, this promotion was clearly inhibited by MAZ51 treatment. In addition, D2-40 has been reported as a specific marker of lymphatic endothelial cells that can be used in the study of lymph node metastasis [α visibly increased D2-40 and PDPN levels in tumor tissues, and these increases were reversed by MAZ51 (Figures Finally, to confirm the effect of TNF-tastasis , 45. The Figures .α significantly increased VEGFC expression in the plasma in vivo, which was reversed by MAZ51 (α visibly increased p-AKT and p-ERK expressions in tumor tissues, but these increases were reversed by MAZ51 (Figures α upregulated the levels of VEGFC and D2-40 in tumor tissues compared with the control group (Figures α had few effects on VEGFR gene expression in tumor tissues (α could promote cervical cancer tumorigenesis, lymphangiogenesis, and lymphatic metastasis in vivo via activating VEGFC-mediated AKT and ERK pathways.Furthermore, ELISA results suggested that TNF-by MAZ51 . Meanwhi Figures . In addi Figures . Meanwhi tissues . In geneα is an important regulator of the inflammatory response [α has been reported to promote tumor development and metastasis [α could promote esophageal cancer cell migration [α could enhance gastric cancer cell migration and invasion via activating NF-κB signaling [α could promote the growth of cervical cancer cells in vivo. Furthermore, this study showed that TNF-α could stimulate cervical cancer cells to secrete VEGFC, which in turn promoted the proliferation, migration, and angiogenesis of HLECs. Therefore, this study was the first to explore the effect of TNF-α on the progression of cervical cancer from the perspective of the TME.TNF-response , 47 thatresponse , 48. Thetastasis , 49. Forigration . In addiignaling . Consistα could upregulate VEGFC expression, promoting lymphangiogenesis and lymphatic metastasis in gallbladder cancer [α-treated cervical cancer cells. CM collected from these cells also promoted the proliferation, migration, and angiogenesis of HLECs; and these changes were reversed by MAZ51, a VEGFR3 inhibitor. Furthermore, TNF-αelevated D2-40 and VEGFC protein expressions in tumor tissues, indicating that TNF-α could promote lymphangiogenesis and lymphatic metastasis of cervical cancer in vivo. Our findings suggest that TNF-α could be apromising target for cervical cancer treatment, as it promote lymphangiogenesis and lymphatic metastasis by upregulating VEGFC.The interaction between cancer cells and lymphatic endothelial cells is crucial in promoting tumor growth and metastasis in the TME, as demonstrated by previous studies , 53. Oner cancer . In our α was found to induce colorectal cancer cell migration and EMT via activating AKT signaling [α could promote triple-negative breast cancer cell metastasis through targeting TNFR2-ERK1/2-EZH2 signaling [α could promote tumor development via modulating AKT and ERK signaling pathways. Additionally, the literature suggested that the AKT and ERK signaling pathways are extensively involved in cervical cancer development [α-treated cervical cancer cells was found to increase p-AKT and p-ERK expressions in HLECs. Moreover, TNF-α could upregulate p-AKT and p-ERK expressions in tumor tissues. However, inhibition of VEGFR3 obviously reversed these changes. All these data suggested that the AKT and ERK signaling pathways are involved in the lymphangiogenesis in cervical cancer.TNF-ignaling . In addiignaling . These felopment –66. For elopment . In addielopment . In the α could inhibit cervical cancer progression by targeting VEGFC-mediated AKT and ERK pathways. Thus, further study is needed to investigate whether TNF-α could affect the progression of cervical cancer via targeting other pathways, such as AMPK/mTOR or the NF-κB signaling pathway [In this study, we only determined that TNF- pathway , 70.αactivatesVEGFC-mediated AKT and ERK pathways, leading to tumorigenesis, lymphangiogenesis, and lymphatic metastasis in vitro and in vivo in cervical cancer. Wehope that our research will provide new directions for the treatment of cervical cancer.To sum up, our studyrevealed that TNF-


The objective of this review is to investigate the commonalities of microvascular  disease in heart failure with preserved ejection fraction (HFpEF) and cerebral small vessel disease (CSVD). Furthermore, the review aims to evaluate the current magnetic resonance imaging (MRI) diagnostic techniques for both conditions. By comparing the two conditions, this review seeks to identify potential opportunities to improve the understanding of both HFpEF and CSVD. There has been significant advancement in the diagnosis and management of heart and brain diseases over the past decades; however, much of this progress has been on macrovascular diseases of the large arteries and veins ,2. MicroHeart failure with preserved ejection fraction (HFpEF) and cerebral small vessel disease (CSVD) are complex conditions that present significant ongoing challenges in both diagnosis and treatment. There are currently limited treatments for either condition, and the lack of effective interventions may be due in part to difficulties understanding the underlying pathophysiology. Additionally, designing clinical trials for HFpEF and CSVD has proven challenging ,6 due toThe objective of this review is to investigate the commonalities of microvascular disease in HFpEF and CSVD. Furthermore, the review aims to evaluate the current magnetic resonance imaging (MRI) diagnostic techniques for both conditions. By comparing the two conditions, this review seeks to gain a greater understanding of both and identify potential opportunities to refine diagnostic approaches.HFpEF has been described as the, “single largest unmet need in cardiovascular medicine,” and comprises 50% of HF cases and admissions ,8. HFpEFThe echocardiographic cutoff LVEF > 50% is somewhat arbitrary  and it is recognised that systolic dysfunction is present in HFpEF; however, LVEF is relatively insensitive to detect mild impairment. Furthermore, recent data  suggest When compared to HF with reduced ejection fraction, HFpEF patients are typically older, female, with a higher burden of diabetes, chronic kidney disease, obesity, and less coronary artery disease. Prognosis is favourable compared to HF with reduced ejection fraction , but theThe underling pathophysiology of HFpEF is incompletely understood with multiple models developed to explain the high diastolic left ventricular stiffness that is a cornerstone finding. Paulus et al.  propose Complex imbalances in nitric oxide pathways associated with both pro-inflammatory stress and mechanical stress   are beliShiattarella et al.  demonstrSeveral modalities, not limited to CMR, are available to assess microvascular disease in the heart. Myocardial biopsy is not routinely undertaken in those presenting with HFpEF, and to date, no modality can directly visualize the coronary microvascular system in vivo. Therefore, current assessment is limited to indirect functional assessment of microvascular function or examination of the downstream structural effects of microvascular disease on the heart.Echocardiography is the standard first-line investigation that assesses structural and functional alterations of HFpEF including left ventricular structure (LVH and remodelling) and function (diastolic relaxation and strain), left atrial dilatation, and elevated pulmonary pressures that occur in response to increased filling pressures. Echocardiography has been used to estimate myocardial blood flow (MBF) through Doppler assessment of the left anterior descending artery on the parasternal short-axis view during hyperaemia and rest, allowing the calculation of myocardial perfusion reserve (MPR) as the ratio between stress and rest. In the PROMIS-HFpEF study , 75% of Stress positron emission tomography (PET) is a well-established method of estimating myocardial blood flow MBF and MPR and has shown similar results to echocardiography with MPR being reduced when compared to controls and hypertensive LVH patients . PET is Invasive coronary angiography is advantageous in excluding significant coronary artery disease and consistently demonstrates impaired MPR (often termed coronary flow reserve)  and elevImaging markers of CSVD  are presCSVD may be asymptomatic and only be identified incidentally on neuroimaging. CSVD has however been associated with various deleterious brain functions including mood disturbance, incontinence, motor and gait disturbance, cognitive impairment, and impaired executive function. Location of the lesion is thought to play an important role in the associated impacted function, with lesions in the left frontotemporal, right parietal, and left thalamus being at greatest risk of subsequent cognitive impairment . CSVD isCSVD is heterogenous and often coexists with Alzheimer’s disease or other forms of dementia (mixed dementia), which can make diagnosis and trial design challenging ,37.Despite advances in neuroimaging and biomarkers, the pathogenesis of CSVD is not well understood, and much of the knowledge has been acquired through animal models and postmortem examination . PatholoDisruption of the BBB  leads toInflammatory responses can lead to enhanced BBB disruption and direct damage of brain tissue . EndotheAs in HFpEF, increased arterial stiffness and elevated pulse pressure may play a role in transferring excess energy to the cerebral microvasculature and tissue. Increased arterial stiffness and elevated pulse pressure have been associated with an increased burden of WMH ,49, cereThe imaging characterisation of CSVD is mainly based on morphological findings that can be made visible by widely available standardized multicontrast MRI protocols, largely avoiding the administration of gadolinium-based contrast agents. Computed tomography (CT) lacks the enhanced soft-tissue characterization of MRI but can reliably identify lacunar infarcts, cerebral atrophy, and the more advanced white matter lesions  and is tOn a broad scale, the heart and the brain share some similarities in both relationship to their histology and vascular supply. Both are terminally differentiated organs with limited ability to replicate or regenerate in the event of injury; furthermore, they require disproportionate amounts of energy and oxygen relative to weight ,53. The Both HFpEF and CSVD are prevalent in the elderly population and are often linked to shared pro-inflammatory cardiovascular risk factors such as hypertension, diabetes, and obesity. A comparison in the pathophysiology uncovers some overlap, suggesting that imbalances in nitric oxide pathways may be a key mechanism in the development of these disorders. Furthermore, a common characteristic in both HFpEF and CSVD is the disturbance in the structure, function, and density of capillaries.A key feature of HFpEF is its female preponderance , and in MRI is widely recognized as the gold-standard technique for assessing CSVD as it provides high-resolution imaging of the brain that reveals the structure changes and abnormalities associated with CSVD including recent small subcortical infarcts (formerly lacunar infarct), white matter hyperintensities (WMH), lacunes, enlarged perivascular spaces, cerebral microbleeds, and cortical cerebral microinfarction. The presumed pathophysiology and imaging features of these are summarized in CMR has established itself as the gold-standard technique for assessing cardiac structure due to its high spatial resolution and freedom from geometric assumptions, which makes it more sensitive to detecting the adverse remodeling seen in HFpEF and provides superior reproducibility compared to echocardiography . The higCMR is a precise and accurate method for assessing LVH, which is a characteristic of HFpEF, being observed in approximately 50–70% of patients. It is also associated with coronary microcirculatory structural and functional abnormalities , and an Although LVEF is preserved (>50%) in HFpEF, this does not exclude significant systolic dysfunction that can be identified through CMR-LV strain analysis. In a cohort of 131 patients with HFpEF, reduced global longitudinal strain was associated with increased MACE on multivariate analysis, while LVEF had no influence . ImpaireAccurate assessment of diastolic function is crucial for the diagnosis of HFpEF, with increasing evidence that it is associated with microvascular disease ,70,71. TWhile many of the parameters assessed by CMR have been associated with microvascular disease, it is important to acknowledge that they do not provide direct measurement of the coronary microvasculature. However, myocardial perfusion CMR is rapidly gaining recognition as a reliable technique for diagnosing, quantifying, and monitoring CMD in patients with HFpEF.The principle underlying myocardial perfusion CMR in HFpEF is that, when there is no significant coronary stenosis, valvular disease, or primary heart muscle disease, inducing hyperaemia through stress or exercise will increase myocardial blood flow (MBF) and that this response is reflective of the function of the coronary microcirculation. The most commonly used stress is adenosine or the A2A adenosine receptor agonist, Regadenason; however, protocols for Dobutamine and exercise exist . During Myocardial perfusion imaging has evolved from a qualitative visual assessment in which reduced MBF was appreciated as a hypointense, to advanced validated quantitative perfusion sequences that automatically segment the myocardium and quantify MBF (in mL/min/g) on a pixel-by-pixel basis ,79. Withp < 0.05) lower in the HFpEF group (2.21 ± 0.6) when compared to hypertensive (3.05 ± 0.7) and healthy controls (3.83 ± 0.7). At-rest MBF was significantly elevated in HFpEF and hypertension when compared to controls, possibly reflecting the increased resting energy demands in these conditions but also their increased left ventricular mass, as the authors did not adjust for that in their estimation of MBF. Also, stress MBF was lower when compared to controls, further reducing MPR.Numerous studies  consistep < 0.01) and higher resting MBF, and lower stress MBF when using a quantitative first-pass perfusion sequence [In another age- and sex-matched cohort, those with HFpEF were found to have significantly reduced global MPR when compared to healthy controls  of obese women with HFpEF and unobstructed coronaries. In relation to diastolic dysfunction, there was a correlation between the stress endocardial/epicardial ratio vs. E/e’  and e’ , indicating that microvascular dysfunction, predominantly of the subendocardium, correlates with diastolic dysfunction, albeit the direction of the relationship is unclear. This finding corroborates with models and invasive study [Markley et al.  recentlyve study ,70, showp < 0.001) in a large (n = 1203) single-centre retrospective cohort [n = 101) prospective cohort [Not only are perfusion abnormalities prevalent in HFpEF, they relate to long-term outcome. Visually assessed perfusion defects are independently associated with MACE (HR 5.6 (3.6–8.6), e cohort . This fie cohort  utilisinDespite advances, there are significant challenges with quantitative perfusion CMR that have limited its widespread clinical adoption; it is not included in any major society guidelines and not available in many nonacademic centres. On a practical level, while the output can be “in-line” and available for review before the end of the scan, time investment is required for quality control of the output . The perp = 0.004) with a reduction in capillary density, indicating that there is an association between MBF and DMF. On CMR, quantification of DMF is achieved through T1 mapping that pre- and post-GBCA can separate the cellular  from the extracellular  compartments, allowing calculation of the extracellular volume fraction (ECV) [p = 0.02), with ECV negatively correlating with MPR , which mirrors the histological correlation of increasing DMF with decreased capillary density [One of the unique capabilities of CMR is to evaluate diffuse myocardial fibrosis (DMF) using T1 mapping and focal fibrosis with LGE sequences. A study of HFpEF patients  using auon (ECV) . Loffleron (ECV)  demonstr density . A simil density , albeit Patients with HFpEF are 10 times more likely to have the presence of nonischaemic focal fibrosis (on LGE) when compared to controls ; howeverAlthough tissue characterisation using CMR provides insight into the potential mechanisms and associations with microvascular disease, the findings from initial studies are not as consistent as for myocardial perfusion CMR. Firstly, there is a current lack of evidence base, and large multicentre prospective studies are required to fully assess the associations between DMF and focal fibrosis on diagnosis, monitoring, and prognosis for HFpEF. For these studies to be successful, trial design will be paramount to overcome the limitations of tissue characterisation via CMR. It is accepted that resolution is limited with a single voxel on CMR incorporating millions of myocytes ; therefoIdentification and quantification of LGE has significant inter- and intra-observer variability, especially when assessing nonischaemic patterns of LGE , which hAccurately characterising CSVD using MRI presents diagnostic challenges that are like those encountered in HFpEF. Nonetheless, MRI has been in use for a longer time and is more deeply integrated into both clinical investigation and research on CSVD. Neuroimaging features associated with CSVD are diverse and nonspecific and are also seen in nonvascular pathologies such as multiple sclerosis or neuroinfection. Inconsistencies in image acquisition protocols, interpretation, and reporting are being tackled by the Standards for Reporting Vascular Changes on Neuroimaging (STRIVE-1 and STRIVE-2) ,33, whicBuilding upon STRIVE, there has been the development of multiple scoring systems for CSVD ,94, in wBrain MRI has several technical advantages over CMR, which allow for improved assessment of brain structure and tissue. Firstly, brain MRI has superior spatial resolution compared to CMR, as it allows for the application of a narrower field of view to the skull when compared to the thorax. Secondly, though internal pulsations exist inside the brain, brain MRI does not have to contend with cardiac motion, which increases precision and reliability and enables superior tissue characterisation compared to CMR. Additionally, no ECG-gating is required, allowing for faster acquisition or the application of longer sequences that would be prohibitively prolonged in CMR. However, complete coverage of the larger brain can be time-consuming and sensitive to minor patient motion, especially as some of the neuroimaging markers are very small and therefore susceptible to patient motion .The previous paragraphs outline some of the advances and advantages of brain MRI over CMR that are relevant to the assessment of CSVD and HFpEF. A comprehensive discussion of standard sequences outlined by STRIVE and the typical neuroimaging features of CSVD is too broad and beyond the scope of this review and can be obtained elsewhere ,97. ThisPerfusion abnormalities are a common finding in CSVD, with an probable inverse relationship between CBF and CSVD severity . There iNote that the abovementioned neuroimaging findings are focal morphological abnormalities (“tip of ice-berg” features) of the brain, while the underlying pathophysiological processes are likely more widely and diffusively spread. To asses more global brain regions, dynamic susceptibility contrast (DSC)  and arteMeta-analysis  combininThe reduction in resting CBF contrasts with the mildly elevated resting MBF detected in HFpEF, which is possibly explained by fundamental functional differences in the heart and brain in relation to perfusion. The brain undergoes neurovascular coupling, which refers to the coordination between neuronal activity and CBF; when neurons are activated, they demand increased oxygen and nutrients, which triggers an increase in CBF to that specific region of the brain. In CSVD, once tissue damage has occurred and neurovascular coupling is impaired , CBF may2-enriched air) [Although HFpEF and CSVD may differ in response to basally required blood flow, they share a significant similarity when it comes to measuring endothelial function in the form of cerebral vascular reserve (CVR) —the ratihed air) ,98 or adAs seen in HFpEF, CVR in CVSD is often reduced when compared to controls. Thrippleton et al.  demonstrAlthough impaired CVR is a promising biomarker in detecting early CSVD before tissue changes and correlates with traditional markers of CSVD severity—presenting a promising parallel with impaired MPR seen in HFpEF—the studies mentioned above have similar limitations as discussed with myocardial perfusion CMR. Many of them are small-scale and single-site, with limited longitudinal follow-up, and there is considerable diversity in the mechanism of inducing stress, image acquisition, and postprocessing. Therefore, although the evidence base is promising, it remains thin, and significant expansion is necessary before drawing firm conclusions.Vessel size (and density) imaging is a research technique that can provide in vivo quantitative estimates of mean vessel diameter and density within a given voxel and is sensitive to large vessels and microvasculature <10 µm in diameter . It explAs with other techniques vessel size imaging is in its infancy for CSVD and further research is required; also, whether this technique can be translated into the heart and overcome motion is to be seen but could be of value.The BBB refers to the properties of the microvasculature of the brain preventing easy exchange between the blood and the tissues, of which there is no analogue inside the myocardium. Pericyte endothelial cells tightly regulate the movement of molecules, ions, and cells between the blood and the brain . As the The main limitations of the technique are the requirement for GBCA, prolonged delay required between GBCA administration and acquisition (>15 min), and the low signal-to-noise ratio of the leakage effect . Newer tDiffusion tensor imaging (DTI) is an advanced imaging technique used to assess the microstructural organization of a tissue by examining the diffusion of water molecules . It provThe comparison between HFpEF and CSVD is becoming increasingly relevant, with some experts suggesting that HFpEF could be considered a “dementia of the heart”,  with botInsights from animal models and autopsy studies ,84,119 iIn both, endothelial dysfunction leading to the disruption in nitric oxide pathways has been identified as a potential common pathway that may be a suitable therapeutic target. Recent CSVD phase 2 study  indicateBoth HFpEF and CSVD face research gaps due to the inability to spatially resolve the microvasculature in vivo. This is critical in the early stages when organ tissue changes and clinical symptoms are not yet present. Functional imaging techniques are emerging as a promising approach to overcome these challenges and provide insights into microvascular functional changes before tissue changes occur.Quantitative perfusion CMR shows promise in reliably assessing the distinctive circumferential subendocardial pattern of impaired MPR in HFpEF . IncorpoImpaired CVR may indicate early CSVD before irreversible tissue damage . This fiComparatively, quantitative perfusion CMR is ahead due to the relative ease of stressing the heart and stress cardiac assessment being a fundamental test of cardiac function with extensive evidence base. It is limited by several factors including: limited availability, lack of standardization, and no formally accepted normative values .Brain MRI excels in tissue characterization, and while this review has focused predominantly on the perfusion changes associated with HFpEF and CSVD, the tissue response is an essential component and cannot be neglected. Future CMR research requires continued effort to characterize myocardial tissue in HFpEF. Given that HFpEF and CSVD may be results of systemic microvascular disease, future research could aim to assess other organs susceptible to microvascular disease or target capillary beds that can be visualized in vivo, such as the retina or sublingually . MicrovaBoth HFpEF and CSVD pose significant challenges in terms of understanding pathophysiology, accurate diagnosis, and development of effective therapies. MRI offers a promising approach for the comprehensive assessment of these disorders, due to its unique noninvasive tissue characterization properties and emerging role in heart and brain functional perfusion imaging. Despite MRI’s potential, there are still limitations that need refinement before translation to clinical practice. MRI needs to be part of a multiparametric clinical assessment, and future research needs to also consider the tissue response to microvascular disease to obtain a complete overview.


Virus-like particles (VLPs) have been proposed as an attractive tool in SARS-CoV-2 vaccine development, both as (1) a vaccine candidate with high immunogenicity and low reactogenicity and (2) a substitute for live virus in functional and neutralization assays. Though multiple SARS-CoV-2 VLP designs have already been explored in Sf9 insect cells, a key parameter ensuring VLPs are a viable platform is the VLP spike yield , which has largely been unreported. In this study, we show that the common strategy of producing SARS-CoV-2 VLPs by expressing spike protein in combination with the native coronavirus membrane and/or envelope protein forms VLPs, but at a critically low spike yield (~0.04–0.08 mg/L). In contrast, fusing the spike ectodomain to the influenza HA transmembrane domain and cytoplasmic tail and co-expressing M1 increased VLP spike yield to ~0.4 mg/L. More importantly, this increased yield translated to a greater VLP spike antigen density (~96 spike monomers/VLP) that more closely resembles that of native SARS-CoV-2 virus (~72–144 Spike monomers/virion). Pseudotyping further allowed for production of functional alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) SARS-CoV-2 VLPs that bound to the target ACE2 receptor. Finally, we demonstrated the utility of pseudotyped VLPs to test neutralizing antibody activity using a simple, acellular ELISA-based assay performed at biosafety level 1 (BSL-1). Taken together, this study highlights the advantage of pseudotyping over native SARS-CoV-2 VLP designs in achieving higher VLP spike yield and demonstrates the usefulness of pseudotyped VLPs as a surrogate for live virus in vaccine and therapeutic development against SARS-CoV-2 variants. COVID-19, the disease caused by the SARS-CoV-2 coronavirus, has led to a significant global burden over the last 3.5 years, totaling >760 M confirmed cases and ~7 M deaths worldwide ,5 of SARIn addition to the need for more effective vaccines, the SARS-CoV-2 vaccine and therapeutic development also face key safety challenges. Working with SARS-CoV-2 virus requires biosafety level 3 (BSL-3) containment, severely limiting the number of experiments that can be performed due to high cost and limited availability of facilities . To addrVirus-like particles (VLPs) represent an attractive platform to serve a dual purpose in SARS-CoV-2 vaccine development, offering several unique advantages both as a vaccine candidate and as a replacement for live virus in assays. SARS-CoV-2 VLPs are commonly produced by co-expressing spike (S), envelope (E), and membrane (M) proteins in a host expression system ,19,20,21While several host expression systems have been investigated to produce SARS-CoV-2 VLPs, including ones utilizing mammalian ,28,29,30In this study, we sought to determine the minimum requirement for native SARS-CoV-2 VLP formation in Sf9 insect cells and quantify the resulting VLP spike yield. Our data demonstrated that co-expressing S protein with either E or M protein resulted in VLP formation, but the VLP spike yield was lower compared to co-expressing all three proteins. However, regardless of the combination, all three showed very low VLP spike yields (<0.1 mg/L). To overcome this limitation, we formed pseudotyped VLPs by co-expressing an S-HA fusion protein with influenza M1, which improved the VLP spike yield approximately fivefold. This improvement further translated to VLPs with a greater antigen density (~96 S monomers/VLP), closely resembling that of native SARS-CoV-2 virus (72–144 S monomers/virion). This pseudotyping strategy also led to the successful production of VLPs for the major circulating variants, including alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529). More importantly, the pseudotyped wild-type and variant VLP spike proteins were all shown to be functional, exhibiting differential affinities for binding with ACE2. Finally, we demonstrated the utility of VLPs to test neutralizing antibody activity in a simple, acellular ELISA assay, highlighting the usefulness of VLPs as SARS-CoV-2 virus surrogates for vaccine and therapeutic development.The majority of SARS-CoV-2 VLP designs reported to date have co-expressed the spike (S), envelope (E), and membrane (M) proteins in insect cells ,19,21,34p > 0.05). Notably, the spike yields for all three native VLP constructs reported here are markedly low, particularly when compared to the influenza VLP hemagglutinin (HA) yield, which typically exceeds 1 mg/L [As the SE and SM VLPs require the Sf9 cells to express one less recombinant protein compared to the SEM VLP, we next examined if this leads to higher spike yields in SE and SM VLPs. The S protein in VLPs was quantified by Western blot analysis using a standard curve generated from purified S protein . The SE,s 1 mg/L . When ths 1 mg/L ,40. Thiss 1 mg/L . Given ts 1 mg/L , we hypoPseudotyping using influenza proteins was previously employed for SARS-CoV-1 VLPs in Sf9 cells, leading to >twofold improvement in VLP spike yield (1 mg/L)  comparedDue to the higher % S protein incorporated into SHAM1 VLPs compared to native SARS-CoV-2 VLPs F, we hypDespite the successful production of several pseudotyped SARS-CoV-2 VLPs using mammalian  and inseVLP designs in insect cells thus far have only incorporated the S protein from the ancestral SARS-CoV-2 strain, and VLPs based on the major circulating variants have yet to be explored using the baculovirus expression vector system in Sf9 cells. Given the rapid emergence of new SARS-CoV-2 variants, we next sought to produce pseudotyped VLPs for several key variants reported to date, including alpha (B.1.1.7), beta B.1.351), delta (B.1.617.2), and omicron (B.1.1.529). These variants contain mutations in the receptor binding domain (RBD) of the S protein, ranging from as few as 1 mutation in alpha to as many as 15 mutations in the case of omicron [1, delta Although all four variants successfully formed pseudotyped VLPs, the S protein band for omicron was much weaker compared to the other three variants C. The WeAs the omicron S RBD has 15 mutations compared to WT A, it is 50  between WT and delta VLPs, in contrast to previous studies using soluble RBDs [SARS-CoV-2 viral infection is initiated by the binding of S protein to its target receptor, angiotensin-converting enzyme 2 (ACE2) . ELISA hble RBDs ,59. A plble RBDs ,61, and ble RBDs ,63. NotaTo eliminate the influence of the avidity effect, we modified the ELISA as depicted in the right panel of 50 values were determined for each of the S:ACE2-Fc interactions. Compared to WT S:ACE2-Fc (EC50 0.7 µg/mL), the delta S:ACE2-Fc EC50 value was ~twofold lower (~0.3 µg/mL), indicating a stronger binding affinity between the delta VLP S protein and ACE2-Fc  was determined to compare the antibody neutralization against both WT and variant VLPs  among all VLPs tested  were grown in Insect XPRESS Media  supplemented with 10 mg/L gentamycin at 27 °C and 135 rpm agitation. DH10Bac cells  were grown in Luria–Bertani (LB) medium containing 50 µg/mL kanamycin, 7 µg/mL gentamicin, and 10 µg/mL tetracycline. Unless otherwise stated, all media and antibiotics were purchased from Thermo Fisher Scientific  and all other chemicals were purchased from Sigma-Aldrich . All primers were purchased from Integrated DNA Technologies .BamHI/HindIII site in plasmid pFastBac Dual to create the intermediary plasmid pFastBac Dual-S. E and M genes were cloned into the XhoI/XmaI site in separate pFastBacDual-S plasmids to create plasmids pFastBacDual-SE and pFastBacDual-SM, respectively. The expression cassette for M including the p10 promoter, M gene, and HSV terminator was then PCR amplified from pFastBac Dual-SM and cloned into the AvrII site of pFastBac Dual-SE to create pFastBac Dual-SEM.The DNA sequences encoding the SARS-CoV-2 S, E, and M proteins were amplified from gBlock fragments purchased from Integrated DNA Technologies. First, the S gene was cloned into the XbaI/HindIII and KpnI/XmaI site in plasmid pFastBac Dual, respectively, to create plasmid pFastBac Dual-H1M1 [XbaI/HindIII site of pFastBac Dual-H1M1, replacing the full-length HA gene to create pFastBac Dual-SHAM1.Previously, influenza HA and M1 genes were cloned into the ual-H1M1 . This plXbaI/ApaI site in pFastBac Dual-SHAM1 plasmid to create pFastBac Dual-SHAM1-Omicron. The templates and primers used for all PCR reactions are listed in Receptor binding domain (RBD) mutations for the SARS-CoV-2 alpha (N501Y), beta , and delta  variants were introduced into separate pFastBac Dual-SHAM1 plasmids using Quikchange mutagenesis  to creatThe recombinant baculovirus genome  was created by transforming each pFastBac Dual plasmid into DH10Bac via transposition. After confirming the recombination events using blue/white colony screening and PCR, the recombinant bacmids were purified using a PureLink HiPure Plasmid Miniprep kit . The purified bacmids were then transfected into Sf9 cells using Cellfectin II (Invitrogen) according to manufacturer’s protocol to generate recombinant baculovirus P1 stocks, which were amplified in Sf9 cells to obtain high-titer P2 baculovirus stocks for use in protein expression and VLP production experiments.Cellular expression of S, E, and M proteins as well as S/HA and M1 was confirmed using Western blot analysis of cell lysates using anti-S , anti-M , anti-E , and anti-M1 . For variant S proteins, anti-S2  was used, as the S2 domain was conserved across all variants. To quantify cellular yields of all S protein constructs, S protein standard  was used. Following primary antibody staining, alkaline phosphatase-conjugated anti-mouse or anti-rabbit IgG secondary antibody (Life Technologies) was used. NBT-BCIP (Thermo Fisher Scientific) was used to develop Western blots. Densitometric analysis of Western blots was performed using a Gel Doc EZ™ Imager  to generate standard curves for S, which were then used to calculate the S cellular expression level.g for 20 min followed by 10,000× g for 20 min. The cleared supernatant was ultracentrifuged at 150,000× g for 2 h, and the pellet containing VLPs was resuspended in PBS containing 40% glycerol. All centrifugation steps were carried out at 4 °C.Influenza VLPs were produced from Sf9 cells infected at an MOI of 3 and harvested 72 hpi. Cell debris was removed from the supernatant by centrifugation at 300× The number of VLPs was quantified using a NanoSight NS300 particle tracking system . Specifically, VLPs were diluted in PBS to manufacturer’s recommended concentrations prior to injection. Videos of 60 s in length were recorded for each sample, and the particle concentration was determined using the nanoparticle tracking analysis (NTA) software provided with the NS300 system. The amount of S in each VLP preparation was quantified by densitometric analysis of Western blots as described in the section above. VLP spike yield (defined as the amount of S protein in VLPs), was determined by densitometric analysis of Western blots as described above. For each VLP construct, the particle concentration and VLP spike yield are represented as the mean of three independent experiments. The spike antigen density (defined as the number of S monomers per VLP) was then determined by using the equation below:Spike is the molecular weight of the spike protein . NA is Avogadro’s number. All appropriate conversion factors were used to calculate the antigen density in units of S monomers per VLP.The MW11 gold nanoparticles/mL for 30 min. Grids were stained with 2% phosphotungstic acid (PTA) and allowed to dry 1 h prior to TEM analysis on a JEM-1400 Transmission Electron Microscope, 80 kV .VLPs were characterized by immunogold labeling analysis using transmission electron microscopy (TEM). Briefly, VLPs were absorbed on Ni grids  and incubated with 20 ng/µL anti-S antibody for 1 h, followed by labeling with protein G—gold nanoparticle (15 nm)—conjugates (Electron Microscopy Sciences) at a concentration of 1050 values were calculated with GraphPad Prism, fitting to a five-parameter logistic curve.VLP binding to ACE2 was analyzed using ELISA. 96-well MaxiSorp plates  were coated with 2 µg/mL of recombinant human ACE2 (carrier free) (BioLegend) overnight at 4 °C. After washing 3× with ELISA wash buffer, wells were blocked with a 1× ELISA diluent  for 2 h at room temperature. VLPs containing 10 µg/mL S protein/well in 1× ELISA diluent were captured for 2 h at room temperature. After washing 3×, recombinant protein ACE2-Fc (BioLegend) was titrated from 0.004–10 µg/mL well for 2 h at room temperature. After washing 3× to remove unbound ACE2-Fc, detection was performed using HRP protein A . After 5× washing, visualization was performed using TMB substrate (BioLegend). Absorbance was read at 620 nm using a SpectraMax M5 plate reader. ECTo confirm that an equivalent amount of VLPs were captured for each sample in the above experiment, three VLP-captured wells for each sample were labeled with anti-S2 , washed 3×, and detected with anti-rabbit IgG-HRP  . Five ti620 signal for VLPs preincubated with and without neutralizing antibody divided by the A620 signal for VLPs preincubated without neutralizing antibody. Following nonlinear fitting, IC50 values were calculated with GraphPad Prism.Neutralization of VLPs was investigated using a similar ELISA setup. First, VLPs containing 5 µg/mL S were incubated with 0.0032–2 µg/mL SARS-CoV-2 (2019-nCoV) Spike-Neutralizing Monoclonal Antibody  overnight at 4 °C. On the same day, plates were coated with 2 µg/mL ACE2/well as described above and stored overnight at 4 °C. The next day, preincubated VLPs were then loaded into the ACE2-coated wells for 2 h. After washing 3× to remove unbound  VLPs, captured VLPs were labeled with anti-S2, detected with anti-rabbit IgG-HRP (1:2000), and visualized with TMB as described above. Percent neutralization of VLPs was calculated as the difference in At test. All data are represented as the mean of three independent experiments and error bars represent the standard error of mean (SE). * p < 0.05, ** p < 0.01, *** p < 0.001; not significant p > 0.05.Statistical analysis was performed using unpaired Student’s


As the COVID-19 pandemic continues to evolve, novel vaccines need to be developed that are readily manufacturable and provide clinical efficacy against emerging SARS-CoV-2 variants. Virus-like particles (VLPs) presenting the spike antigen at their surface offer remarkable benefits over other vaccine antigen formats; however, current SARS-CoV-2 VLP vaccines candidates in clinical development suffer from challenges including low volumetric productivity, poor spike antigen density, expression platform-driven divergent protein glycosylation and complex upstream/downstream processing requirements. Despite their extensive use for therapeutic protein manufacturing and proven ability to produce enveloped VLPs, Chinese Hamster Ovary (CHO) cells are rarely used for the commercial production of VLP-based vaccines.Using CHO cells, we aimed to produce VLPs displaying the full-length SARS-CoV-2 spike. Affinity chromatography was used to capture VLPs released in the culture medium from engineered CHO cells expressing spike. The structure, protein content, and glycosylation of spikes in VLPs were characterized by several biochemical and biophysical methods. In vivo, the generation of neutralizing antibodies and protection against SARS-CoV-2 infection was tested in mouse and hamster models.We demonstrate that spike overexpression in CHO cells is sufficient by itself to generate high VLP titers. These VLPs are evocative of the native virus but with at least three-fold higher spike density. In vivo, purified VLPs elicit strong humoral and cellular immunity at nanogram dose levels which grant protection against SARS-CoV-2 infection.Our results show that CHO cells are amenable to efficient manufacturing of high titers of a potently immunogenic spike protein-based VLP vaccine antigen. Virus-like particles (VLPs) have a structure that is similar to viruses but they cannot cause infection or illness. If VLPs are injected into the body they produce an immune response similar to that seen following infection by a virus. This means that VLPs can be used as vaccines against viruses that cause illness in people. Many drugs, named biologics, are manufactured using living cells, including cells that were originally derived from Chinese Hamster Ovaries (CHO cells). We developed a simple method to produce VLPs similar to the SARS-CoV-2 virus in CHO cells. We show that vaccination of rodents with these VLPs prevents them from becoming ill following infection with SARS-CoV-2. These VLPs could become a part of an alternative, easily produced vaccine for the prevention of COVID-19 in humans. Alpuche-Lazcano et al. produce virus-like particles (VLPs) by the sole expression of the full-length SARS-CoV-2 spike protein in Chinese Hamster Ovary (CHO) cells. Administration of adjuvanted VLPs to mice and hamsters induces the production of neutralizing antibodies, providing effective protection against SARS-CoV-2 infection. Purified and adjuvanted antigen vaccines, which can consist of S proteins (full-length or fragments) such as Nuvaxovid (Novavax) and VidPrevtyn (Sanofi/GSK), or S-decorated VLPs like Covifenz (Medicago), were slower to develop but have gained prominence with recent approvals by regulatory authorities. In this regard, Novavax NVX‑COV2373, containing the ancestral S sequence, has shown after three doses to generate more robust geometric mean titers (GMT) of neutralizing antibodies against the Omicron BA.1 sub-lineage than the BNT162b mRNA vaccine2. Similarly, compared to the original Comirnaty (Pfizer BioNTech), booster doses of VidPrevtyn in different clinical trials were shown to restore immunity against different SARS-CoV-2 variants with higher neutralizing activity against Omicron BA.13. VLPs displaying S protein are structurally reminiscent of wild-type viruses4, promoting high immunogenicity due to their repetitive array of antigens which induces strong Th1/Th2 responses. Additional advantages of VLPs as vaccine antigens include storage stability, strong recognition and uptake by peripheral antigen-presenting cells (APCs); VLPs also eliminate the risk of revertant strains and the potential loss of key neutralizing epitopes associated with attenuated and inactivated viruses8.In pursuit of safe, economical, and effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an array of antigen production and delivery strategies has been explored. In USA and Canada, the first approved vaccines were based on mRNA- or adenoviral vector-induced expression of spike (S) antigen by host cells9, a common approach for inducing VLP formation in mammalian cells. Medicago (Covifenz) achieved regulatory approval in Canada for VLPs produced in N. benthamiana plants by expression of a chimeric construct consisting of the S ectodomain fused to the transmembrane and C-terminal domains of influenza HA11. VLPs can also be produced by co-expression of the coronavirus structural proteins membrane (M), envelope (E), nucleocapsid (N), and S, an approach that results in VLPs more closely resembling the natural virion and which is also undergoing clinical evaluation12.VLPs displaying S antigens are increasingly well-regarded as candidates for clinical SARS-CoV-2 vaccine development. VBI vaccines have completed Phase I trials of a vaccine based on VLPs based on retroviral Gag core protein co-expressed with S protein15, we explored whether these cells, the predominant mammalian cell host for therapeutic protein manufacturing, could also be used to effectively produce SARS-CoV-2 VLPs.The promise of VLPs as SARS-CoV-2 vaccine antigens is counterbalanced by common pitfalls including manufacturability challenges, poor particle yields, low S protein density on particles, and heterologous expression platform-driven non-human-like glycosylation. For these reasons, and because of our success at expressing the S protein at very high levels in Chinese Hamster Ovary (CHO) cellsHerein, we describe how the expression of native full-length SARS-CoV-2 S protein in CHO cells induces the release of abundant S-coated VLPs (S-VLP) without requiring the co-expression of any additional viral structural proteins. These S-VLPs can be produced in CHO cells engineered for abrogated endogenous retrovirus-like particle (ERVLP) production, which could otherwise contaminate the vaccine candidate. Effective purification of S-VLPs was achieved from cell culture supernatants using a one-step affinity chromatography protocol. A variety of biochemical and biophysical assays were performed to characterize these particles in detail, including S protein concentration, particle size, S surface density, and N-glycans identity. Finally, we demonstrated that purified S-VLPs are very potent as vaccine antigens in pre-clinical rodent models, providing effective protection at sub-microgram dose levels with licensed adjuvants. We believe that their good manufacturability, stability, and efficacy features make these VLPs promising antigens for next-generation COVID-19 vaccines.2353™ cell lines have been described previously17. CHO2353 is a clone derived from CHO-DXB11 cells which were selected based on its high production yields of recombinant proteins. A complete description of the origin and selection of this clone can be consulted in ref. 15. Clone CHO-C2 was derived from the parental CHO2353 by targeting and disrupting retroviral gag and env sequences responsible for ERVLP formation and release using CRISPR-Cas9 technology similarly as described in ref. 18. Cells were maintained in a proprietary media formulation in polycarbonate Erlenmeyer flasks with 0.2 µm vent cap (Corning) with constant orbital shaking at 120 rpm in a humidified incubator with 5% CO2 at 37 °C. All cells used in this study were negative for Mycoplasma.NRC CHO13 but without C-terminal epitope tags. All gene sequences were synthesized by GenScript, Piscataway, NJ, USA, and sub-cloned in pTT5® plasmid between EcoRI and BamHI restriction sites. Plasmids were transformed in E. coli DH5α bacteria (Invitrogen) and amplified in CircleGrow medium  supplemented with 100 µg/mL ampicillin (Gibco). Plasmids were purified using a proprietary anion exchange chromatography method followed by isopropanol precipitation, ethanol wash, and 0.22 µm filtration. DNA quantification was carried out using a DeNovix DS-11+ spectrophotometer. Sequences were verified by Sanger sequencing using an ABI 3500xl genetic analyzer.The coding sequences for SARS-CoV-2 S, M, and E were obtained from the genome ancestral sequence NC_045512. All S protein sequences contained mutations at the furin cleavage site , stabilizing prolines , and C-terminal HA epitope-tag (YPYDVPDYA). Expression constructs for spikes were prepared by re-synthesizing and replacing restriction fragments encompassing mutations present in Beta B.1.351 , Delta B.1.617.2  and Omicron B.1.1.529  variants. The SmT2v3 construct consists of the ectodomain of the SARS-CoV-2 ancestral strain S fused to T4 foldon and is identical to the ECDm-T4-Fib construct previously described13. Individual structural SARS-CoV-2 gene transfections were performed in CHO media at 85% (w:w) of pTT5-Spike-HA, 10% Bcl-XL plasmid (anti-apoptotic effector) and 5% GFP plasmid. Co-transfection of structural SARS-CoV-2 genes was performed using a mixture of plasmids comprising 42.5% pTT5-membrane-envelope (M-E), 42.5% pTT5-Spike-HA, 10% Bcl-XL and 5% GFP diluted in CHO complete media.CHO cell transfection methods were performed as described in ref. 2, and incubated for 1 h at 37 °C. For VLP sedimentation, treated supernatants were centrifuged over a 15% Optiprep (w/v) (Sigma-Aldrich)19 cushion  at 5300 × g for 16 h at 4 °C. After centrifugation, supernatants were discarded and pellets resuspended in DPBS (Cytiva).Five days post-transfection, cell suspensions (viability ≥95%) were centrifuged for 15 min at 3200 rpm on a Sorvall legend RT centrifuge (Thermo Fisher Scientific). Cell supernatants were then treated with 10 U/mL of Denarase (C-LEcta), 5 mM of MgCl2 pH 6.0 followed by desalting into DPBS using NAP25 columns. Desalted VLPs were filter-sterilized and tested for endotoxins  prior to in vivo experiments. The SmT2v3 soluble S ectodomain was produced from stable CHO cells as described previously14 and purified using the NGL COVID-19 S Affinity resin (Repligen) following the manufacturer’s instructions.Denarase-treated CHO-C2 supernatants were directly purified on AVIPure-COV2S VLP (AVIpure) resin (Avitide) packed in a BioRad PolyPrep chromatography column. AVIpure column was first equilibrated with 5 column volumes (CV) of DPBS. Next, the treated supernatant was loaded at a constant flow rate of 1 mL/min. The column was then washed with 5 CVs of DPBS. VLP was eluted with 50 mM Bis–Tris, 1 M MgClSedimented or purified VLPs were lysed with RIPA buffer  supplemented with protease inhibitor cocktail . VLP samples in RIPA buffer were incubated at 4 °C for 20 min followed by 15 min centrifugation at top speed in a benchtop centrifuge (Eppendorf 5427R). Pellets were discarded and supernatants recovered. Protein quantification was performed with Pierce bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific). In brief, 200 µL of BCA working reagent prepared according to the manufacturer’s protocol was added to a 96-well plate and mixed with 25 µL of bovine serum albumin (BSA) standards (Thermo Fischer Scientific), sedimented or purified VLP lysates, and incubated for 30 min at 37 °C. Absorbance at 562 nm was measured using a SpectraMax 340PC spectrophotometer.20 using a ChemiDoc MP imaging system (Bio-Rad) and Image Lab 6.1 software.Following protein quantification, 0.7 µg of total protein from VLPs or 7.5 µL of Denarase-treated supernatants (unless otherwise indicated) were mixed (3:1 [v:v]) with XT sample buffer 4X (Bio-Rad) supplemented with 200 mM dithiothreitol (DTT), heat-denatured at 70 °C for 10 min under reducing conditions followed by separation of proteins by SDS–PAGE using NuPAGE 4–12% Bis–Tris gels. Total protein staining (Coomassie Blue) was performed using standard methods. VLP S protein concentration was determined by the comparison to an NRC Metrology soluble S trimer standard (STD) Reference Material SMT1-1For immunoblotting, proteins separated by SDS–PAGE were transferred to nitrocellulose membranes using a Trans-Blot Turbo (Bio-Rad) apparatus with default settings. Membranes were blocked for 30 min with 5% milk in DPBS with 0.1% Tween 20 (DPBS-T) followed by three 5-min washes with DPBS-T. Membranes were then incubated for 1 h at room temperature with anti-S (S1) (ProSci #9083), anti-membrane (ProSci #3529), or a mouse anti-ERVLP-Gag p30 (produced in-house) at a 1:1000 dilution. Primary antibody incubation was followed by three 5-min washes with DPBS-T and incubation for 1 h with horseradish peroxidase-conjugated secondary antibodies at a 1/10,000 dilution for goat anti-rabbit (Jackson 111-035-003) and 1/5000 dilution for goat anti-mouse (Sigma A2304). After three additional 5-minute washes with DPBS-T, the bands were visualized with Clarity Western ECL Substrate (Bio-Rad) and ChemiDoc MP imaging system. All uncropped gel/blot images can be consulted in Supplementary Fig The ELISA method to detect S-VLP binding to hACE2 is provided in the CHO HCP quantification was determined by ELISA using the CHO Cygnus kit (Cat # F550-1) according to the manufacturer’s protocol.Observation of VLPs by transmission electron microscope  equipped with bottom-mounted AMT NanoSprint 12MP camera and operating at 80 kV in high-contrast mode, was performed using negative staining. TEM grids  were freshly glow-discharged using EMS GloQube-D, Dual chamber glow discharge system (Electron Microscopy Sciences) in negative mode with a plasma current of 25 mA during 45 s. Such grids were floated on 10 µL sample aliquots on Parafilm for 5 min. The excess droplets were subsequently wicked away from the edge of the grid with filter paper strips (Whatman 541). The grid was then rinsed three times with three droplets of double distilled water each time removing the excess. Immediately after the last rinse, the grid was exposed to the staining solution (1% uranyl formate) for 60 s and the stain was carefully removed using a fresh piece of filter paper. Finally, the grid was dried at room temperature for 2 h and used for TEM analysis.21 with a Leica-EM GP2 plunger. TEM grids  were treated with a Harrick PDC-32G plasma cleaner with a plasma power of 18 W for 60 s. Such grids were picked up with adapter-attached forceps and loaded in an environmental chamber (humidity ~80%) of the Leica-EM GP2 unit. 5 µL of the aqueous sample was applied on the carbon-film side of the TEM grid and blotted with a filter paper (Whatman #1) for 1.5 s. The grid was then plunged into liquid ethane with a temperature of −180 °C and transferred to a cryo-sample box in liquid nitrogen (LN2). The sample box was further transferred in LN2 and loaded to a pre-cooled (−180 °C) Gatan 914 cryo-specimen holder in a workstation filled with LN2. The holder with the cryo-grid protected under an anti-frost cover was inserted in the TEM column. Observation of the cryo-plunged VLPs sample was carried out on a JEOL 2200 FS TEM, operating at 200 kV, equipped with a field emission filament. Image collections were under low dose mode22 to mitigate beam damage. A 10 eV wide energy filter (with only zero energy loss electrons contributing to image formation) was used to enhance image contrast. Each image was collected with an exposure time between 0.5–1 s based on the local vitreous ice thickness and the microscope settings. Images were processed with the built-in functions  of Gatan DigitalMicrograph software.S-VLP samples were prepared by cryo-plungeS protein quantification at the VLP surface and modeling method are described in the  S-VLPs purified material and mock samples contained in DPBS were analyzed by NTA with a NanoSight NS 5000  equipped with a laser wavelength of 532 nm and a 565 nm long-pass filter. Two sets of data were acquired. Parameters for NTA measurements of samples were performed as follows: VLPs and polystyrene beads were captured 3 times (1 independent preparation) at a temperature of 20 °C with the camera Level at 16 . The following analysis settings were used to process the acquired data; Detection Threshold 3; Blur Size and Max Jump Distance were Auto. VLP samples were diluted in DPBS at a minimal final volume of 1 mL to allow replicate injection. Aspiration of the sample into the system was done using the integrated peristaltic pump connected to the tubing. A minimal volume of 1.8 mL was prepared for each sample to allow technical replicate analysis. NanoSight NS 5000 calibration was carried out by loading 100 nm polystyrene beads, 1/100,000 (Thermo Fisher Scientific) with a predetermined acceptance criteria of 15–90 particles per frame and a ≤15% of CV between injections from the same preparation.Mock samples were diluted in DPBS at 1:475. S-VLPs were diluted at 1:500, 1:1000, 1:1250, 1:1500, and 1:2000. Image processing, particle size distribution, and concentration calculations were performed with NanoSight NTA 3.2 Analytical Software  with a finite track length adjustment (FTLA) fitting model.S protein glycosylation analysis method for S-VLPs or S protein is described in detail in the Female C57BL/6 mice (6–8 weeks old) and Syrian golden hamsters (81–90 g) were purchased from Charles River Laboratories . Animals were maintained at the animal facility of the National Research Council Canada (NRC) in accordance with the guidelines of the Canadian Council on Animal Care. All procedures performed on animals in this study were approved by our Institutional Review Board  and covered under animal use protocol 2020.06 and 2020.10. All experiments were carried out in accordance with the ARRIVE guidelines. Findings in the present manuscript do not apply to a particular sex, but female rodents were preferred because they tend to be less aggressive during manipulation and socialize better between them.n = 10 per group) of mice and four groups (n = 6 per group) of hamsters were immunized with different formulations to evaluate the immunogenicity of S-VLPs. In brief, affinity-purified S-VLPs and adjuvant vaccine components were admixed and diluted in PBS (Thermo Fisher Scientific) prior to administration in a final volume of 50 µL per dose. Adju-phos (Invivogen) dose levels were based on data from previous studies with 50 µg Al3+ included per dose23. AS01b  based on the saponin QS-21 and TLR4 agonist monophosphoryl lipid A (MPL) was used at 1/20th of the human dose.Eight groups  and monitored for body weight change for the next five consecutive days. On day 40, animals were euthanized by exposure to CO2 (following anesthesia by isofurane) and lung tissues were collected for assessment of viral load by plaque assay and viral RNA detection and quantification by RT-qPCR.Animals were immunized by intramuscular (i.m.) injection (50 µL) into the left tibialis anterior (T.A.) muscle on days 0 and 21. Vaccine formulations of 3 µg of S-VLPs alone, or 3, 0.3, and 0.06 µg of adjuvanted S-VLPs were administrated to mice. Vaccine formulations for hamster studies consisted of: PBS vehicle (naïve), 0.3 µg of S-VLPs, AS01b alone, and 0.3 µg S-VLPs+AS01b. S-VLP quantities are based on the amount of S protein present in the purified particles and adjuvant amounts were kept constant regardless of antigen dose. On day 28, mice were anesthetized with isoflurane and then euthanized by cervical dislocation prior to the collection of spleens for measurement of cellular immune responses by IFN-γ ELISpot. Mice were bled via the submandibular vein on days 20 and 28 with recovered serum used for quantification of antigen-specific IgG antibody levels and neutralization assays. On day 35 hamsters were challenged intranasally by 1 × 1023 and can be consulted in detail in the The method to determine total anti-S IgGs has been described previously24 and is described in the The surrogate neutralization assay was performed as in ref. 25.Viral RNA (vRNA) was isolated from lung tissue homogenized in 1 mL RNA/DNA Shield (Zymo Research). vRNA was extracted under BSL-3 conditions with Quick-RNA Viral Kit (Zymo Research). Viral genomic RNA was quantified by real-time-PCR to the target viral envelope gene as previously described23. Briefly, the whole left lungs from each hamster were individually homogenized in 1 mL PBS and centrifuged. The clarified supernatants were then diluted 1 in 10 serial dilutions in infection media . Next, Vero E6 cells were infected for 1 h at 37 °C before the inoculum was removed and overlay media was added, which consisted of infection media with 0.6% ultrapure, low-melting point agarose. Infected cells were incubated at 37 °C with 5% of CO2 for 72 h. After incubation time, cells were fixed with 10% formaldehyde and stained with crystal violet. Plaques were enumerated and PFU was determined per gram of lung tissue.This assay was carried out in containment level 3 facilities (CL3) as previously describedn = 10 per group) of mice and four groups (n = 6 per group) of hamsters were assessed for in vivo experiments. A one-way ANOVA with Tukey’s multiple comparison tests were performed to assess the significance of total anti-S IgG and quantification of spike-specific T cells analyses. A one-way ANOVA with Dunnette’s multiple comparison tests was performed to assess the significance of cell-based surrogate neutralization assays, virus quantification by plaque assay, and viral RNA quantification in hamster lungs. p ≤ 0.05 was regarded as statistically significant.All graphs and statistical analyses in the figures presented in this manuscript were performed by GraphPad Prism 9.3.1 (GraphPad Software). Eight groups  averaged 8.5 ± 0.4 mg/L , based on S protein content of iodixanol-sedimented particles  and in-house preparations of beads conjugated to S antibodies or recombinant human ACE2 (hACE2). All of the tested resins effectively bound VLPs, but their elution under non-denaturing conditions was quite inefficient. To address this issue, an S affinity resin with lower ligand density was generated by Avitide (AVIPure COV2S VLP). This resin permits effective capture and gentle elution of S-VLPs, which could then be buffer-exchanged and sterilized by 0.2 µm filtration for in vivo testing. Compared to iodixanol sedimented samples, the affinity-purified S-VLPs contain visibly lower levels of non-S proteins by total protein staining and show a high particle concentration with preserved morphology by TEM Fig. . To eval3 ppm HCPs  (Table 10), this would correspond to 0.17 µg HCPs in S-VLPs. By comparison, the ChAdOx1 nCov19 vaccine (AstraZeneca) has been reported to contain substantially more HCPs per dose32. Our CHO-C2-based production method followed by one-step affinity purification yielded 2.4 ± 0.3 mg/L (SEM) of VLPs based on S protein content  with much lower total levels of S protein (0.015 mg of S in VLPs purified from one liter of HEK293 supernatant31). In such cases, Gag is by far the most abundant protein in the final, purified VLP product (compared to S-VLPs which are composed of >80% S protein); although Gag-S-based VLPs have been shown to be effective immunogens in several studies, we believe that the higher S density and reduced levels of non-relevant structural proteins are a likely advantage of our VLP platform.Using a CHO host cell protein (HCP) ELISA kit as an orthogonal assay to assess purity, we determined that one-step affinity-purified VLP preparations contain approximately 47 × 10ent Fig. . Notably13 particles with a median width of 119–128 nm. In comparison, purified SARS-CoV-2 VLPs generated in insect cells by the expression of M + E + S or Gag-S VLPs from HEK293 cells yielded up to 5.8 × 1011 and 6.03 × 1010 particles per 1 L of culture respectively33. Of note, S-VLP particle concentrations remained constant during storage for at least 100 days at 4 °C, indicating very good storage stability  and assessed stability during storage at 4 °C. S-VLPs purified from a 1 L culture contained 1.38 × 1034. In this regard, antigen density on the pathogen or particle’s surface along with its glycosylation pattern may impact immunogenicity and thus, the resulting immune response35. Negative stain TEM imaging indicates that S-VLPs contain more S on their surface , we then calculated the real Euclidian distance which generated equidistant points between spikes  injection favors rapid recognition and uptake of vaccine antigens by peripheral APCsace Fig.  than SAR nm Fig. , similarkes Fig. . This al38. Glycan composition might not necessarily impact S protein structure or receptor binding but could drastically change its thermal stability or potency to generate neutralizing antibodies39. Importantly, non-stabilized S proteins (similar to the ones expressed by ChAdOx1 nCoV19) have shown different glycosylation patterns compared to stabilized recombinant trimeric S and S of SARS-CoV-2 virions all derived from human cells, which might also influence antigenicity and other biological properties40.The capability of CHO cells to provide glycoproteins with human-like glycosylation could be advantageous for vaccine antigen candidates compared to those produced in non-mammalian cell hosts 42.To investigate possible glycosylation discrepancies between CHO-produced soluble S ectodomain and S-VLP, we conducted LC-HCD MS/MS assays, and profiled glycans present at 20 out of 22 potential N-glycosylation sites of the S protein present on S-VLPs or produced as a soluble, recombinant ectodomain trimer (SmT2v3), also from CHO cells. For both sample types, N-glycosylation sites were mostly sialylated and core-fucosylated with complex-type glycans which tended to be biantennary or triantennary. High mannose was commonly observed at specific sites and low levels of hybrid glycans were also observed at some other sites Fig. . A majorNicotiana benthamiana plants used for the production of VLPs by Medicago generate S with glycans known as putative plant allergens45. Also, insect cells such as Sf9, which are used to manufacture Novavax and Sanofi S-based vaccines, produce glycoproteins with primarily high mannose, oligomannose, and paucimannose glycans 48. More studies will be necessary to elucidate the impact of these glycan structures on the S protein immunogenicity. APC processing and presentation to B cells of an S protein containing immunogenic glycans or glycans not normally found on SARS-CoV-2 virions may divert immune response towards epitopes that do not protect against the natural virus.In contrast, S glycosylation can differ notably for proteins and VLPs produced in other production hosts used for vaccine manufacturing. Taken together, these results demonstrate that CHO-produced S-VLPs display a higher density of S proteins on their surface than the native virus produced in Vero cells with glycosylation similar to that reported previously for S produced by human and Vero cells (as soluble ectodomain or associated with virus particles).23 demonstrated substantial variability in neutralization activity with serum from the VLP and VLP+Adju-phos groups , or at different doses  with Adju-phos or AS01b adjuvants. Analysis of day 28 serum samples demonstrated that VLPs induced anti-S antibodies even without adjuvant, but as low as 60 ng of VLPs induced titers >4-fold higher than the antigen alone when adjuvanted with Adju-phos and >23-fold higher with AS01b Fig. . IFN-γ Eups Fig. . Conversups Fig. . Therefop = 0.0003)   Fig. . Differe15) Fig. , likely 9. VLPs generated by the assembly of 4 structural proteins (S-M-E-N) of SARS-CoV-2 need 2.83 μg of adjuvanted antigen to produce neutralizing antibodies (EC50) in mice4. ABNCoV2 using split-protein Tag/Catcher technology, where S RBD produced in insect cells is linked to a bacteriophage AP205 capsid-like particle produced in E. coli, induces neutralizing activity in preclinical and clinical trials using 6.5 or ≥ 6 µg of adjuvanted antigen, respectively51. Moreover, effective vaccine dosage in humans of Medicago-Covifenz required a prime dose of 3.75 µg and a similar booster dose of VLP-S10. Remarkably, our study indicates that in mouse and hamster models, S-VLPs require as low as 0.06–0.3 µg dose of S protein when adjuvanted with AS01b to induce potent neutralizing activity along with concomitant protection against SARS-CoV-2 challenge.Taken together, these results suggest S-VLPs + AS01b could be more immunogenic than most VLP vaccines currently in preclinical or clinical phases. VBI-2902a used 0.2 and 1 µg doses of adjuvanted S protein from Gag-S-based VLPs to induce neutralizing antibodies and grant protection in murine models, respectively52, we have discovered that expression of SARS-CoV-2 S alone in CHO cells is sufficient to induce assembly and release of abundant, high-density S-coated VLPs. Compared to alternative VLP technologies, S-VLPs not only show similar or better in vivo potency but also permit simplified upstream production and downstream purification processes, which should facilitate low-cost, large-scale manufacturing. We expect that CHO-produced S-VLPs could provide a cost-effective and agile vaccine platform to continue combating the current pandemic including future SARS-CoV-2 variants of concern.In summary, in contrast to previously published work using other cell-based expression platformsPeer Review FileSupplementary informationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting summary