Mycobacterium tuberculosis (Mtb) remains a major global health threat. The only available vaccine Bacille Calmette-Guérin (BCG) does not prevent adult pulmonary TB. New effective TB vaccines should aim to stimulate robust T cell responses in the lung mucosa to achieve high protective efficacy. We have previously developed a novel viral vaccine vector based on recombinant Pichinde virus (PICV), a non-pathogenic arenavirus with low seroprevalence in humans, and have demonstrated its efficacy to induce strong vaccine immunity with undetectable anti-vector neutralization activity.Tuberculosis (TB) caused by Using this tri-segmented PICV vector (rP18tri), we have generated viral vectored TB vaccines  encoding several known TB immunogens . A P2A linker sequence was used to allow for the expression of two proteins from one open-reading-frame (ORF) on the viral RNA segments. The immunogenicity of TBvac-2 and TBvac-10 and the protective efficacy of TBvac-1 and TBvac-2 were evaluated in mice.Mtb lung tissue burden and dissemination in an aerosol challenge mouse model.Both viral vectored vaccines elicited strong antigen-specific CD4 and CD8 T cells through intramuscular (IM) and intranasal (IN) routes as evaluated by MHC-I and MHC-II tetramer analyses, respectively. The IN inoculation route helped to elicit strong lung T cell responses. The vaccine-induced antigen-specific CD4 T cells are functional, expressing multiple cytokines as detected by intracellular cytokine staining. Finally, immunization with TBvac-1 or TBvac-2, both expressing the same trivalent antigens , reduced via the use of P2A linker sequence and elicit strong systemic and lung T cell immunity with protective efficacy. Our study suggests the PICV vector as an attractive vaccine platform for the development of new and effective TB vaccine candidates.The novel PICV vector-based TB vaccine candidates can express more than two antigens  The onlpulation , 5. Withpriority . TB vaccMtb infection is generally believed to require cellular immune responses  was approved by the Food and Drug Administration (FDA) in 2019  rP18tri, which can accommodate two additional open-reading frames (ORFs) , 22. PICy fluids . For they fluids  also mity fluids . The rP1y fluids . The livy fluids , possibly fluids . These fMtb antigens including Ag85B, EsxH, and ESAT-6/EsxA. We characterized the quantity, phenotype, and functionality of T cells elicited by these viral vectored vaccines and evaluated protection in a virulent Mtb challenge mouse model. Our study suggests the PICV vector as an attractive vaccine platform for development of new effective TB vaccine candidates.Here, we provide a proof-of-concept that rP18tri-based multivalent TB vaccine candidates can stimulate robust systemic and lung T cell immune responses with protective efficacy in mice. We generated three viral vectored vaccines TBvac-1, TBvac-2, and TBvac-10 expressing multiple 22.1Research conducted for this manuscript was approved by the Institutional Biosafety Committee at the University of Minnesota, Twin Cities, under the protocol ID 2008-38359H and 2010-38519H. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of University of Minnesota, Twin Cities, under the protocol ID 2006-38175A and 2005-38161A.2.2Mycobacterium tuberculosis Erdman is a fully virulent isolate and a BSL-3 agent. Mtb was routinely cultured at 37˚C with aeration in Middlebrook 7H9 liquid medium (Difco), which was supplemented with 10% albumin-dextrose-saline (ADS), 0.5% glycerol and 0.1% Tween-80.BHK21 Baby hamster kidney cells and Vero African green monkey kidney cells were grown in DMEM media (Fisher Scientific) with 10% fetal bovine serum (FBS) (Sigma) and 50 μg per ml penicillin and streptomycin (Invitrogen-LifeTechnologies). BSRT7-5 cells, obtained from K.K. Conzelmann , are BHK-21 cells stably expressing T7 RNA polymerase. BSRT7-5 cells were grown in minimal essential medium (MEM) (Invitrogen-LifeTechnologies) with 10% FBS, 1 μg/ml Geneticin (Invitrogen-LifeTechnologies), and 50 μg/ml penicillin-streptomycin. Recombinant PICV-vectored TB vaccines were amplified in BHK-21 cells. Infectious virus titer was determined by viral plaque assay in Vero cells as described previously . Mycobac2.3Mtb proteins, peptide arrays, and antibodies were obtained from BEI Resources, NIAID, NIH. These include purified Ag85B (NR-14857) and ESAT-6/EsxA (NR-49424) proteins from Mtb strain H37Rv, peptide arrays of Mtb Ag85B (NR-34828) and ESAT-6 (NR-50711), rabbit polyclonal anti-ESAT6 (NR-13803) and anti-Ag85 Complex (NR-13800) antisera. Phycoerythrin (PE)-conjugated EsxH MHC-I tetramer (H-2K(b) IMYNYPAM), PE-conjugated MHC-II control and Ag85B tetramers were obtained from the NIH Tetramer Core Facility at Emory University. Antibodies used for T cell analysis of immunized mice were purchased from commercial sources, including allophycocyanin (APC)-labeled anti-CD3 (17A2) (Biolegend), fluorescein isothiocyanate (FITC)-labeled anti-CD4 (Biolegend), peridinin chlorophyll protein (PerCP)-Cy5.5-labeled anti-CD8 (53-6.7) (eBioscience), brilliant violent (BV) 510-labelled anti-CD44 (Biolegend), APC-Cy7 anti-CD69 (Biolegend), and BV421 anti-CD103 (BD Biosciences).2.4Mtb antigens Ag85B (NP_216402), EsxH/TB10.4 (NP_214802), ESAT6/EsxA (YP_178023), Rv1733c (NP_216249), and RpfA (NP_215382) were obtained from GenBank. Gene fragments encoding Ag85B single gene and dual antigens  linked by a P2A linker sequence (GSGATNFSLLKQAGDVEENPGP) -based TB vaccines were constructed as described previously , 22. SeqVEENPGP)  were codAg85B NP_6402, Esx2.5Vero cells in six-well plates were washed and infected with 0.5 ml of viruses in 10-fold serial dilutions. After incubation for 1 h at 37°C, the infection medium was removed. Fresh MEM supplemented with 0.5% agar and 10% FBS were added to the cells, which were cultured for 4 days at 37°C. Plaques were stained overnight with neutral red solution diluted (1:50) in 0.5% agar–MEM–10% FBS.2.6Vero cells grown on coverslips were mock infected or infected with the rP18tri vectors alone or respective TBvac viruses at MOI of 0.1. At 24 hours post infection (hpi), cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and washed three times with phosphate buffer saline (PBS) (Invitrogen-LifeTechnologies). Cells were treated with 0.1% Triton X-100 for 12 min followed by incubation with rabbit polyclonal antibody against Ag85B or ESAT6 (BEI) for 1 h. After washing, cells were incubated with goat anti-rabbit Alexa Fluor-488 (Invitrogen) for 1 hr at room temperature. The cells were washed three times and mounted on a glass slide for examination by confocal microscopy.2.75 PFU of TBvac-2, TBvac-10, rP18tri vector, or phosphate buffered saline (PBS) as a mock control. To evaluate immune responses after primary immunization, blood was collected into lithium heparin tubes (Greiner Bio-One) via the facial vein. Peripheral blood mononuclear cells (PBMCs) were isolated by first lysing red blood cells (RBC) using 1X RBC lysis buffer (eBiosciences) and then washing in PBS with 2% fetal bovine serum (FBS) (Sigma Aldrich). Three weeks after primary immunization, mice were boosted IN or IM with the same vaccine candidate. Seven days after the boost, mice were euthanized by CO2 inhalation and the spleen and lung collected into sterile PBS. To isolate splenocytes, spleens were gently crushed through a 40 µm cell strainer and washed with RPMI-1640 medium with L-glutamine (Cytiva HyClone). Red blood cells were lysed using 1X RBC lysis buffer and splenocytes resuspended in PBS with 2% FBS. To isolate lung lymphocytes, lungs were cut into small pieces and incubated for 1 hour at 37C in PBS+5% FBS with 1.5 mg/ml type I collagenase  and 2 μg/ml DNase I (New England BioLabs). Then, lungs were gently crushed through a 40 µm cell strainer, washed with RPMI-1640/1% FBS and 10mM HEPES (ThermoFisher). Cells were resuspended in 44% Percoll , underlain with 67% Percoll, centrifuged, and the lymphocyte layer collected. Lymphocytes were resuspended in PBS with 2% FBS.For immunogenicity studies, female six- to eight-week-old C57BL/6 mice were obtained from Jackson Laboratories and housed for at least one week for acclimatization. Groups of three to five mice were immunized intranasally (IN) or intramuscularly (IM) depending on the study protocol with 1x102.85 splenocytes were added per well and incubated with medium only or with peptide pools of Ag85B or ESAT6/EsxA (2 µg/ml) (BEI) for 18 hours at 37°C and 5% CO2. After incubation, biotinylated anti-mouse IFNγ detection antibody was added and incubated overnight at 4°C. The following day, streptavidin-alkaline phosphatase and BCIP/NBT were added for color development. Spots were recorded using CTL ImmunoSpot Reader and quantified using immunospot Fluox suite software.ELISpot was performed using Mouse IFNγ ELISpot Development Module  following the manufacturer’s instruction. Briefly, 96-well PVDF-bottom Immunospot plates were coated with anti-mouse IFNγ capture antibody overnight at 4°C. The next day, plates were washed and blocked in 1% bovine serum albumin (BSA)/5% sucrose/PBS. 1x102.9The PE-labeled MHC-I tetramer H-2K(b)/EsxH epitope IMYNYPAM was provided by the NIH Tetramer core facility at Emory University . Isolated PBMCs, splenocytes, and lung lymphocytes were incubated with PE-labeled EsxH/H-2K(b) MHC-I tetramer, fixable viability stain 780 (BD Biosciences), APC-labeled anti-CD3 (Biolegend), PerCP-Cy5.5-labeled anti-CD8 (eBioscience), FITC-labeled anti-CD4 (Biolegend), and BV510-labeled anti-CD44 (Biolegend) for one hour at room temperature. Cells were washed three times with PBS/2% PBS. Sample acquisition was performed on FACSCelesta and data analyzed using FlowJo.2.10The PE-labeled MHC-II Ag85B tetramer I-A(b)/FQDAYNAAGGHNAVF and control tetramer I-A(b)/PVSKMRMATPLLMQA were provided by the NIH Tetramer core facility at Emory University . Isolated PBMCs, splenocytes, and lung lymphocytes were incubated with fixable viability stain 780 (BD Biosciences), APC-labeled anti-CD3 (Biolegend), PerCP-Cy5.5-labeled anti-CD8 (eBioscience), FITC-labeled anti-CD4 (Biolegend), and BV510-labeled anti-CD44 (Biolegend), together with either control or Ag85B PE-labeled MHC-II tetramer. To detect tissue-resident CD4 T cells in the lung, lung lymphocytes were incubated with the same cocktails, together with APC-Cy7-labeled anti-CD69 (Biolegend). Cells were washed three times with PBS/2% PBS. Sample acquisition was performed on FACSCelesta and data analyzed using FlowJo.2.116 cells per well and incubated with medium alone or with purified Ag85B/ESAT-6 proteins (each 10 µg/ml) obtained from BEI for 2 hours at 37°C. Cells were further incubated for six hours at 37°C with anti-CD28 antibody and GolgiStop (BD Biosciences). Cells were collected, washed with PBS/2% FBS, and stained with fixable viability dye 780, PerCP-Cy5.5-labeled anti-CD3, FITC-labeled anti-CD8, APC-Cy7-labeled anti-CD4, and PE-Cy7-labeled anti-CD44 for 45 minutes on ice. Cells were then fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences) and stained with BV421-labeled anti-TNFα, PE-labeled anti-IL-2, and APC-labeled anti-IFNγ for 40 minutes on ice. Sample acquisition was performed on FACSCelesta and data analyzed using FlowJo.Isolated cells were seeded in leukocyte medium (complete RPMI) at 1x102.125 PFU of rP18tri vector, TBvac-1, or TBvac-2 in a prime-boost strategy with a 21-day interval. At 28 days after immunization, vaccinated mice were challenged with ~400 CFU of virulent Mtb Erdman, using a Glas-Col Inhalation Exposure System, in a biosafety level 3 (BSL3) laboratory, as previously described  were sacrificed; lungs and spleen were collected for bacterial quantification. Tissues were homogenized (BioGen Pro200) in 3 ml of PBS containing 0.05% Tween-80 (PBS-T), serially diluted in PBS-T and plated on Middlebrook 7H10 agar (Difco) supplemented with 0.5% glycerol, 10% oleic acid-albumin-dextrose-catalase  and 100 μg/ml cycloheximide. Plates were incubated for 3-4 weeks at 37˚C before counting CFU.Female six to eight-week-old C56BL/6 mice were immunized IM with 1x10escribed . A group33.1The genome of wild-type PICV consists of two RNA segments, each encoding two genes in opposite orientations . The L sMtb antigens EsxH/TB10.4 and Ag85B as they have well-established major histocompatibility complex (MHC) class I (MHC-I) and MHC-II tetramer assays to conveniently evaluate the antigen-specific CD8 and CD4 T cells, respectively, in mice  and that have been included in other candidate TB vaccines  assay, which detects production of IFNγ from lymphocytes after antigen stimulation. We have previously shown that the rP18tri vector-based influenza vaccine induces even stronger antibody and T cell responses upon boost than after prime , so we e3.3via both routes generated a significantly higher percentage of tetramer-positive CD8 T cells in the lung than mock or vector alone, with a trend toward higher responses in mice immunized by the IN route compared to IM, though this did not achieve statistical significance  or mucosal (IN) inoculation using an established MHC-I EsxH tetramer assay. BL6 mice were immunized with PBS/mock, rP18tri vector alone, or TBvac-2 through either IM or IN route , which has been shown to result in greater protection against nea pigs . We evalTaken together, these results show that two different rP18tri-based TB vaccines (TBvac-2 and TBvac-10) administered systemically, mucosally, or a combination of the two, elicit strong antigen-specific CD8 T cells in the spleen, peripheral blood, and lungs. As EsxH is expressed after the P2A linker in both TBvac-2 and TBvac-10, these results also demonstrate that P2A-linked antigens in the rP18tri viral vector can stimulate robust immune responses.3.4+CD4+CD44h population and lung lymphocytes were further gated by CD69+, which is a tissue-resident cell surface marker , TBvac-2, or TBvac-10 by either the IM-IN or IN-IN routes (e marker . TetrameMtb-specific CD4 and CD8 T cells with tissue-resident memory phenotypes are associated with protection (+ (a tissue-resident cell marker) population of the CD4 T cells (CD3+CD4+CD44hCD69+) for quantification of tetramer-positive cells. Both TBvac-2 and TBvac-10 stimulated high levels of tetramer-positive CD69+ CD4 T cells in the lung (+CD44hCD4 T cells than TBvac-2 (Recent studies suggest that tissue-resident memory T cells (Trm) play an important role in the primary defense against infection  and thatotection . To deteMtb  and that TBvac-10 may induce a stronger CD4 T cell immunity than TBvac-2.3.5Mtb low-dose aerosol challenge mouse model  upon antigen stimulation and express the CD69 tissue-resident marker in the lung. While CD4 T cells are traditionally thought to exert control over Mtb , 48. Thuvia the IM-IN routes induced a similarly high level of CD4 T cells in the lung as via the IN-IN routes, suggesting that a mucosal boost immunization of the PICV-vectored vaccine is sufficient to induce effective lung T cell immunity. Our results are consistent with a published study showing that a systemic IM prime and IN boost of a viral vector vaccine elicited efficient T cell homing to the lungs  , which moduction , 52. A p in NHPs  and mice in NHPs . Trm-medosal BCG  or a recosal BCG . Functioosal BCG . The impicit Trm . Multiplicit Trm , 47, 54.icit Trm , 58.Mtb  expressing the same three Mtb antigens , decreased bacterial load in the lung and the spleen by ~ 1 log at the 4-week time point in Mtb-infected mice compared to the viral vector alone. TBvac-1 (TBvac-2 not tested) also reduced the bacterial load in the lung but not in the spleen at the 12-week time point. This might be explained by the delayed Mtb dissemination from the lung to the spleen, or less durable protection in the spleen than in the lung. Future studies to analyze the long-lived memory cells at both lung and spleen infection sites will help address this question. A relatively high dose (~400 CFU) of virulent Mtb was used in the challenge model, which might have obscured some protective effect of the vaccines. As a mock-immunized group was not included in the protection experiment, we could not determine whether general immune stimulation induced by the PICV vector could further contribute to protection. Such vector-induced protection has been documented with other viral vectors  immunization of PICV-based TB vaccine candidates could partially protect mice from virulent  vectors  and will vectors , 46. The for BCG .Mtb infection in a virulent mouse model. Our study supports the continued development of multiantigen TB vaccine candidates using the PICV vector platform.In summary, we have shown that novel PICV-based TB vaccines elicit strong systemic and lung T cell immunity, and confer protection against The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of University of Minnesota, Twin Cities.HL, AT, and YL contributed to conception and design of the study. NK, SV, QH, MR, AB, DW, SN, and HM generated data. NK, AT, and YL contributed to data analysis. NK and YL performed statistical analysis. NK and YL drafted the manuscript. AT and YL revised the manuscript. HL and MR contributed to manuscript edition. All authors contributed to the article and approved the submitted version.


PROteolysis TArgeting Chimeras (PROTACs) has been exploited to degrade putative protein targets. However, the antitumor performance of PROTACs is impaired by their insufficient tumour distribution. Herein, we present de novo designed polymeric PROTAC (POLY-PROTAC) nanotherapeutics for tumour-specific protein degradation. The POLY-PROTACs are engineered by covalently grafting small molecular PROTACs onto the backbone of an amphiphilic diblock copolymer via the disulfide bonds. The POLY-PROTACs self-assemble into micellar nanoparticles and sequentially respond to extracellular matrix metalloproteinase-2, intracellular acidic and reductive tumour microenvironment. The POLY-PROTAC NPs are further functionalized with azide groups for bioorthogonal click reaction-amplified PROTAC delivery to the tumour tissue. For proof-of-concept, we demonstrate that tumour-specific BRD4 degradation with the bioorthogonal POLY-PROTAC nanoplatform combine with photodynamic therapy efficiently regress tumour xenografts in a mouse model of MDA-MB-231 breast cancer. This study suggests the potential of the POLY-PROTACs for precise protein degradation and PROTAC-based cancer therapy. Proteolysis targeting chimeras (PROTACs) have emerged as promising cancer therapy agents but have suffered from systemic toxicity issues. Here, the authors report on the creation of polymeric PROTAC nanoparticles for tumour targeting delivery and demonstrate protein degradation in vivo, in combination with photodynamic therapy. PROTACs are generally composed of a warhead that binds to the protein of interest (POI), a ligand-hijacking endogenous E3 ubiquitin ligase, and a linker connecting the warhead and the ligand6. PROTACs can label the POI with ubiquitin by recognising the E3 ligase and subsequently degrade the POI through the ubiquitin-proteasome system (UPS)9. Compared to small molecule inhibitors, PROTACs can potentially degrade any intracellular protein, including undruggable targets 12. Furthermore, PROTACs are potent agents that can circumvent acquired drug resistance by degrading whole proteins after a short drug exposure time and a low dosage14. Despite being promising, conventional small molecular PROTACs generally display unfavourable pharmacokinetics and lack tumour specificity, which might cause systemic toxicity due to their nonspecific distribution in normal tissues16. Thus, it remains a formidable challenge to achieve tumour-specific delivery and potentiate the antitumor potency of conventional PROTACs.Heterobifunctional PROteolysis TArgeting Chimeras (PROTACs) hold promising potential for cancer therapy, as they can degrade oncoproteins, particularly undruggable targets22. These decorated PROTACs have shown increased cellular uptake in vitro. In particular, aptamer-PROTAC conjugates have displayed increased tumour accumulation and antitumor potency in vivo compared to conventional PROTACs22. Nevertheless, these ligand-PROTAC conjugates suffer from low serum stability, limited tumour penetration and heterogeneous expression of the receptors in different tumour cells and cancer types. Furthermore, opto-PROTACs were developed for ultraviolet light-inducible protein degradation24. These photoactivatable PROTACs have been demonstrated to spatiotemporally control protein degradation in vitro27. However, clinical translation of opto-PROTACs is restricted by the poor tissue penetration profile of ultraviolet light with short wavelength . Therefore, precise PROTAC delivery to the tumour and efficient POI degradation inside tumour cells remain formidable challenges.To achieve tumour-targeted delivery of PROTACs, several ligand modification strategies  have been investigated in recent yearsIn this work we rationally engineer a polymeric PROTAC (POLY-PROTAC) nanoplatform for tumour-targeted degradation of the bromodomain and extraterminal (BET) protein BRD4. We first synthesise four von Hipel-Lindau (VHL)-based small molecular PROTACs and then design a series of reduction-activatable POLY-PROTACs and self-assemble into micellar nanoparticles (NPs) for systemic PROTAC delivery Fig. . We subs28. To design a BRD4-targeted POLY-PROTAC, we selected the VHL ligand for PROTAC synthesis since the hydroxyl group of VHL can be reversibly caged via a disulfide spacer. The ability of the modified PROTACs to bind VHL can be restored by GSH-triggered cleavage of the disulfide bond in the cytosol of tumour cells.BET family proteins, in particular BRD4, have been investigated as promising antitumor targets due to their crucial role in gene transcription30. Western blot assays validated that all four PROTACs remarkably degraded BRD4 in MDA-MB-231 breast cancer cells in vitro and consequently suppressed downstream c-Myc expression. Noticeably, the compounds ARV771 and MZ1 reduced BRD4 expression more efficiently than their analogues. The half-degradation concentration (DC50) of ARV771 was 1.9-fold lower than that of mARV771  of the PROTACs were tens of times lower than that of the BRD4 inhibitor JQ1   and mass spectrometry (MS) measurements  Fig.  to rever33, and reduction-triggered delivery of the BRD4 PROTACs. The resultant POLY-PROTACs were termed according to the PROTAC molecule used: PD7 (based on Me-ARV771) or PDM (based on Me-MZ1). An MMP-2-cleavable GPLGLAG (GG) heptapeptide spacer35, was introduced into the PD7 POLY-PROTAC  to promote tumour-specific accumulation and cellular uptake of the POLY-PROTAC NPs  (termed PGDA) diblock copolymer was synthesised by grafting PPa onto the pendant hydroxyl groups of PGDH  and the acid-ionisable 2-(diisopropylamino)ethyl methacrylate (DPA) monomer for intracellular acidity 37. At neutral pH (7.4), dynamic light scattering (DLS) data and transmission electron microscopy (TEM) examinations revealed an average hydrodynamic diameter of ~55 nm and a spherical morphology of the POLY-PROTAC NPs with a narrow particle size distribution (polydispersity index (PDI) < 0.2). In contrast, amorphous aggregates appeared at pH 6.0 due to acid-induced protonation of the DPA groups and dissociation of the POLY-PROTAC NPs , which mimics the acidic microenvironment of endosome organelles (pH = 5.8~6.5)  Fig. . This phHPLC examinations showed that in the absence of GSH, ARV771 was marginally released from the PGD7 NPs under both neutral and acidic conditions (pH = 7.4 and 6.0) Fig. . In cont39. MDA-MB-231 breast tumour cells were then incubated with PDA7 or dePEGylated PGDA7 NPs for the desired time durations. Flow cytometry measurements identified an ~2.8-fold higher intracellular fluorescence signal in the PGDA7 group than in the PDA7 group  multicellular spheroid (MCS) MDA-MB-231 tumour model in vitro. CLSM images showed that after 12 h of incubation, the MMP-2-insensitive PDA7 NPs were primarily entrapped in the peripheral areas and marginally penetrated into the deep areas of the MCSs Fig. . In cont50 of the PGD7 and PGDM NPs, which were 1.9- and 3.1-fold lower than their MMP-2-insensitive PD7 and PDM counterparts, respectively, and comparable to those of free ARV771 and MZ1  and the POLY-PROTAC NPs  Fig. , verifyi3. In vivo fluorescence imaging displayed clear tumour distribution of the PGDA7 NPs over time  injection at an identical ARV771 dose of 10 mg/kg and PPa dose of 5.0 mg/kg when the tumour volume reached 200 mmime Fig. , which cion Fig. .Fig. 4StThe tumour-specific distribution of the PGDA7 NPs was confirmed by ex vivo fluorescence imaging of the major organs  and the tumour tissue at 48 h post-injection Fig. . CLSM ex3 and i.v. injected with PBS, ARV771 or the PGD7 NPs at an identical ARV771 dosage of 10 mg/kg. The treatments were repeated every three days for a total of five injections  staining of the tumour sections Fig. . Immunoh42, we next designed an extracellular tumour acidity-activatable pretargeted NPs for tumour-targeted delivery of the dibenzocyclooctyne (DBCO) groups   diblock copolymer  Fig.  and with3PGDH)  NPs. The azide group on the surface of the N3@PGD7 NPs was expected to perform in-situ click reaction with DBCO groups in the acidic tumour microenvironment. Both the PED and N3@PGD7 NPs displayed good serum stability in 10% (V/V) foetal bovine serum-containing neutral buffer solution (pH 7.4)  and PDT dramatically regressed 95% of tumour growth, which was 1.5-fold more efficient than BRD4 degradation alone caused by PED + N3@PGDA7 treatment.The antitumor performance of combining PDT with BRD4 degradation was next investigated in vivo. MDA-MB-231 tumour-bearing BALB/c nude mice were randomly grouped when the tumour volumes reached ~ 100 mmser Fig. . The N3@3@PGDA7 and PDT prolonged the survival of the tumour-bearing mice by 40% compared to those in the PED + N3@PGDA7 group, with 40% of the animals surviving 100 days post-treatment  than N3@PGDA7 NP alone, validating that the increased intratumoural distribution of ARV771 via the in situ bioorthogonal reaction contributed to BRD4 degradation in vivo  and degraded BRD4 (3.0-fold) than PED + N3@PGD7, verifying the synergistic apoptosis-induction profile caused by combined PDT and BRD4 degradation , and these are both under investigation in our laboratory for potentiated antitumor performance.In comparison with their small molecular PROTAC counterparts, the POLY-PROTAC NPs reported in this study possess several distinct advantages. First, the POLY-PROTAC NPs with MMP-2-liable PEG coronas elongated the blood circulation of the small molecular PROTACs, while the PEG coronas were cleaved via intratumoural MMP-2 to facilitate PROTAC tumour-specific accumulation and retention. Second, the POLY-PROTAC NPs can disintegrate in the intratumoural acidic microenvironment. The PROTAC prodrug can thus be restored in the cytosol via GSH-mediated cleavage of the disulfide bond. Furthermore, we demonstrated that the POLY-PROTAC NPs can be adapted for bioorthogonal click reaction-enforced tumour specificity. Remarkably, the azide-modified POLY-PROTAC NPs showed 3.9-fold higher tumour accumulation than their small molecule counterparts via an in situ click reaction with the pretargeted NPs, which therefore further boosted PROTAC-based cancer therapy. Moreover, other kinds of stimuli-activatable chemical bonds In summary, a POLY-PROTAC prodrug strategy was developed for the tumour-specific delivery of PROTACs in this study. We demonstrated that this POLY-PROTAC nanoplatform can be activated by multiple stimuli , which can achieve increased tumour accumulation, deep tumour penetration and enhanced protein degradation performance compared with its small molecule counterpart. The POLY-PROTAC nanoplatform with an extracellular tumour acidity-triggered bioorthogonal click reaction remarkably enhanced tumour-specific PROTAC delivery. This clickable POLY-PROTAC nanoplatform can be further applied with PDT for protein degradation and combination therapy, as this combinatorial treatment suppressed over 95% of MDA-MB-231 TNBC tumour growth. The extracellular acidic microenvironment containing MMP-2 has been well documented in various solid tumours. Therefore, this acid-activatable bioorthogonal POLY-PROTAC nanoplatform can be further employed for combination therapy to treat a broad spectrum of cancers by integrating multiple therapeutic regimens . Taken together, the proposed POLY-PROTAC nanoplatform might provide a novel avenue to potentiate PROTAC-based cancer therapy.113-NH2) and azide-poly(ethylene glycol) amine (N3-PEG113-NH2) were purchased from Seebio Biotech. Co., Ltd . Pyropheophorbide a (PPa) and Bis(4-nitrophenyl) carbonate were purchased from Dibai biotechnology CO., LTD. . Tert-butyl ((2 S)−1-((4 R)−4-hydroxy-2-(((S)−1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)−3,3-dimethyl-1oxobutan −2-yl) carbamate was obtained from Ruozhi Chemical Technology Co., LTD. .1-(3-Dimethylaminopropyl)−3-ethylcarbodiimide hydrochloride (EDCI), 1-Hydroxybenzotriazole (HOBT), 4-Dimethylaminopyridine (DMAP), Trifluoroacetic acid (TFA), N, N-Diisopropylethylamine (DIEA), N,N-Dimethylacetamide , Dichloromethane , Dimethyl sulfoxide , N,N-Dimethylformamide , 2,2’-Azobis (2-methylpropionitrile (AIBN), and methanol  were purchased from J&K Chemicals . 4-Cyano-4-(dodecylsulfanylthiocarbonyl) sulfanylpentanoic acid (CDP), 2-(diisopropylamino) ethyl methacrylate (DPA), IV Gelatinase, Triethylene glycol and tert-butyl (S)−2-(4-(4-chlorophenyl)−2,3,9-trimethyl-6H-thienotriazolodiazepin-6-yl)acetate (JQ1) were obtained from Sigma-Aldrich . Bis(2-hydroxyethyl) disulfide, Triphosgene, 2-bromoethanol, 2-hydroxyethyl methacrylate, Methacryloyl chloride and methacryloyl chloride N-ethylpropan-1-amine were purchased from TCI . Tert-butyl 2-(3-(2-aminoethoxy)propoxy)acetate, tert-butyl-2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy) acetate were all purchased from Shanghai Tebo Chemical Technology Co., LTD. Fmoc-protected heptapeptide Gly-Pro-Leu-Gly-Leu-Ala-Gly (Fmoc-GPLGLAG) was synthesised by GL Biochem. Co., Ltd . Methoxy poly(ethylene glycol) amine (mPEGDulbecco’s modified eagle medium (DMEM), 0.25% trypsin-EDTA, penicillin/streptomycin solution, PBS buffer solution (1×), bovine serum albumin , BCA protein quantification kit, protein marker (10-190 kDa), TBST buffer solution (10×), HEPES buffer solution (2×), 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI), Anti-fade solution, Lyso-green DND-189, singlet oxygen fluorescent probe SOSG, reactive oxygen species fluorescent probe DCFH-DA, cell counting kit-8 (CCK-8), MG132 proteasome inhibitor and ECL luminescence reagent were purchased from Meilun Biotech Co., Ltd . Foetal bovine serum (FBS) was purchased from Gibco . BD Matrigel, Tris-Base, glycine, SDS, Peroxidase-Conjugated Goat Anti-Rabbit IgG (H + L) (CAT:33101ES60) and Peroxidase-Conjugated Goat Anti-Mouse IgG (H + L) (CAT:33201ES60) were purchased from YEASEN . The anti-BRD4 antibody (ab128874), anti-c-Myc antibody (ab32072), anti-caspase-3 antibody (ab 184787), anti-GAPDH antibody (ab8245), anti-β-actin antibody (ab8226), anti-β-tubulin antibody (ab78078) were all purchased from Abcam (Shanghai). All other reagents and solvents were analytical grade and obtained from SinoPharm Chemical Reagent Co., Ltd. .2. All cell culture studies were performed in the logarithmic phase of cell growth in vitro.MDA-MB-231 human breast cancer cell line was obtained from the cell bank of Chinese Academy of Sciences . The cells were incubated in complete DMEM cell culture medium containing FBS 10% (V/V), penicillin G sodium and streptomycin sulfate (100 U/mL), and incubated at 37 °C under a humidified atmosphere containing 5% of COBalb/c nude mice  were obtained from Shanghai Experimental Animal Center . Animals were housed under SPF conditions in groups of 4-5 mice per cage, and maintained at a temperature of ~25 °C in a humidity-controlled environment with a 12 h light/dark cycle, with free access to standard food and water. All animal procedures were carried out under the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Institute of Material Medica, Chinese Academy of Sciences.113-GALGLPG-b-P(DPA42-r-ARV7712) (2.0 mg) and mPEG113-GALGLPG-b-P(DPA48-r-HEMA7) (1.0 mg) were dissolved in 100 μL of DMF, and the mixture was added into 1 mL of DI water under ultrasonication to obtain the POLY-PROTAC nanoparticles (termed as PGD7 nanoparticles). The organic solvent was removed by dialyzing against DI water. One set of the POLY-PROTAC nanoparticles were prepared by following the same procedure described above:Briefly, mPEG113-b-P(DPA46-r-ARV7712) + mPEG113-b-P(DPA52-r-HEMA6);POLY-ARV77 nanoparticles (PD7): mPEG113-GALGLPG-b-P(DPA46-r ARV7712) + mPEG113-GALGLPG-b-P(DPA48-r-PPa4);MMP-2-liable POLY-ARV77 nanoparticles (PGDA7): mPEG113-b-P(DPA46-r-ARV7712) + mPEG113-b-P(DPA52-r-PPa4);PPa-modified POLY-ARV771 nanoparticles (PDA7): mPEG113-b-P(DPA42-r-MZ12) + mPEG113-b-P(DPA52-HEMA6);POLY-MZ1 nanoparticles (PDM): mPEG113-GALGLPG-b-P(DPA46-r-MZ12) + mPEG113-GALGLPG-b-P(DPA48-r-HEMA7);MMP-2-liable POLY-MZ1 nanoparticles (PGDM): mPEG113-b-P(EPA60-r-DBCO4) (namely PED) and mPEG113-b-P(EPA60-r-PPa1);DBCO-loaded pretargeted nanoparticles (PEA): mPEG3@PGDA7): mPEG113-GALGLPG-b-P(DPA42-r-ARV7712) (1.7 mg) + mPEG113-GALGLPG-b-P(DPA48-r-PPa4) (0.85 mg) + N3-PEG113-GALGLPG-b-P(DPA41-r-mPEG113-GALGLPG-b-P(DPA48-r-PPa4) (0.45 mg) were dissolved into 100 μL of DMF, and added into 1 mL of DI water under ultrasonication. Then, DMF was removed via dialyzing against DI water to obtain the N3@PGDA7 nanoparticles.To prepare the azide-functionalized POLY-ARV771 nanoparticles  and TEM examination .The fluorescence emission profile of the PPa-labelled micelle suspension was examined by microplate reader , and the fluorescence images were recorded by IVIS imaging system .2O = 1:1, v/v) for pre-determined time duration at 37 °C, the concentration of ARV771 released was examined by HPLC measurement .To demonstrate reduction sensitivity of the POLY-PROTAC nanoparticles, ARV771 release from ARV771 methyl methacrylate (Me-ARV771) was investigated in vitro. Briefly, Me-ARV771 (50 μM) was incubated with 10 mM of DTT  Tween 80-containing buffer solution at 37 °C . ARV771 concentration in the buffer solution was examined by HPLC measurement at the predetermined time points .To investigate PROTAC-induced protein degradation in vitro, MBA-MB-231 tumour cells were incubated with free PROTACs or POLY-PROTAC nanoparticles at the predetermined condition. The cells were then lysed in RIPA lysis buffer  including 1 mM of phenylmethanesulfonyl fluoride (PMSF). The protein lysate was obtained by centrifugation at 12000 g for 15 min at 4 °C. Then, the lysates were denatured at 100 °C and resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were blotted onto PVDF membrane (Merck Millipore) and blocked by 5% BSA buffer for 2 h at room temperature. After incubated with the indicated primary antibodies  at 4 °C overnight, the membrane was washed several times with TBST buffer, incubated with corresponding secondary antibody (Peroxidase-Conjugated Goat Anti-Rabbit IgG (H + L), CAT:33101ES60, 1:5000 and Peroxidase-Conjugated Goat Anti-Mouse IgG (H + L), CAT:33201ES60, 1:5000) in 5% BSA solution for 1 h at room temperature, washed several times with TBST buffer, and then imaged with Bio-Rad Chemi Doc XRS imaging system.4 MDA-MB-231 cells were cultured in 96-well cell culture plate for 24 h, the cells were then treated with free PROTAC or POLY-PROTAC nanoparticles at predetermined condition for 72 h. The cell viability was then examined by CCK-8 assay.To investigate the cytotoxicity of the POLY-PROTAC nanoparticles in vitro, 1 × 10To investigate cellular uptake profile of the POLY-PROTAC nanoparticles, MDA-MB-231 cells cultured in 24-well tissue culture plate were incubated with various POLY-PROTAC nanoparticles at the identical PPa concentration of 5.0 μM for the desired time durations , the intracellular fluorescence intensity were then examined by flow cytometry . The intracellular distribution of the POLY-PROTAC nanoparticles was investigated by CLSM examination.3D multicellular spheroid (MCS) of the MDA-MB-231 breast tumour was employed to illustrate the tumour penetration profiles of the POLY-PROTAC nanoparticles in vitro. Briefly, the 3D tumour spheroids were prepared by culturing MDA-MB-231 cells in 48-well tissue culture plate (2000 cells/well) on 1% agarose gel. The 3D tumour spheroids were then incubated with the PGDA7 and PDA7 (MMP-2 pretreated) nanoparticles for 12 h. Nanoparticle distribution inside the tumour spheroids was then investgiated by CLSM examination at the predetermined time points.2) for 1 min, and the fluorescence intensity of SOSG were detected by microplate reader .To investigate photoactivity of the PPa-modified POLY-PROTAC nanoparticles, 671 nm laser irradiation-induced ROS generation was examined. Briefly, the PGDA suspension with different PPa concentrations  was incubated in pH 6.0 or pH 7.4 buffer solution for 30 min, and then added with singlet oxygen sensor green . The micellar suspension was then irradiated with 671 nm laser at different photodensity , and the intracellular fluorescence intensity of DCF was analysed by flow cytometry. Laser irradiation induced intracellular ROS generation was validated by CLSM examination.To examine the photoactivity in vitro, MDA-MB-231 cells were culture in 24-well tissue culture plate for 24 h, incubated with the PGDA nanoparticles for 12 h, and then incubated with DCFH-DA (10 μM) for 30 min. The cells were thereafter irradiated with 671 nm laser for 1 min at varied photodensity  into the fat pad of the Balb/c nude mice . The tumour-bearing mice were randomly grouped when the tumour volume reached ∼200 mm3 and intravenously (i.v.) injected with PPa-labelled POLY-PROTAC nanoparticles at an identical PPa dose of 5.0 mg/kg. Then, fluorescence imaging was collected with the IVIS imaging system at predetermined time points . The mice were sacrificed at 48 h post-injection, the major organs and tumour tissue were harvested for fluorescence imaging ex-vivo. Afterwards, the tumour tissues were fixed, sectioned and stained with identified antibodies and DAPI, the fluorescence signal of the tumour sections were measured by CLSM examination.MDA-MB-231 breast tumour model was established by subcutaneously injecting 100 μL of cell suspension (1 × 103 (n = 3). The DBCO-loaded pretargeted (PED) nanoparticle was i.v. injected at a DBCO dose of 1.0 mg/kg, and the N3@PGDA7 nanoparticle was i.v. injected 2 h post PED administration at an ARV771 dose of 10 mg/kg. The biodistribution of PED and N3@PGDA7 was then examined by IVIS fluorescence imaging systems in vivo.To investigate whether in-situ click reaction increased tumour accumulation of the POLY-PROTAC nanoparticles, the tumour-bearing nude mice were divided into three groups when the tumour volume reached ~ 200 mmTo evaluate in-situ bioorthogonal click reaction enhanced tumour distribution of PROTAC, the tumour-bearing mice were i.v. injected with the POLY-PROTAC nanoparticles and sacrificed at the predetermined time points . The major organs  and tumour tissue were harvested and homogenised. ARV771 was extracted with methanol, examined by HPLC measurement and normalised with tissue mass.7 cells, DMEM: Matrigel = 1:1, v/v). The tumour volume reached 100 mm3 at 15-days post inoculation. The tumour-bearing mice were randomly grouped (n = 5), and i.v. injected with PBS, ARV771, or PGD7 nanoparticle at an identical ARV771 dose of 10 mg/kg for five cycles at a time interval of 3 days. The tumour volume was monitored for 30-days post treatment.The anti-tumour performance of the POLY-PROTAC nanoparticle was evaluated in MDA-MB-231 orthotropic tumour model in vivo. The female Balb/c nude mice were s.b. injected with 100 μL of cell suspension (1 × 103 (n = 5), the tumour-bearing mice were then i.v. injected with PBS, ARV771, PGD7, or PED + N3@PGD7 every three days at an identical ARV771 dose of 10 mg/kg, and DBCO dose of 1.0 mg/kg for five cycles (PED was injected 2 h pre- POLY-PROTAC nanoparticle administration).To investigate the antitumor performance of the bioorthogonal POLY-PROTAC nanoparticles, the tumour-bearing mice were randomly grouped when the tumour volume reached 100 mm3 (n = 5), and then i.v. injected with PBS, ARV771, PED + N3@PGDA, PED + N3@PGD7 or PED + PGDA7 at the identical ARV771 dose of 10.0 mg/kg, PPa dose of 5.0 mg/kg and DBCO dose of 1.0 mg/kg, respectively (PED was i.v. injected 2 h pre-administration of the N3@PGD7 nanoparticles). Thirty-six hours post nanoparticle injection, the mice of the PED + N3@PGDA and PED + PGDA7 groups were treated with 671 nm laser irradiation at photodensity of 400 mW/cm2 for 5 min. The treatment was repeated for five cycles at a time interval of three days. The tumour volume and body weights of the animals were monitored during the whole experimental period. The mice were considered death when the tumour volume reached 1500 mm3 according to the animal ethics of our institute and animal welfare. The tumours and the major organs  of mice were harvested at the end of the antitumor studies, and examined by H&E and TUNEL staining. BRD4 degradation in the tumour tissue was validated by western blot assay of the tumour lysate. The tumour volume was calculated with the formula below:To investigate the antitumor performance of POLY-PROTAC nanoparticle-performed combinatory BRD4 degradation and PDT, the POLY-PROTAC nanoparticles were labelled with PPa for PDT. The tumour-bearing mice were grouped when the tumour volume reached 100 mmAll data were given as Mean ± SD. The GraphPad Prism software 6.01 was used for the statistical analyses. Two-tailed Student’s t-test was used for the statistical comparison between the two groups. One-way analysis of variance (ANOVA) with a Tukey post hoc test was used for statistical comparison among multiple (more than two) groups. Two-sided log-rank (Mantel-Cox) test was used for the statistical comparison of the survival study.Further information on research design is available in the ChemDraw files of chemical structureSupplementary Information Engineered Bioorthogonal POLY-PROTAC Nanoparticles for Tumor-Specific Protein Degradation and Precise Cancer TherapyReporting summaryPeer Review File


This cohort study compares the antispike antibody titers of the mRNA-1273 and BNT162b2 vaccines for SARS-CoV-2 in incrementally immunosuppressed patients. This study compares antispike antibody titers after the 2-dose mRNA-1273 and BNT162b2 vaccines in incrementally immunosuppressed patients.Both mRNA-1273 and BNT162b2 SARS-CoV-2 vaccines elicit immune responses consistent with viral neutralization in most immunocompetent persons.4 Participants receiving 2 SARS-CoV-2 messenger RNA (mRNA) doses between December 16, 2020, and July 6, 2021, were included. This study followed the STROBE reporting guideline for observational studies and was approved by the Johns Hopkins Institutional Review Board. Patients provided informed consent electronically.Patients with RMDs and SOTRs without prior positive SARS-CoV-2 test results were recruited to this cohort study as previously described.6 Participants were stratified by increasing intensity of immunosuppression: patients with RMDs not receiving immunosuppression or receiving hydroxychloroquine or intravenous immunoglobulin, patients with RMDs receiving immunosuppression, SOTRs not receiving mycophenolic acid or mycophenolate mofetil, and SOTRs receiving mycophenolic acid or mycophenolate mofetil. Immunosuppression intensity was defined by the presence or absence of baseline immune dysregulation and data on SARS-CoV-2 vaccination antibody response.4 We compared response rates between mRNA-1273 and BNT162b2 recipients using modified Poisson regression weighted for age, time since vaccination, and number of immunosuppressive medications and repeated this analysis for positive response thresholds of 50, 100, and 250 U/mL.Semiquantitative testing for antibodies against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein was performed using the Roche Elecsys anti–SARS-CoV-2 S enzyme immunoassay 15 to 45 days after the second dose. Anti-RBD titers were divided into 50, 100, and 250 U/mL or greater based on levels associated with incremental plasma neutralizing capacity in patients convalescing from COVID-19 and a modeling study across COVID-19 vaccine trials.P < .05 was considered significant. Analyses were performed using Stata/SE, version 17.0 (StataCorp LLC).Participant characteristics were compared using the Wilcoxon rank sum test for continuous and Fisher exact test for categorical variables. A 2-sided P = .69). However, mRNA-1273 recipients had higher rates of anti-RBD titers of 250 U/mL or greater than BNT162b2 recipients among the 938 patients with RMDs receiving immunosuppression , 260 SOTRs not receiving mycophenolic acid or mycophenolate mofetil , and 437 SOTRs receiving mycophenolic acid or mycophenolate mofetil . Similar trends were observed with the other cutoffs (This cohort included 1158 participants with RMDs and 697 SOTRs: 647 patients with RMDs (55.8%) and 367 SOTRs (52.7%) received BNT162b2; the remainder received mRNA-1273 . Among t cutoffs .We observed similar seroresponses to mRNA-1273 and BNT162b2 vaccines in patients not receiving immunosuppression. However, patients receiving immunosuppression had higher mRNA-1273 vs BNT162b2 vaccination seroresponses. Differential immunogenicity was highest in the most immunosuppressed participants (SOTRs receiving mycophenolic acid or mycophenolate mofetil), with a 2.6-fold higher rate of achieving anti-RBD titers of 250 U/mL or greater.5 correlated anti-RBD levels with neutralization capacity in vaccinated individuals.Limitations include self-reporting of SARS-CoV-2 infection and lack of virus neutralization testing. However, this self-report correlated well with prevaccination antibody levels, and a previous studyIn conclusion, mRNA-1273 was more likely to induce stronger humoral immunogenicity compared with BNT162b2 in immunosuppressed patients; this effect was more pronounced with greater immunosuppression. These findings suggest that choice of mRNA vaccine platform is important in optimizing immune responses to SARS-CoV-2 vaccination and can help inform strategies for booster doses in high-risk, immunosuppressed populations.


Both parental and Delta variant of concern (VOC) strains induced Hippo pathway. The chemical inhibition and gene knockdown of upstream kinases MST1/2 and LATS1 resulted in significantly enhanced SARS-CoV-2 replication, indicating antiviral roles. Verteporfin a pharmacological inhibitor of the Hippo pathway downstream transactivator, YAP, significantly reduced virus replication. These results delineate a direct antiviral role for Hippo signaling in SARS-CoV-2 infection and the potential for this pathway to be pharmacologically targeted to treat COVID-19.SARS-CoV-2, responsible for the COVID-19 pandemic, causes respiratory failure and damage to multiple organ systems. The emergence of viral variants poses a risk of vaccine failures and prolongation of the pandemic. However, our understanding of the molecular basis of SARS-CoV-2 infection and subsequent COVID-19 pathophysiology is limited. In this study, we have uncovered a critical role for the evolutionarily conserved Hippo signaling pathway in COVID-19 pathogenesis. Given the complexity of COVID-19 associated cell injury and immunopathogenesis processes, we investigated Hippo pathway dynamics in SARS-CoV-2 infection by utilizing COVID-19 lung samples, and human cell models based on pluripotent stem cell-derived cardiomyocytes (PSC-CMs) and human primary lung air-liquid interface (ALI) cultures. SARS-CoV-2 infection caused activation of the Hippo signaling pathway in COVID-19 lung and  SARS-CoV-2 primarily causes severe lung injury with diffuse alveolar damage resulting in the development of acute respiratory distress syndrome (ARDS)4. The virus is also known to infect heart, kidney, brain and digestive systems due to the widespread expression of host cell surface membrane receptor angiotensin converting enzyme 2 (ACE2) and other receptors, including NRP1, AXL and CD14711. Therefore, patients develop additional systemic complications including acute kidney injury14, vascular inflammation (endotheliitis), and cardiac effects16. Viral replication-mediated epithelial cell injuries result in a vigorous immune response leading to organ failure, and possibly death in severe cases. Patients with various underlying conditions such as cardiovascular diseases, diabetes, and obesity are linked with increased risk of COVID-19 mortality18.Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is a betacoronavirus of zoonotic origin having similarity with bat SARS-CoV-like viruses. SARS-CoV-2 is responsible for the outbreak of the Coronavirus Disease 2019 (COVID-19) pandemic leading to over 5 million deaths and 270 million cases worldwide19. COVID-19 pathogenesis is a complex process involving crosstalk with multiple cellular pathways. To understand the fundamental principles underlying RNA viral mediated cell injury processes, we hypothesize that the conserved cellular circuitry of the Hippo signaling pathway, which is involved in tissue growth, immune response, and inflammation, can contribute to regulating SARS-CoV-2 replication and COVID-19 pathogenesis. Previously, we observed that the Zika Virus (ZIKV), a member of the Flaviviridae family, dysregulates this pathway20. The core components of the Hippo pathway in mammals are STE20-like kinases STK4/MST1 and STK3/MST2 (Hpo in Drosophila), large tumor suppressors , MOB kinase activator 1A and 1B (Mats in Drosophila), Neurofibromatosis 2 (NF2), and Salvador 1 (SAV1)21. MST1/2 and LATS1/2 are serine/threonine kinases, whereas NF2 and SAV1 are adaptors or co-factors. Upon activation by stimuli, MST1/2 phosphorylate and activates the downstream serine/threonine kinase LATS1/2. LATS1/2 can be phosphorylated by MAP4Ks independent of MST1/2 axis. The phospho-LATS1/2, along with SAV1, then phosphorylates YAP (Yes-associated protein), a transcription co-activator and oncogene. Phospho-YAP, bound to a 14-3-3 protein and retained in the cytoplasm, is subsequently ubiquitinated and degraded by proteasomes. While the Hippo pathway is inactive, the unphosphorylated YAP and its homologous partner TAZ translocate to the nucleus, where they bind to the TEAD family of transcription factors and mediate expression of target genes involved in cell survival and proliferation 22. As the Hippo signaling pathway controls progenitor cell proliferation and differentiation, and the size of organs27, the pathway is closely linked to mitochondrial energy metabolism and biogenesis. Studies suggest that the Hippo pathway modulates host antiviral immune responses33, where YAP inhibits antiviral defense mechanisms by antagonizing the function of pro-innate immune factor TBK134. Inherited autosomal recessive mutations of MST1 (STK4) in human have been attributed to primary immunodeficiency with T- and B-cell lymphopenia, neutropenia, and defective regulatory T cells37. Thus, we postulate that Hippo signaling pathway plays a critical role in SARS-CoV-2 antiviral response and COVID-19 pathogenesis.To replicate, viruses hijack cellular machinery and signaling pathways. Thus, understanding the molecular basis of dysregulated signaling circuitry can provide potential therapeutic targets. Utilizing a library of kinase inhibitors, we previously demonstrated critical pathways that are important for SARS-CoV-2 virus replication such as the DNA-Damage Response (DDR), mTOR-PI3K-AKT, and ABL-BCR/MAPK pathwaysp <0.01) (in situ hybridization (RNA-FISH), SARS-CoV-2 viral genomes were detected in the COVID-19 lung tissue  data sets of lung samples from COVID-19 patients (n=5). KEGG pathway analysis indicates that many genes (45 genes) in the Hippo pathway were differentially regulated (p <0.01) , and 33 g tissue . In addig tissue . The COVg tissue .39. The ciliated and secretory airway epithelial cell types in the ALI culture were verified using immunostaining for the cell type-specific markers, acetylated tubulin and Mucin5AC, respectively . Human lung airway basal stem cells (ABSC) harvested from the trachea and bronchi of healthy lungs were differentiated into a ciliated pseudostratified columnar epithelium in a collagen-coated transwell membrane culture systemectively . The apiectively . In addiectively . Next, wectively . We obse43. To further understand how the Hippo signaling pathway may be modulated by SARS-CoV-2 and the host response, we studied the pathway in cardiomyocytes. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been shown to be efficient at recapitulating cardiovascular diseases at a cellular level45, have demonstrated susceptibility to SARS-CoV-2 replication46, and have been used as a platform to study potential therapeutics against SARS-CoV-2 infection19. Transcriptomic analysis of the SARS-CoV-2 infected hPSC-CM system showed that many genes in the Hippo pathway are significantly dysregulated compared to uninfected mock hPSC-CM control (p <0.01)  . While cp <0.01)  and of ip <0.01) , we obsep <0.01) . In addip <0.01) . Simultap <0.01) . Furtherp <0.01)  and the p <0.01) . PhosphoIn order to uncover the functional significance of the Hippo pathway, we utilized both genetic and pharmacological ablation approaches. The core Hippo signaling pathway is a kinase cascade where MST1/2 kinases and Sav1 form a complex to phosphorylate and activate LATS1/2 kinases. After activation, LATS1/2 can phosphorylate and inhibit YAP and TAZ transcription co-activators. YAP/TAZ, when dephosphorylated, interacts with TEAD1–4 and additional transcription factors to stimulate gene expression for cell proliferation and to inhibit apoptosis. Therefore, the Hippo pathway is regulated at multiple levels. MST1/2 and LATS1/2 are regulated upstream by other molecules like Merlin and KIBRA, and YAP/TAZ is regulated downstream by protein ubiquitination. Thus, we postulated that inhibition of MST1/2 and LATS1/2 would lead to an increase in YAP/TAZ activity, and consequently an increase in SARS-CoV-2 replication. To test this mechanism, we performed shRNA-mediated gene silencing experiments in hPSC-CM. For knockdown experiments, we transduced the cells with lentiviral vectors expressing non-specific control shRNA or shRNAs targeting YAP/TAZ or LATS1. 72 hours post-transduction, the cells were infected with SARS-CoV-2 at MOI of 0.01. At 48 hpi, the cells were fixed for IHC or harvested in RIPA protein lysis buffer for Western blot analysis. Immunohistochemical analysis revealed that the shRNA-mediated partial knock down of YAP1 gene in hPSC-CM cells resulted in significantly reduced SARS-CoV-2 infection, whereas LATS1 knockdown, increased SARS-CoV-2 infection  and D. T47. Therefore, XMU-MP-1, a selective and reversible inhibitor, increases tissue repair and regeneration properties following tissue injuries48. XMU-MP-1 increases cardiomyocyte survival and reduces apoptosis following oxidative stress48. In mice, XMU-MP-1 has been shown to increase intestinal repair, liver repair and regeneration47, and preserve cardiac function and inhibit cardiomyocyte apoptosis in mice with transverse aortic constriction (TAC)48. Infection of cardiomyocytes and Calu-3 lung cells pre-treated with the MST1/2 inhibitor, XMU-MP-1 (10 μM), led to an increase in SARS-CoV-2 replication  in Calu-3 cells  approved drug used in patients with predominantly classic, subfoveal choroidal neovascularization (CNV) caused by AMD Age-related Macular Degeneration (VAM)-3 cells . Therefo-3 cells  for 24 h-3 cells  as well -3 cells . Overallin vitro cultures are efficiently infected by SARS-CoV-2, consequently causing activation of immune, and inflammatory responses, and altered Hippo signaling cascade. shRNA-mediated partial knockdown of LATS1 and pharmacological modulation of core upstream MST1/2 kinases resulted in enhanced SARS-CoV-2 replication. Loss of function mutations in these antiviral kinases could enhance susceptibility to SARS-CoV-2 infections in human. While Verteporfin, a pharmacological inhibitor targeting downstream transactivator, YAP, significantly reduced viral replication and production. Moving forward, additional mechanistic investigations are required to elucidate the underpinnings of the Hippo pathway role in antiviral responses to RNA viruses, and the preclinical animal safety and efficacy studies of Verteporfin therapy for COVID-19. Taken together, our results indicate a direct antiviral role for Hippo signaling in SARS-CoV-2 infection, thus creating potentially new avenues this pathway can be pharmacologically targeted to treat COVID-19.In conclusion, our results show that SARS-CoV-2 infection caused activation of the Hippo signaling pathway in pluripotent stem cell-derived cardiomyocytes (PSC-CMs), human primary lung air-liquid interface (ALI) cultures, and human airway epithelial cells . These For human tissue procurement, large airways and bronchial tissues were acquired after lung transplantations at the Ronald Reagan UCLA Medical Center from deidentified normal human donors following Institutional Review Board (IRB) exemption. Pluripotent stem cell related work received UCLA ESCRO-IRB approval. All the SARS-CoV-2 live virus experiments were performed at the UCLA BSL3 High containment facility.™)] or DSMZ . Cells were cultured in EMEM growth media containing 10% fetal bovine serum (FBS) and penicillin (100 units/ml). Human lung adenocarcinoma epithelial cell line  was purchased from ATCC (ATCCHTB-55) and cultured in Dulbecco’s Modified Eagles Medium (DMEM), supplemented with 20 % fetal bovine serum (FBS), 1 % L-glutamine (L-glu) and 1 % penicillin/streptomycin (P/S). Cells were incubated at 37°C with 5% CO2. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) were derived from hESC line H9 using the previously described method51. Temporarily, hPSCs were maintained in mTeSR1 (STEMCELL Technology). Shortly after, RPMI1640 was supplemented with B27 minus insulin (Invitrogen) and used as differentiation medium. From Day 0–1, 6 μM CHIR99021 (Selleckchem) was added into differentiation medium. Next, Day 3–5, 5 μM IWR1 (Sigma-Aldrich) was added to differentiation medium. After Day 7, RPMI 1640 plus B27 maintenance medium was added. From Day 10–11, RPMI 1640 without D-glucose supplemented with B27 was transiently used for metabolic purification of CMs52. After selection, hPSC-CMs were replated for viral infection. Air-liquid interface (ALI) cultures derived from primary human proximal airway basal stem cells (ABSCs) were used as described previously39. 24-well 6.5mm trans-wells with 0.4mm pore polyester membrane inserts were used for culturing ALI cells. 500 μl ALI media  was used in the basal chamber for ALI cultures and cells were cultured at 37°C with 5% CO2. Use of these established cell lines for the study was approved by the Institutional Biosafety Committee at UCLA.Vero E6 cells were obtained from ATCC .Calu-3 cells were seeded at 30 × 1053 methodology for quantifying TCID50.10 ×103cells/well of Vero E6 cells were seeded in 96-well plates.. The next day, culture media samples of supernatant collected from Calu-3 cells were at 48 hour time point, was subjected to 10-fold serial dilutions (101 to 108) and inoculated onto Vero E6 cells. The cells were incubated at 37°C with 5% CO2 for 3 to 4 days to evaluate for presence or absence of viral CPE. After measuring the percent infected dilutions immediately above and immediately below 50%, the TCID50 was calculated based on the method of Reed and Muench.Viral production by infected cells was measured by quantifying TCID50 as previously describedCOVID 19 patient autopsy lung samples were procured from the UCLA Translational Pathology Core Lab for research use. Samples were processed and sectioned, after a pathologist confirmed the section quality by H&E staining, and subsequent confirmation of COVID-19 positivity by RNAscope V-nCoV2019-S probe . Immunohistochemistry stainings were also performed on this lung tissue: Paraffin-embedded sections were cut at 4μm thickness and paraffin was removed with xylene and the sections were rehydrated through graded ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 min. Heat-induced antigen retrieval (HIER) was carried out for all sections in AR9 buffer (AR9001KT Akoya) using a Biocare decloaker at 95°C for 25 min. The slides were then stained with YAP (S127) antibody  and CD68 antibody  at 4 degree overnight, the signal was detected using Bond Polymer Refine Detection Kit  with a diaminobenzidine reaction to detect antibody labeling and hematoxylin counterstaining.Cells were fixed with 4%PFA for 30–60 minutes or methanol (incubated in −20°C freezer until washed with 1XPBS). Cells were washed 3 times with 1x PBS before permeabilizing with blocking buffer  for 1 hour at room temperature. After adding specific primary antibodies Refer to , cells w−1 aprotinin, 2 mg ml−1 leupeptin and 0.7 mg ml−1 pepstatin or Laemmli Sample Buffer . Cell lysates were resolved by SDS-PAGE using 10% gradient gels and transferred to a 0.2 μm PVDF membrane. Subsequently, the membranes were blocked with 5% skim milk and 0.1% Tween-20 in 1x TBST (0.1% Tween-20) at room temperature for 1 hour. The membranes were then probed with respective monoclonal antibodies and detected by Thermo Scientific SuperSignal West Femto Maximum Sensitivity Substrate.Cells were lysed in 1x RIPA 50 mM Tris pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, 150 mM NaCl, 1 mM Na3VO4, 20 Mm or NaF, 1mM PMSF, 2 mg mlhttps://imagej.nih.gov/ij/index.html). Total cell count was then used for normalization and to calculate the percentage of individual marker positive cell populations in respective images. The mean percentage of positively stained cells from 3–8 independent images  were quantified and presented as graph format.The confocal images were obtained using the Leica Application Suite X (LAS X) and/or the Zeiss LSM 700 Confocal Microscopy by Zeiss Software Program with maximum intensity projection feature. Using a double blinded approach to count the positively stained cells, Image J’s plugin Cell Counter program was used. 3–4 independent images per condition of the hPSC-CM were analyzed. For all the analysis, we used confocal images acquired at 63X objective and fluorescent microscope images acquired at 20 and 40x. Total number of cells in each image was obtained by manually counting all the DAPI stained nuclei using ImageJ program  if they were supported by a false discovery rate (FDR) p<0.01. The heatmaps were prepared using shinyheatmap web interface56. RNA-seq data generated from hiPSC-CM used in this study was retrieved at Gene Expression Omnibus with accession number GSE15039257.High throughput sequencing dataset of five healthy and autopsy lung samples from COVID-19 patients  deceased due to SARS-CoV-2 infection were obtained from the NCBI Gene Expression Omnibus (GEO) under the accession number GSE150316https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE150316). The raw read counts per gene were used for differential expression gene analysis using v1.28.1 in R v4.0.3. DEG were considered if they supported by a FDR p<0.01. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database was used to examine the hippo signaling pathway in the DEG datasets58.High throughput sequencing dataset of five healthy and autopsy lung samples from three patients  deceased due to SARS-CoV-2 infection were obtained from GEO under the accession number GSE150316 (t-test was used to compare two groups (uninfected vs. infected). GraphPad Prism software, version 8.1.2  was used.All statistical testing was performed at the two-sided alpha level of 0.05. To test statistical significance, unpaired student’s 1


Scientific Reports 10.1038/s41598-018-20176-9, published online 29 January 2018Correction to: The Article contains an error in Figure 2, where the western blot showing tubulin has 7 lanes instead of 6 lanes.The correct Figure


We used nasopharyngeal swab samples of patients with a symptomatic (n = 82) or asymptomatic (n = 20) coronavirus disease 2019 (COVID-19) diagnosis to assess the ability of antigen detection tests to infer active  or inactive  infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using the subgenomic RNA (sgRNA) as an active replication marker of SARS-CoV-2, 48 (76.2%), 56 (88.9%), and 63 (100%) of 63 samples with sgRNA positive results tested positive with the SD BIOSENSOR STANDARD Q COVID-19 Ag (Standard Q), the SD BIOSENSOR STANDARD F COVID-19 Ag FIA (Standard F), or the Fujirebio LUMIPULSE G SARS-CoV-2 Ag (Lumipulse) assay, respectively. Conversely, 37 (94.9%), 29 (74.4%), and 7 (17.9%) of 39 samples with sgRNA negative results tested negative with Standard Q, Standard F, or Lumipulse, respectively. Stratifying results by the number of days of symptoms before testing revealed that most antigen positive/sgRNA positive results were among samples tested at 2–7 days regardless of the assay used. Conversely, most antigen negative/sgRNA negative results were among samples tested at 16–30 days only when Standard Q or Standard F were used. In conclusion, based on our findings, a negative antigen test, especially with the Lumipulse assay, or a positive antigen test, especially with the Standard F assay, may suggest, respectively, the absence or presence of replication-competent SARS-CoV-2. Reverse transcription-PCR (RT-PCR)-based molecular assays offer a reliable means of detecting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic RNA (gRNA) in respiratory tract samples from patients who develop coronavirus disease 2019 (COVID-19) within 1 to 2 weeks of SARS-CoV-2 exposure , may be Only one study so far , to the Study design and clinical samples. This retrospective study was carried out at the Fondazione Policlinico Universitario A. Gemelli IRCCS hospital of Rome, Italy, and approved by the Institutional Ethics Committee (reference no. 0040205/21), during a 2021 one-month (24 November through 24 December) period. We randomly selected NPS samples from patients who had a laboratory-confirmed COVID-19 diagnosis, which relied on a positive RT-PCR result through a Panther Fusion SARS-CoV-2 assay . Residual 2-mL aliquots from originally collected samples in 3 mL of universal transport medium  were put into anonymized blank vials and immediately processed for or kept at −80 °C until testing (see below). One hundred and two NPS samples from patients diagnosed with asymptomatic (n = 20), mild (n = 14), moderate (n = 17), severe (n = 32), or critical (n = 19) COVID-19, according to a median (interquartile range) RT-PCR Ct value of 24.6 (18.6–30.2) for SARS-CoV-2 gRNA detection, were included (https://www.covid19treatmentguidelines.nih.gov accessed on 10 May 2022).included . Only foSARS-CoV-2 RNA and antigen testing. Following diagnostic RT-PCR , samples  were used to detect SARS-CoV-2 gRNA , sgRNA, and antigen. For gRNA, Ct values for three SARS-CoV-2 targets, such as envelope (E), RNA-dependent RNA polymerase (RdRP)/spike (S), and nucleoprotein (N) encoding genes, were determined using an updated version of the Allplex™ 2019-nCoV assay [https://www.sdbiosensor.com accessed on 10 May 2022), STANDARD F COVID-19 Ag FIA , and LUMIPULSE G SARS-CoV-2 Ag  immunoassays  according to the respective manufacturer’s instructions. Briefly, the Standard Q is a lateral flow assay that detects SARS-CoV-2 N antigen in a cassette-based format with a visual read-out, allowing interpretation of results as positive  or negative . The Standard F is a fluorescence immunoassay that detects SARS-CoV-2 N antigen with STANDARD F Analyzer, allowing interpretation of results as positive  value of ≥1.0) or negative , according to the manufacturer’s instructions (https://www.sdbiosensor.com accessed on 10 May 2022). The Lumipulse uses a CLEIA (chemiluminescent enzyme immunoassay) technology to measure SARS-CoV-2 S antigen by a LUMIPULSE® G System automated instrument, allowing interpretation of results as positive (>10–>5000 pg/mL), gray-zone positive (1.34–10 pg/mL), or negative (<1.34 pg/mL) [oV assay , now desoV assay , which woV assay . For ant4 pg/mL) .Statistical analysis. For SARS-CoV-2 antigen or sgRNA assays’ results, differences between a priori established groups were assessed using the chi-square test or the Wilcoxon test, as appropriate. Percent agreement values, with their respective confidence intervals (CIs), were calculated comparing results from antigen assays with those from the sgRNA assay, which was used as the reference method. Statistical analysis was conducted using Stata 15  or GraphPad Prism 7  software. p < 0.05 was considered statistically significant.For the 102 patients studied, ages ranged from 45.7 to 78.8 years (median 62.8), and 53 (52.0%) out of 102 were male . Excludip value < 0.001 for all comparisons), sgRNA RT-PCR Ct values differed significantly between antigen positive and antigen negative samples’ groups . Seven (6.9%) samples with Lumipulse antigen (and sgRNA) negative results had a diagnostic RT-PCR Ct median value of 35.1, which was higher than the median value of 32.4 noticed in 39 (38.2%) samples with sgRNA negative results. Except for seven samples, detectable sgRNA levels were noticed in the group of 52 (51.0%) samples with Standard Q antigen negative results  or the group of 36 (35.3%) samples with Standard F antigen negative results .Of the 102 samples studied , 95 (93.Compared to sgRNA results , 48 (76.Stratifying antigen results by number of days of symptoms before sample collection  showed tWe comparatively assessed SARS-CoV-2 antigen detection tests, i.e., three among a myriad of commercially available tests , whose aPrevious studies’ findings supported us to adopt SARS-CoV-2 sgRNA RT-PCR as the reference method for our antigen tests’ assessment ,15,16,17Our study has some intrinsic limitations. First, the sample size is relatively small, particularly for patients without COVID-19 compatible symptoms who represent only 19.6% of the patients included in the study. This precludes our results from being informative for study populations that primarily consist of asymptomatic patients. Second, antigen assays studied by us are very different platforms, implying their analytical sensitivities are bound to be different. This was especially noted for asymptomatic patients, but it could also explain the high rates of Standard Q or Standard F disagreement with the sgRNA RT-PCR assay that occurred for samples tested after 7 days or more of symptoms. Third, our results suggest that antigen assays can be very helpful at the onset of symptoms, which is pretty much expected.In conclusion, our findings reinforce the potential of SARS-CoV-2 antigen tests to serve as a marker for active SARS-CoV-2 replication. However, further studies are required before using such tests to predict SARS-CoV-2 infectivity and, thus, inform the discontinuation of COVID-19 transmission-based precautions.


Correction: J Genet Eng Biotechnol 20, 115 (2022)https://doi.org/10.1186/s43141-022-00392-7Bacillus sp. BNPI-92‘ while it should read ‘Bacillus sp. LPPI-18‘ and affiliation 3 should be removed and in addition Seid Mohammed is only affiliated with affiliation 1. The correct Table Following publication of the original article , the autThe original article  has been


In the original publication , there wFigure 1. Emerging therapeutic approaches against COVID-19 disease: (1) antibody-based therapeutics against the S protein (either via adoptive transfer of vaccination) to avoid progression of severe infections; (2) application of protease inhibitors against serine protease (TMPRSS2) prevents cleavage of S protein; (3) SRS-CoV-2 virus-specific memory CD8+ T cells from vaccination or earlier infection; (4) a new treatment approach targets the symptoms of cytokine storm, wherein the blood of infected patients is passed through customized filtration columns to capture pro-inflammatory cytokines before the pureblood returns to patients. Adapted and modified from [63].Figure 2. Cytokine pathogenesis of SARS-CoV-2 infection. Intensive care or appropriate treatment is recommended for hospitalized severe SARS-CoV-2 patients who exhibit elevated levels of biomarkers in blood plasma: alveolar cell (AC), angiotensin-converting enzyme 2 (ACE2), atopic dermatitis (AD), ancestry clusters (AC1-4) granulocyte-macrophage colony–stimulating factor (GM–CSF), granulocyte colony–stimulating factor (G–CSF or GCSF), interleukin (IL), interferon (INF), monocyte chemoattractant protein 1 (MCP1), macrophage inflammatory proteins (MIP1α), natural killer (NK), polymorphonuclear granulocyte (PMN), T-effector cell (TEFF cell), tumor necrosis factor (TNF), regulatory T cell (Treg cell). Adapted and modified from [116].Table 1. Mechanism of types of drugs and therapies useful in the treatment of SARS-CoV-2-infected patients .The authors apologize for any inconvenience caused and state that the scientific conclusions are unaffected. These corrections were approved by the Academic Editor. The original publication has also been updated.


Litopenaeus vannamei and its Drosophila orthologue Ectoderm-expressed (Ect4) are also involved in immune defense against pathogen infection. However, the functional mechanism of the protective effect remains unclear. In this study, we show that Ect4 is essential for the viral load in flies after a Drosophila C virus (DCV) infection. Viral load is increased in Ect4 mutants resulting in higher mortality rates than wild-type. Overexpression of Ect4 leads to a suppression of virus replication and thus improves the survival rate of the animals. Ect4 is required for the viral induction of STAT-responsive genes, TotA and TotM. Furthermore, Ect4 interacts with Stat92E, affecting the tyrosine phosphorylation and nuclear translocation of Stat92E in S2 cells. Altogether, our study identifies the adaptor protein Ect4 of the Toll pathway contributes to resistance to viral infection and regulates JAK/STAT signaling pathway.Sterile alpha and HEAT/Armadillo motif-containing protein (SARM) is conserved in evolution and negatively regulates TRIF-dependent Toll signaling in mammals. The SARM protein from  Anotignaling . Deficieignaling .Drosophila, JAK/STAT signaling is initiated by three cytokines of the Unpaired (Upd) family . The binding of Upd induces Domeless (Dome) dimerization and activation of the receptor-associated JAK molecules (termed Hopscotch). Activated Hopscotch then phosphorylates Dome, creating a docking site for the single Drosophila STAT family transcription factor, Stat92E. Phosphorylated Stat92E migrates into the nucleus in dimers, promoting target genes transcription (virus-induced RNA 1 (vir-1) and stress response genes Turandot A and M (TotA and TotM)  . Althougallenges , 19.Drosophila and Pacific white shrimp (Litopenaeus vannamei), the production of antimicrobial peptides (AMPs) was downregulated by Ect4 and LvSarm  protein is evolutionarily conserved from arthropods to mammals . The mamd LvSarm , 24, sugfections . The pos22.11118w flies were used as wild-type control. The IRw; L811fsXdcr-2 mutant flies have been previously described . For the generation of Ect4 mutant lines, transgenic U6:3-gRNA-Ect4 flies were crossed with the nos-Cas9 flies to get male F0  that were crossed with 1118w; TM3, Sb/TM6B, Tb to obtain F1 progenies. Singular F1 flies were crossed with 1118w; TM3, Sb/TM6B, Tb. PCR products amplified from F1 flies before being cloned into the pMD19-T vector according to the manufacturer’s instructions (TAKARA) for mutation identification.escribed . ubi-Galescribed . Ect4-IR2.2pAC5.1-Ect4-Flag was made by cloning Ect4 cDNA into pAC5.1-Flag vectors. For pAC5.1-Ect4-GFP constructs, the EGFP fragment was amplified from pEGFP-C1 and assembled with the Ect4 fragment into pAC5.1-V5 vectors using ClonExpress MultiS One Step Cloning Kit (Vazyme). Stat92E cDNA was inserted in pAC5.1-HA to generate pAC5.1-Stat92E-HA. The hop (or Dome) cDNA was inserted in pAC5.1-V5 to generate pAC5.1-hop-V5 (or pAC5.1-Dome-V5). For the truncated Ect4 constructs, ARM domain (aa 318-701), SAM domain (aa 680-826), and TIR domain (aa 829-1360) were amplified from pAC5.1-Ect4-Flag before assembled into pAC5.1-Flag empty vector, respectively.2.3S2 cells were cultured at 25°C in Sf-900™ III SFM (Gibco). All S2 cell transfection experiments were carried out with the Effectene Transfection Reagent (QIAGEN). For a co-immunoprecipitation assay, S2 cells were transfected with different plasmids. After 48h, cells were collected and lysed in lysis buffer . Lysates were incubated overnight at 4°C with Anti-Flag M2 affinity gel (Sigma) or EZview Red Anti-HA Affinity Gel (Sigma). After centrifugation, pellets were washed with 1ml lysis buffer three times before resuspension in 2X Laemmli SDS-PAGE buffer and detection by Western blot. Western blot was performed according to standard procedures.Primary antibodies: Mouse anti-V5 ; mouse anti-HA ; mouse anti-α-Tubulin ; goat anti-Stat ; mouse anti-FLAG ; rabbit anti-DCV ; mouse anti-PY20 . Secondary antibodies: HRP-linked anti-mouse IgG ; HRP-linked anti-rabbit IgG ; HRP-linked anti-goat IgG (1:5000 Millipore AP106P); Alexa Fluor 555 goat anti-mouse IgG .2.4RpL32 , vir-1 , DCV , TotA , TotM , Ect4 , Vago .According to the manufacturer’s instructions, total RNA was extracted from infected flies using RNAiso Plus (TAKARA), and cDNA was synthesized with the HiScript II Q RT SuperMix (Vazyme). The ChamQ SYBR qPCR Master Mix (Vazyme) was used for quantitative. Expression of the gene of interest was normalized to the Rpl32 RNA level. The following primers were used for qPCR: 2.52 and injected with PBS (Gibco) or virus suspension intra-thoracically using the Nanoject II injector (Drummond). Infected flies were monitored daily for survival rate or frozen for RNA analysis at the indicated time points.Virus stocks were prepared as described previously . All fly2.6pAC5.1-Ect4-GFP and pAC5.1-Stat92E-HA plasmids, and approximately 1×106 cells were transferred to 24 well plates containing coverslips 48 h after transfection. Twelve h later, cells were washed in 0.5ml PBS and fixed with 4% formaldehyde in PBS for 15 min, then washed twice in 0.5 PBT (PBS containing 0.1% Tween-20) before blocking with 5% BSA in TBST for 1 h. Cells were then incubated with primary antibody (anti-HA 1:1000) overnight at 4°C before 2×5 min TBST washes. The secondary antibody was incubated for 4 h at room temperature. Nuclei were stained with PBS with 10 μg/ml DAPI for 5 min. Immunostaining samples were photographed with a Zeiss confocal microscope.S2 cells were transfected with d old, raised at 25°C) of each indicated genotype were homogenized in methanol . After centrifugation, the supernatants were measured for absorbance at 480 nm.For eye pigment assay, the heads of 50 female flies  pre-incubated for 15 min was added to S2 cells to induce tyrosine phosphorylated Stat92E for 30 min. Cell lysates were prepared with the lysis buffer before immune precipitation with an anti-Stat92E antibody at 4°C and incubated with Pierce Protein A/G Plus Agarose (Thermo Scientific) beads. Co-immuno-precipitated proteins were detected with an anti-Stat92E antibody or anti-PY20 antibody.dsRNA targeting 2.8t-test. Grey value analysis was performed by the ZEN 2012 (blue edition) system. P-values below 0.05 were considered significantly different.Survival data were analyzed by the Kaplan-Meier method using GraphPad Prism. Quantification of immunoblots was performed with ImageJ 1.51p. Altered protein levels were presented as normalized fold change compared to the control value. Statistical analysis was performed using the Student’s 33.1Ect4 in Drosophila antiviral defense, an Ect4 mutant line was generated with the CRISPR/Cas9 system. The mutation, 17Ect4, covers a genomic deletion of 17 bp in the coding region of Ect4  position 598, located in the last exon, has the strongest effect on DCV susceptibility , thus limiting the effect of discordance in the SNP profile between different fly lines to the difference in DCV resistance.Variation in the pastrel gene is associated with natural resistance to DCV infection in tibility . SequencEct4 transcription in 17/+Ect4 heterozygotes was reduced by 45% compared with the wild-type flies . Therefore eye pigmentation of a Dcr-2 null mutant (L811fsX/dcr-2L811fsXdcr-2) in GMR>UAS-wIR background is red, whereas Dcr-2 heterozygous mutants (L811fsX/+dcr-2) display pale orange  acts as the first line of defense against viruses in osophila . There wosophila , 34. Sine orange . Our res Dicer-2 , did not3.4Drosophila (via the JAK/STAT pathway including virus-induced RNA-1 (vir-1), the stress-induced genes Turandot A and M (TotA and TotM) . As previously reported, DCV infection induced a strong up-regulation of vir-1, TotA, and TotM in wild-type flies , which encodes a hyperactive JAK kinase due to a G341E substitution  . To exampe flies . Howevertitution . Reducin hopTum-l, which e hopTum-l, which e hopTum-l, which e3.5Ect4 and JAK/STAT pathway, we examined whether Ect4 interacted with any known components of the JAK/STAT pathway. Differentially tagged forms of JAK/STAT pathway components and Ect4 were expressed in S2 cells, and co-immunoprecipitation studies were performed. As shown in To unravel the molecular mechanism underlying the relationship between Ect4 protein harbor three different domains: ARM (Armadillo motif) domains followed by two SAM (Sterile Alpha motif) domains and TIR (Toll -Interleukin-1 receptor) domain  was not detected by immunostaining, likely due to the transient activity of p-Stat92E dimers. Therefore, is Ect4 required for the nuclear translocation of Stat92E? As expected, reduced nuclear translocation of Stat92E in response to the pervanadate stimulus was detected in cells treated with Ect4 RNAi  and L. vannamei (LvSarm) were required to express antimicrobial peptides. Depletion of both led to decreased survival of the animals upon bacterial infections , and t stress , 46. TheEct4 reduced phosphorylated Stat92E upon pervanadate treatment in S2 cells, which suggests a role of Ect4 in regulating cell death through the modulation of JAK/STAT via the interaction with Stat92E.Ect4 interacts with Stat92E in S2 cells through the highly conserved TIR domain of SARM family origin, which have roles in cell death and neuronal destruction in mammals . As an ain vivo study revealed that Ect4 mutants showed enhanced mortality and increased viral load upon DCV challenge. We seek to further elucidate the unknown mechanisms of antiviral response in Drosophila by assessing whether Ect4 affects host resistance to viral infection by regulating cell death.In addition to antiviral immune defense, apoptosis is a conserved mechanism of programmed cell death that can prevent the infection before viral replication is completed , 51. OurDrosophila Ect4 in regulation of JAK/STAT signaling pathway and protection against DCV infection. It is still unclear if Ect4 also participated in the control of other virus infection. The contribution of JAK/STAT signaling to Drosophila antiviral protection is virus-specific. Although JAK/STAT pathway can be activated by RNA viruses, including DCV, CrPV, FHV, and DXV, it is only required for resistance against two Dicistroviridae family members, DCV and CrPV  polymerase PARP9 was recently reported as a noncanonical sensor for RNA viruses that depends on the PI3K/AKT3 pathway to produce antiviral type I interferon . PARP9 iThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.ZH and PX carried out experiments. SG contributed to data collection. YH analyzed confocal images. YL, SY, and FS conducted statistical analysis. KA and W-MD revised the manuscript. JL and RJ contributed to the conception and design of the experiments. WW wrote the first draft of the manuscript. JC directed the studies. All authors contributed to the article and approved the submitted version.