C. elegans after injection. In some cases, fluorophores are used to identify transgenics and to propagate animals with extra-chromosomal or integrated arrays  . In other cases, fluorescent markers are used as visual markers to identify and later select against array animals. Such negative selection is used generate single-copy transgene insertions by plasmid injection, e.g., Mos1-mediated single-copy insertion (MosSCI) or CRISPR/Cas9 . These methods generate targeted double-strand breaks, and transgenes are inserted by homologous recombination into specific locations. Selection schemes that rely on positive  and negative   selection markers are frequently used to identify single-copy insertions. Negative peel-1 selection is under control of a heat-shock promoter (Phsp-16.41); after heat-shock, animals with arrays or dual insertions with the peel-1 transgene rapidly die . peel-1 is convenient, but this selection has two significant drawbacks: the transgene is toxic in the absence of heat shock, resulting in fewer F1 progeny , and the induced lethality is often not fully penetrant resulting in false positives. Regardless of whether the peel-1 selection is used, we have advocated for the inclusion of all three red fluorescent markers expressed in neurons (Prab-3), body-wall muscle (Pmyo-3), and pharynx (Pmyo-2) because a single or even two markers were inefficient at avoiding false positives. These three selection markers are widely used and have been requested more than 300 times each from Addgene. However, adding three fluorescent markers to every injection mix is increasingly inconvenient as the mixes also contain plasmids encoding enzymes (Mos1 transposase or Cas9), sgRNAs, repair templates, and sometimes additional markers. Pharyngeal expression of the Pmyo-2 fluorophore is the easiest to identify on a fluorescence dissection microscope due to early embryonic expression and brightness, but the transgene is frequently toxic and is therefore not commonly injected at high concentrations. For these reasons, we sought to identify an improved co-injection marker. An ideal marker would be bright, non-toxic, expressed in a clearly identifiable tissue, and only require a single marker in the injection mix. Here we demonstrate that fluorescent transgenes containing the pan-muscular promoter from myosin light chain 1 (mlc-1) fulfill these criteria. The mlc-1 and the related mlc-2 promoter may also be generally useful to identify all  muscles or for robust expression of transgenes .Fluorescent markers are useful for identifying transgenic et al. 2010). We identified two putative muscle-specific myosin light chain genes, mlc-1 and mlc-2, that are highly expressed from a single divergent locus. RNA expression levels based on Fragments Per Kilobase Million reads (FPKM) are comparatively high for these two genes in young adults, with 500 FPKM (mlc-1) and 1159 FPKM (mlc-2) compared to 230 FPKM (unc-54), 14 FPKM (myo-2), and 29 FPKM (myo-3). We selected unc-54, mlc-1, and mlc-2 as candidates and amplified 1.9 kb, 1.3 kb, and 1.2 kb promoters, respectively. Due to an early splice site (immediately after the start codon), Pmlc-1 also included the first six amino acids of mlc-1. To quantify the relative expression level of the three promoters, we generated otherwise identical transgenes with a codon-optimized gfp under each promoter’s control and made single-copy insertions at the ttTi5605 site using MosSCI. All promoters drove GFP expression in the body wall, stomato-intestinal, and anal depressor muscles . However, only Punc-54 was not expressed in pharyngeal  and vulval muscles  in agreement with previous promoter analysis . Using a Copas large-particle flow cytometer, we quantified GFP expression in young adults from a mixed population of the MosSCI strains and found that Pmlc-1:gfp resulted in the highest expression, with approximately two-fold more expression than Punc-54 . The fluorescence signal matched our subjective visual impression from transgenic animals carrying extra-chromosomal arrays. We verified that the Pmlc-1 and Pmlc-2 strains harbored only single transgene insertions , excluding the possibility that transgene copy-number caused the observed difference in fluorescence.We focused on promoters from genes expected to have muscle-specific expression and manually checked their relative expression levels based on RNA-seq from the ModENCODE project . Higher concentrations thus make it less likely to generate array animals lacking the fluorescent marker, which is essential to avoid false positives . Therefore, we quantified the toxicity of Pmlc-1::mCherry and Pmyo-2::mCherry transgenes when used as co-injection markers at increasing concentrations . We quantified the number of injected P0 animals that gave F1 transgenic animals and stable F2 transgenic lines . We did not observe apparent toxicity from either promoter at low concentrations (2.5 ng/ul and 10 ng/ul). At high concentration (25 ng/ul), we observed a reduction in plates with rescued F1s and fewer stable lines for Pmyo-2, suggesting some toxicity. In contrast, even at high concentrations, Pmlc-1 did not show any apparent toxicity.Co-injection markers are incorporated into extra-chromosomal arrays in proportion to their concentration in the injection mix (Mello mlc-1 at high concentration (25 ng/ul). We inserted a germline-expressed Psmu-1::gfp transgene  by MosSCI using cbr-unc-119 as the positive selection and a Pmlc-1::tagRFP-T transgene as the visual negative selection marker (in addition to Phsp-16.41:peel-1). We injected ten animals and recovered six plates with extra-chromosomal arrays in the F2 or F3 generation. We heat-shocked these plates and identified four plates with rescued animals  but with no visible red fluorescence on a dissection fluorescence microscope. To rule out false positives, we picked a single worm from each plate and checked for stable germline expression from a transgene insert in the next generation . All four independent lines expressed GFP in the germline and we observed no tagRFP expression from the fluorescent co-injection marker at high magnification. These results indicate that we could use a single fluorescence marker to select against false positives. Homologous repair relies on unique sequences in the extra-chromosomal array. Therefore, we have generated a set of green (gfp and mNeonGreen) and red (mCherry and TagRFP-T) Pmlc-1 fluorescent markers with cytoplasmic and nuclear expression to avoid cross-talk with the repair machinery.Our goal was to use a single, bright co-injection marker to generate MosSCI insertions, and the lack of toxicity allowed us to test Pmlc-1 and mlc-2 that can drive bright fluorescent expression in all muscles. We have used mlc-1 to generate co-injection markers that are non-toxic at high concentrations. The use of a single fluorescent co-injection marker solves a minor inconvenience when generating MosSCI injection mixes and would, presumably, also be useful for other methods that rely on similar selection schemes. More generally, mlc-1 and mlc-2 promoters should be generally useful when a bright pan-muscle expression is desired, e.g., for transgene rescue, for expressing optogenetic effectors , or for isolating cells or nuclei from all muscles . Pmlc-2 is expressed at slightly lower levels but may be preferable in the cases where the first six amino acids of mlc-1 might perturb experiments. To facilitate the use of these reagents, we have deposited fluorescent transgenes and Gateway-compatible promoter vectors at Addgene for distribution.Here, we describe two promoters from ReagentsCo-injection markers available at AddgenePmlc-1::mNeonGreen(2xNLS)::cbr-tbb-2 UTR (Addgene #159895)pSEM228 – Pmlc-1::mNeonGreen::cbr-tbb-2 UTR (Addgene #159896)pSEM229 – Pmlc-1::GFP(2xNLS):: cbr-tbb-2 UTR (Addgene # 159794)pSEM230 – Pmlc-1::GFP::cbr-tbb-2 UTR (Addgene #159897)pSEM231 – Pmlc-1::TagRFP-T(2xNLS)::cbr-tbb-2 UTR (Addgene #159898)pSEM232 – Pmlc-1::TagRFP-T::cbr-tbb-2 UTR (Addgene #159899)pSEM233 – Pmlc-1::mCherry(2xNLS)::cbr-tbb-2 UTR (Addgene # 159795)pSEM234 – Pmlc-1::mCherry::cbr-tbb-2 UTR (Addgene #159900)pSEM235 – Promoter – Pmlc-1 (w ATG) (Addgene #159888)pCFJ2113 – [4-1] – Promoter – Pmlc-2 (no start) (Addgene #159889)pSEM223 – [4-1] – Annotated plasmid sequences are available at www.wormbuilder.org and https://www.addgene.org/Christian_Froekjaer-Jensen/Molecular biologyWe generated all co-injection markers by three-fragment multisite Gateway reactions (Invitrogen Cat #12538200). The pENTR[4-1] vectors were made by amplifying the promoters and assembled by BP Gateway reaction (Invitrogen Cat #11789013) into a custom pDONR4-1 vector (pCFJ1509). All PCRs were done using high fidelity Phusion polymerase (F530S) and validated by Sanger sequencing.Promoter quantificationmlc-1, Pmlc-2, or Punc-54 fused to a codon-optimized gfp were inserted by MosSCI into the EG4322 ) strain following standard protocols. The injection mixes were composed of 25 ng/ul repair template, 20 ng/ul pCFJ104 (Pmyo-3::mCherry), 20 ng/ul pGH8 (Prab-3::mCherry), 5 ng/ul pCFJ90 (Pmyo-2::mCherry), 20 ng/ul pMA122 (peel-1::HS), 20 ng/ul pNP403 (histamine selection), 20 ng/ul pCFJ1532 (Psmu-1:Mos1 transposase). After injection, we singled each worm onto a standard NGM plate seeded with HB101 and placed animals at 25º Celsius. Starved plates with unc-119 rescued animals were placed for one hour at 37º Celsius (heat-shock) and “chunked” to a new NGM plate. Twenty-four hours later, we picked a single worm with unc-119 rescue and without visible fluorescence from co-injection markers to generate a stable line. Once a line was generated, we placed worms onto standard NGM plates seeded with OP50 and grew at 25º Celsius.Transgenes with PPlasmids used to generate MosSCI insertion strainsPunc-54::gfp::cbr-tbb-2 UTRpSEM218 – Pmlc-2::gfp::cbr-tbb-2 UTRpSEM219 – Pmlc-1::gfp::cbr-tbb-2 UTRpSEM220 – MosSCI strainskstSi26[Punc-54 | gfp | cbr-tbb-2 3′ UTR] II; unc-119(ed3) IIICFJ62: kstSi27[Pmlc-2 | gfp | cbr-tbb-2 3′ UTR] II; unc-119(ed3) IIICFJ63: kstSi28[Pmlc-1 | gfp | cbr-tbb-2 3′ UTR] II; unc-119(ed3) IIICFJ64: smu-1::gfp) for germline expression, 25 ng/ul pSEM233 (Pmlc-1::TagRFP-T), 20 ng/ul pMA122 (peel-1 ::HS), 20 ng/ul pCFJ1532 (Psmu-1:Mos1 transposase), GeneRuler 1kb plus DNA ladder (ThermoFisher SM1331) for a final concentration of 100 ng/ul.To test if a single co-injection marker was sufficient to select for MosSCI insertions, we used the same strategy but with a germline-expressed transgene and a modified injection mix. The mix consisted of 25 ng/ul pCFJ1805 .We placed six worms onto NGM plate seeded with OP50 (four plates per strain) and placed animals at 25º Celsius. After five days, worms were washed off growth plates with M9 and washed twice to remove bacteria. We quantified fluorescence using a Copas flow cytometer (Union Biometrica). A mixed population was analyzed, but we only quantified the fluorescence of young adult worms  and GeneRuler 1kb plus DNA ladder (ThermoFisher SM1331) for a final concentration of 100 ng/ul. Ten worms were injected per condition, singled onto NGM plates seeded with OP50, and placed at 25º Celsius. 48 hours after, we determined the number of plates with transgenic F1s and several days later determined if the plates had stable arrays lines in the F2 or F3 generation.Transgenic worms were generated by injection into N2 (wild type) worms. The injection mixes were composed of PPlasmids used for toxicity assaysmlc-1::mCherrypSEM235 – Pmyo-2::mCherrypCFJ90 – PMicroscopyWe imaged animals on a Leica DM2500 microscope equipped with a Leica DFC7000 GT camera at 20X. Worms were immobilized using an M9 solution containing 50 mM of sodium-azide and mounted on 2% agarose pads. We analyzed the fluorescence signal using ImageJ and Affinity designer software.


The COVID-19 pandemic has already caused over 1 million deaths. Therefore, effective vaccine concepts are urgently needed. In search of such a concept, we have analyzed a measles virus-based vaccine candidate targeting SARS-CoV-2. Using this well-known, safe vaccine backbone, we demonstrate here induction of functional immune responses in both arms of adaptive immunity, yielding antiviral efficacy in vivo with the desired immune bias. Consequently, no immunopathologies became evident during challenge experiments. Moreover, the candidate still induces immunity against the measles, recognized as a looming second menace, when countries are forced to stop routine vaccination campaigns in the face of COVID-19. Thus, a bivalent measles-based COVID-19 vaccine could be the solution for two significant public health threats. + and CD4+ T cell responses with S protein-specific killing activity were detected. Upon challenge using a mouse-adapted SARS-CoV-2, virus loads in vaccinated mice were significantly lower, while vaccinated Syrian hamsters revealed protection in a harsh challenge setup using an early-passage human patient isolate. These results are highly encouraging and support further development of MeV-based COVID-19 vaccines.The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and has spread worldwide, with millions of cases and more than 1 million deaths to date. The gravity of the situation mandates accelerated efforts to identify safe and effective vaccines. Here, we generated measles virus (MeV)-based vaccine candidates expressing the SARS-CoV-2 spike glycoprotein (S). Insertion of the full-length S protein gene in two different MeV genomic positions resulted in modulated S protein expression. The variant with lower S protein expression levels was genetically stable and induced high levels of effective Th1-biased antibody and T cell responses in mice after two immunizations. In addition to neutralizing IgG antibody responses in a protective range, multifunctional CD8 Coronaviridae family and emerged toward the end of 2019 as causative agent of pneumonia in the Hubei province in China (0 of at least 2 to 2.5 (https://www.who.int/emergencies/diseases/novel-coronavirus-2019), while worldwide shutdowns of social life and economy to confine the spread of this respiratory virus have considerable impacts.Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) belongs to 2 to 2.5 . Due to http://www.cepi.net/research_dev/our-portfolio).After the emergence of SARS in 2002 and then MERS in 2012, vaccine development efforts have been initiated, including the use of recombinant measles virus (MeV) vaccine as a platform concept , to deve−/−-CD46Ge mice induced strong and functional humoral and cellular immune responses directed against both MeV and SARS-CoV-2 S protein biased for Th1-type T cell and antibody responses. The induced immunity translated in antiviral efficacy in two different challenge models, that is, vaccinated hamsters and mice, thereby illustrating the potential of MeV platform-based COVID-19 vaccine candidates.Here, we explored the potential of recombinant MeV as vectors for the expression of the SARS-CoV-2 spike protein (S) as successfully applied for the development of MERS , 8 and Svac2 genome, either downstream of the P (post P) or H (post H) gene cassettes (7 50% tissue culture infectious doses (TCID50)/mL. The stability of the viral genomes was demonstrated via sequencing after RT-PCR of viruses in P2 or P10. In parallel to Sanger sequencing of the ATU region encompassing the SARS-CoV-2-S gene, the full genome was sequenced using next-generation sequencing (NGS) with a coverage of 4 to 29,683 reads of each position  in P2. Both methods revealed no mutations across the whole vaccine genomes but a single A to G substitution on position 9 of the noncoding trailer region of the MeVvac2-SARS2-S(H) clone used for in vivo studies (GenBank accession no. MW090971).Since the SARS-CoV and MERS-CoV S proteins have been shown to potently induce humoral and cellular immune responses, the SARS-CoV-2 S protein was chosen as an appropriate antigen to be expressed by the recombinant MeV vaccine platform. A codon-optimized full-length gene encoding SARS-CoV-2 S protein was cloned into two different additional transcription units (ATUs) in the vaccine strain MeVassettes A. Recombvac2-SARS2-S was performed. The S protein expression was slightly attenuated when cells were infected with viruses encoding the antigen in the ATU post-H compared to the post-P constructs (vac2-ATU(P) control virus revealed that the MeVvac2 encoding full-length SARS-CoV-2 S gene in post-P position grew remarkably different than the control virus, with ∼100-fold reduced maximal titers. In contrast, growth of MeVvac2-SARS2-S(H) was much closer to MVvac2-ATU(P), with only a slight trend for lower titers  was accompanied by a hyperfusogenic phenotype , which was also observed for the post-H vaccine candidate, but to a lesser extent. Therefore, fusion activity was quantified and compared to the parental MVvac2-ATU(P) as well as the MVNSe-GFP(N), which is known for its hyperfusogenic phenotype due to a V94M substitution in the F2 subunit of the MeV fusion protein (vac2-ATU(P) induced fusion of 16.8 ± 0.8 (mean± SD) Vero cells 30 h after infection. MeVvac2-SARS2-S(P) revealed approximately fourfold enhanced fusion activity (syncytia including 70 ± 8 cells), while MeVvac2-SARS2-S(H) just fused 41 ± 6 cells, thereby representing an intermediate phenotype. However, fusion activity of the latter was surpassed by MVNSe-GFP(N) that fused 56 ± 4 cells in 30 h under the same conditions .The impaired growth of MeVSI Appendix, Fig. S3).To investigate whether this increased fusion activity is due to SARS-CoV-2 S protein-mediated cell-to-cell fusion, we expressed the SARS-CoV-2 S protein by transfection of the eukaryotic expression plasmid pcDNA3.1-SARS2-S into SARS-CoV-2 receptor hACE2-negative 293T as well as into receptor-positive Vero cells. Indeed, expression of SARS-CoV-2 S protein induced syncytia of Vero, but not of 293T cells (vac2-SARS2-S(H) was chosen for further characterization in vivo.These data demonstrate that the hyperfusogenic phenotype of the SARS-CoV-2 S-encoding MeV is linked to expression of a fusion-active form of the SARS-CoV-2 S protein, indicating that cells infected by the vaccine candidates express a functional S protein. Thus, cloning and rescue of MeVs expressing correctly folded SARS-CoV-2 S was achieved successfully. Since higher S protein expression levels impaired viral replication, MeVvac2-SARS2-S(H) in vivo, genetically modified IFNAR−/−-CD46Ge mice were used, since they are the prime small animal model for analysis of MeV-derived vaccines (5 TCID50 of MeVvac2-SARS2-S(H) or empty MVvac2-ATU(P) as a control. As positive control, recombinant SARS-CoV-2 S protein adjuvanted with aluminum hydroxide gel (Alum) was injected subcutaneously, and medium-inoculated mice served as mock controls. Twenty-one days after the second immunization, sera of immunized mice were analyzed in comparison to prebleed and postprime immunization sera, by ELISA on antigen-coated plates, for total IgG antibodies binding to MeV bulk antigens (vac2-SARS2-S(H) contained IgG antibodies that bound to SARS-CoV-2 S protein (vac2-SARS2-S(H)  C.vac2-SARS2-S(H) cohort initially reacted only weakly, and another mouse reacted not at all, reflecting individual differences in response to immunization. All animals had developed neutralizing antibodies after the second immunization, and a threefold increase was observed upon the second immunization  after the second immunization, and reached a titer of 15 to 80 in three out of seven mice . No VNTs against MeV or SARS-CoV-2 were detected in control mice inoculated with medium alone. Interestingly, the Alum-adjuvanted recombinant S protein did not induce any neutralizing antibodies despite higher binding IgG levels in ELISA, indicating that these antibodies bind to other epitopes of S or with lower affinity than those induced by the MeV-based vaccine candidate. In summary, the SARS-CoV-2 S protein-expressing MeV elicited robust neutralizing antibody responses against MeV and SARS-CoV-2.We next determined the neutralizing antibody responses against SARS-CoV-2 D–F or Meven mice F. These vac2-SARS2-S(H) to induce SARS-CoV-2−specific cellular immune responses, splenocytes of vaccinated animals were analyzed for antigen-specific IFN-γ secretion by Enzyme-Linked Immunosorbent Spot (ELISpot) assay. Toward this, antigen-specific T cells were restimulated by cocultivation with the syngeneic murine dendritic cell (DC) cell lines JAWSII or DC2.4 stably expressing the SARS-CoV-2 S protein. For JAWSII cells, bulk cultures of transduced cells were obtained by flow cytometric sorting. For DC2.4 cells, single-cell clones were generated by limiting dilution of sorted bulk cultures. Antigen expression by transduced DCs was verified by Western blot analysis and flow cytometry .To assess the ability of MeV6 splenocytes after immunization with MeVvac2-SARS2-S(H) −vaccinated mice was confirmed by stimulation with transgenic JAWSII-SARS2-S bulk cells. These cells also stimulated in excess of 1,400 IFN-γ secreting cells per 1 × 106 splenocytes in animals receiving the recombinant SARS-CoV-2 vaccines, whereas only slight background stimulation was observed by the respective controls. The differences between MeV control and MeVvac2-SARS2-S(H) vaccinated mice were statistically significant for both cell lines. Mice vaccinated with Alum-adjuvanted S protein showed no specific reactivity in IFN-γ ELISpot.ELISpot assays using splenocytes of vaccinated animals in coculture with DC2.4-SARS2-S cells revealed more than 1,400 IFN-γ secreting cells per 1 × 10RS2-S(H) . In contckground . To rule6 splenocytes of MVvac2-ATU(P)−vaccinated animals, splenocytes of MeVvac2-SARS2-S(H)−vaccinated animals were in a similar range of 400 to 1,400 IFN-γ secreting cells per 1 × 106 splenocytes. Splenocytes of all animals revealed a similar basic reactivity to unspecific T cell stimulation, as confirmed by numbers of IFN-γ secreting cells upon concanavalin A (ConA) treatment at the limit of detection. Remarkably, stimulation of splenocytes by DC2.4 expressing SARS-CoV-2-S resulted in at least similar or even higher numbers of IFN-γ+ cells than after stimulation by MeV bulk antigens, indicating an extremely robust induction of cellular immunity against this antigen. Taken together, these data show that MeVvac2-SARS2-S(H) induces not only humoral but also strong SARS-CoV-2 S protein-specific cellular immune responses.Cellular immune responses upon stimulation with MeV bulk antigens were detected in animals that had been vaccinated with any recombinant MeV virus, as expected. While MeV bulk antigens stimulated about 300 to 1,400 IFN-γ secreting cells per 1 × 10+ and CD4+ positive CD3+ T cells upon restimulation with SARS-CoV-2 S-presenting DC2.4-SARS2-S cells by intracellular cytokine staining (ICS). As a positive stimulus for T cell activation, ionomycin and phorbol myristate acetate (Ionomycin/PMA) were used. Exocytosis of cytokines was blocked by addition of brefeldin A (10 μg/mL) during stimulation. Cells were permeabilized, labeled, and fixed for flow cytometry. The gating strategy excluded duplicates , selected for living cells , and separated CD8+ and CD4+ T cells on CD3+ cell populations . Selected T cells were then analyzed for their expression of IFN-γ, TNF-α, or IL-2, double-positive , or triple-positive  cells as exemplarily shown for CD4+ T cells after restimulation with PMA and ionomycin .To gain more detailed insights into the quality of the observed T cell responses, we further characterized the responsive T cell populations by flow cytometry, determining the expression of IFN-γ, TNF-α, and IL-2 in CD8vac2-SARS2-S(H) induced a significant amount of SARS-CoV-2 S-specific CD8+ T cells expressing either IFN-γ (Left), IL-2 (Middle), or TNF-α (Right), with means between 0.02% and 0.5% of positive cells for each of these cytokines. Among those, a significant fraction of cells proved to be multifunctional, with a mean of 47% of the reactive CD8+ cells expressing two cytokines or 13% of responsive CD8+ cells being positive for TNF-α, IL-2, and IFN-γ . A much lower portion of responsive CD4+ T cells was observed, varying between 0.01% and 0.07% of CD4+ T cells. Among the responsive CD4+ cells, 40% expressed two cytokines and 11% were positive for TNF-α, IL-2, and IFN-γ. Moreover, vaccination induced a significant fraction of vector-specific CD4+ T cells expressing IFN-γ (Left), IL-2 (Middle), or TNF-α (Right) upon restimulation with MeV bulk antigen. Among those, multifunctional CD4+ T cells expressing two or all three cytokines were induced with a mean of about 23% and 6% polyreactive T cells , respectively. To conclude, vaccination with MeVvac2-SARS2-S(H) induces not only IFN-γ, TNF-α, or IL-2 expressing T cells directed against SARS-CoV-2 and MeV but also a significant fraction of multifunctional cytotoxic T cells specific for SARS-CoV-2 S and CD4+ T cells specific for MeV antigens, illustrating that a broad and robust SARS-CoV-2−specific immune response is induced by vaccination with MeVvac2-SARS2-S(H).Vaccination with MeVer IFN-γ B, Left, t), IL-2 B, Middleor TNF-α B, Right,ng IFN-γ A, Left, t), IL-2 A, Middleor TNF-α A, Right + and CD8+ T cells upon stimulation with S-presenting DCs only among the splenocytes of MeVvac2-SARS2-S(H)−vaccinated animals .The reactivity of these T cells also became apparent in a proliferative response to antigen-specific stimulation. Using carboxyfluorescein succinimidyl ester (CFSE)-labeled splenocytes and flow cytometry, we detected significant proliferation of both CD4green-SARS2-S target and EL-4red control cells (ratio ∼1:1), only T cells from MeVvac2-SARS2-S(H)−vaccinated mice significantly shifted the ratio of live SARS-CoV-2 S protein-expressing target cells to control cells in a dose-dependent manner (To demonstrate the effector ability of induced cytotoxic T lymphocytes (CTLs), a killing assay was performed to directly analyze antigen-specific cytotoxicity . Splenoct manner B. This at manner A.vac2-SARS2-S(H)−vaccinated mice are capable of lysing cells expressing SARS-CoV-2 S. Neither splenocytes of control mice restimulated with DC2.4-SARS2-S nor splenocytes of SARS-CoV-2 S protein vaccinated mice restimulated with control DC2.4 cells showed such an antigen-specific killing activity, demonstrating that MeVvac2-SARS2-S(H) induces fully functional antigen-specific CD8+ CTLs.These results indicate that CTLs isolated from MeVvac2-SARS2-S(H) is reassuring, the SARS-CoV-2 vaccine development has to proceed with some caution because of the potential risk of immunopathogenesis observed in some animal models, such as antibody-dependent enhancement (ADE) and enhanced respiratory disease (ERD) which seem to correlate with a Th2-biased immune response. Since, in mice, IgG1 is a marker for Th2 bias and risk of ADE development, whereas IgG2a antibodies indicate a favorable Th1 bias, IgG subtype-specific ELISA was performed with the sera collected at different time points. Animals vaccinated with Alum-adjuvanted SARS-CoV-2 S protein, a vaccine concept known for its Th2 bias (vac2-SARS2-S(H) induced 100-fold fewer IgG1 antibodies, but at least 10-fold higher IgG2a levels . Most likely due to the low number of S-reactive T cells in animals that had been vaccinated with recombinant SARS-CoV-2 S protein and Alum, no, or minimal, constant cytokine levels were measurable in the supernatants of restimulated splenocytes (vac2-SARS2-S(H) reacted specifically with the secretion of IFN-γ, TNF-α, and IL-2 upon restimulation by DC2.4-SARS2-S , which indicates a relatively low risk for putatively Th2-mediated immunopathologies.Thus, both humoral and cellular responses reveal a Th1-biased immunity induced by MeVSI Appendix, Fig. S10 A and B) that necessitated killing four out of six animals within 5 d to 6 d postinfection . To take advantage of this model, hamsters were vaccinated with MeVvac2-SARS2-S(H), MVvac2-ATU(H) as vector control, Alum-adjuvanted SARS-CoV-2 Spike protein as vaccine control, or medium (mock) in a prime−boost scheme for challenge. These hamsters showed induction of anti-MeV and anti-SARS-CoV-2 humoral immune responses with neutralizing antibody titers against SARS-CoV-2 between 15 and 80 VNT corresponding to 20 to 213 PRNT50 , exceeding those of the Alum-S vaccine control group . Upon challenge, the initial weight loss observed in all groups was stopped in the MeVvac2-SARS2-S(H) cohort on day 3 postinfection, and animals started to gain weight, whereas all other groups revealed progressive weight loss : The hamsters vaccinated with MeVvac2-SARS2-S(H) had a more than 10-fold reduction in virus genomes in lungs and nasal turbinates than all other groups (vac2-SARS2-S(H) significantly ameliorated virus loads in infected animals and protected them against severe pathology in this remarkably aggressive challenge model. In this model, SARS-CoV-2 neutralization antibody titers in MeVvac2-SARS2-S(H)−vaccinated hamsters showed a tendency to reversely correlate with weight loss and RNA copy numbers in lung and nasal turbinates, indicating some role of neutralizing antibodies in fighting disease progression , and revealed a tendency for reduced titers in nasal turbinates, as well (vac2-SARS2-S(H).Our second animal model confirmed these observations. Virus loads of the mouse-adapted SARS-CoV-2 MA  were sig as well E, in thevac2-SARS2-S(H) replicated comparably to MeV vaccine strain viruses and was genetically stable over extended passaging. Upon vaccination of mice, it induced robust humoral immune responses of the IgG2a subtype directed against the SARS-CoV-2 spike glycoprotein S with neutralizing activity in a range already shown to be protective by others. In addition, considerable amounts of SARS-CoV-2 S-specific CD4+ and CD8+ T cells were induced, the major fraction of which were secreting two or even all three cytokines when analyzing for IFN-γ, TNF-α, or IL-2 upon antigen-specific restimulation. These T cells proliferated and specifically depleted antigen-positive target cells in a mixed population. Importantly, all responses were skewed toward Th1-biased immunity. In parallel, the capacity to induce measles-specific immune reactivity remained conserved. Finally, the induced immunity conferred protection in two different animal models of SARS-CoV-2 infection, Syrian golden hamsters inoculated with a low-passage patient isolate and mice challenged with a mouse-adapted recombinant SARS-CoV-2 MA.In this study, we aimed to analyze the efficacy of MeV-derived vaccine candidates encoding the Spike glycoprotein S of SARS-CoV-2 to induce functional immune responses to protect against COVID-19. We show that MeVvac2-SARS2-S(H) is a live-attenuated vaccine that encodes the full-length, functional version of the SARS-CoV-2 S protein as the main target for functional antibodies, but also for induction of T cell responses. Vero cells revealed homogenous expression of the SARS-CoV-2 S antigen by Western blot analyses and positive immunostaining of syncytia after infection by MeVvac2-SARS2-S(H). Stable antigen expression is a prerequisite for the immune system to encounter the specific antigen to mount robust immune responses and for industrial production of a vaccine. Indeed, IFNAR−/−-CD46Ge mice vaccinated with MeVvac2-SARS2-S(H) in a prime−boost protocol showed uniform induction of antibodies directed against MeV bulk antigens or SARS-CoV-2 S, which had considerable neutralizing activity against both pathogens. We observe antibody responses in these animals at a level that correlates with protection in mouse challenge models −vaccinated animals developed VNT. On the other hand, all mice, including the three improperly immunized ones, revealed significant, multifunctional T cell responses against SARS-CoV-2 S, which were still recordable 3 wk after the second vaccination, when we already expect constriction of antigen-specific T cell effector populations. These data suggest that anti-S antibody responses mature after repeated vaccination, but, on the other hand, that a one-shot vaccination regime will already induce especially functional memory T cell immune responses, the protective efficacy of which, as well as their duration, has to be demonstrated in future challenge experiments. Moreover, anti-measles immunity is generally stable  . This genetic stability indicates that the slight impairment seen in multistep growth curves when compared to a vaccine-strain MeV is not critical for the vaccine’s amplification and therefore crucial for product safety. In accordance with its genetic stability, the minor enhancement of fusion activity can also be regarded as noncritical, especially when considering the fusion activity of MeV used in clinical trials for treatment of tumors. These so-called oncolytic MeV have been used in 15 phase 1 and phase 2 clinical trials, so far. Thereby, advanced-stage tumor patients suffering from different tumor entities have been treated. Despite constituting, in principle, a vulnerable patient collective, application of high doses of nontargeted, fusion-active MeV (up to 1 × 10 TCID50)  systemic TCID50)  was acco TCID50) . Therefo TCID50) . Likewis TCID50) , 24, havIn any case, generation of MeV-derived COVID-19 vaccines encoding a less fusion-active variant of the SARS-CoV-2 S glycoprotein might be beneficial to enhance titers of the vaccine virus. In the meantime, stabilized S variants have become available that have attenuated or no cell−cell fusion activity. One variant has a deletion of the multibasic cleavage motif for furin-like proteases at the S1/S2 boundary that facilitates preactivation of S . A seconThe induction of the “right” antibodies and T cell responses is especially crucial when taking potential complications into consideration that can be observed when coronavirus encounters “wrong” immune responses that can give rise to immunopathologies after infection. In some infected cats, infection with feline coronavirus causes feline infectious peritonitis, a deadly disease characterized by viral infection of macrophages during the acute phase. Interestingly, the switch of pathology after infection from a rather moderate pathogenesis into an acute, devastating disease can be triggered by vaccination of persistently infected cats and has been attributed to the induction of antibodies that mediate enhancement of the disease, a process called ADE. During COVID-19, ADE might be the cause of the severe cases currently observed. Some case reports indicate that severe disease appeared more frequently in patients with high SARS-CoV-2 IgG levels . ADE hasvac2-SARS2-S(H) compared to the animals immunized with alum-adjuvanted recombinant S protein. Moreover, the cytokine profile of splenocyte cultures of immunized mice after restimulation of S-specific T cells reveals a respective preferable Th1 bias. Since SARS-CoV-2 and SARS-CoV use the same primary attachment receptor for cell entry, hACE2, and selected hACE2-transgenic mice show differential pathology after inoculation with SARS-CoV-2  is a promising vaccine candidate that warrants further investigation.In conclusion, the bivalent MeV/SARS-CoV-2 vaccine candidate has a number of desirable properties with respect to its immunogenicity and efficacy against SARS-CoV-2. Furthermore, the concurrent induction of anti-MeV immunity would allow its use in the context of routine measles immunization schedules. Such an MeV-based COVID-19 vaccine could be included in the currently applied MMR  vaccine, providing additional protection against SARS-CoV-2. While there is controversy regarding to what extent it occurs, children do become infected and shed the virus, despite them rarely being severely affected. In any case, preventing or reducing infection or virus shedding from vaccinated children can also help to contain the disease and protect vulnerable patient groups. On the other hand, the vaccinated children themselves could be protected against newly described side effects of COVID-19 infections, such as Multisystem Inflammatory Syndrome, affecting also the cohort of (very) young patients . TherebySI Appendix, Supplementary Extended Materials and Methods.Detailed descriptions of the materials, methods, and equipment used in this work, including cells, plasmids, production of lentiviral vectors and generation of antigen-expressing dendritic cell lines, viruses, MeV genome sequence analysis, NGS library preparation and sequencing, RNA sequence analysis, immunoperoxidase monolayer assay, Western blot analysis, animal experiments, total IgG and IgG1-/IgG2a quantification, Th1/Th2 cytokine multiplex assay, virus neutralization test, plaque reduction neutralization test, IFN-γ ELISpot analysis, ICS, T cell proliferation assay, CTL killing assay, virus titers in organs of infected animals, RNA preparation, quantitative RT-PCR, and statistical analyses, are provided in Supplementary FileSupplementary File


Scientific Reports 10.1038/s41598-020-58870-2, published online 04 February 2020Correction to: Supplementary Tables 1 and 2 are omitted from the Supplementary Information which accompanies this Article. Tables


PRPF8 mutations have been identified in human patients. However, the cellular and molecular consequences of their expression in vivo in specific tissue contexts remain largely unknown. Here, we establish a Drosophila melanogaster model for RP13 by introducing the nine distinct RP mutations into the fly PRPF8 ortholog prp8 and express the mutant proteins in precise spatiotemporal patterns using the Gal4/UAS system. We show that all nine RP-Prp8 mutant proteins negatively impact developmental timing, albeit to a different extent, when expressed in the endocrine cells producing the primary insect moulting hormone. In the developing eye primordium, uncommitted epithelial precursors rather than differentiated photoreceptors appeared sensitive to Prp8 malfunction. Expression of the two most pathogenic variants, Prp8S>F and Prp8H>R, induced apoptosis causing alterations to the adult eye morphology. The affected tissue mounted stress and cytoprotective responses, while genetic programs underlying neuronal function were attenuated. Importantly, the penetrance and expressivity increased under prp8 heterozygosity. In contrast, blocking apoptosis alleviated cell loss but not the redox imbalance. Remarkably, the pathogenicity of the RP-Prp8 mutations in Drosophila correlates with the severity of clinical phenotypes in patients carrying the equivalent mutations, highlighting the suitability of the Drosophila model for in-depth functional studies of the mechanisms underlying RP13 etiology.Retinitis pigmentosa (RP) represents genetically heterogeneous and clinically variable disease characterized by progressive degeneration of photoreceptors resulting in a gradual loss of vision. The autosomal dominant RP type 13 (RP13) has been linked to the malfunction of PRPF8, an essential component of the spliceosome. Over 20 different RP-associated This article has an associated First Person interview with the first author of the paper. Summary: A Drosophila melanogaster disease model of retinitis pigmentosa type 13 to study the mechanisms underlying the loss of tissue homeostasis and degeneration caused by mutations in the core component of the spliceosome Prp8. Intriguingly, nearly one-quarter of these genes encode core components of the spliceosome, a macromolecular RNA-protein complex that removes introns from nascent pre-mRNAs, generating mature transcripts. The spliceosome consists of five small nuclear ribonucleoprotein particles (snRNPs) – U1, U2, U4, U5 and U6, – and more than 100 associated proteins  and the U5 small nuclear ribonucleoprotein 200 kDa DEAD-box RNA helicase (SNRNP200 or Brr2)  represents a heterogeneous group of hereditary eye disorders characterized by a progressive degeneration of the light-sensing photoreceptor cells in the retina. Early RP symptoms include night blindness and gradual loss of peripheral vision due to the loss of rod photoreceptors which ensure achromatic, low-light vision. As the disease advances, the rod elimination is followed by the death of cones, ultimately resulting in complete blindness . The ratproteins . Each ofproteins . Remarkaor Brr2) . How malPre-mRNA processing factor 8 (PRPF8/Prp8) is the largest and the most conserved protein of the spliceosome involved in nearly all functions of the U5 snRNP. These include the splice site and branch region recognition, assembly and stabilization of the U4/U6.U5 tri-snRNP, exon alignment and activation of the catalytic core of the spliceosome. Prp8 does so through precisely controlled interactions with the U5 snRNA and several proteins of the splicing machinery including Brr2, and a GTPase EFTUD2 (Snu114) .PRPF8 mutations have been identified so far in patients suffering from RP13. The majority of these mutations map to the terminal exon 43 encoding the Jab1/MPN domain  (RP12), rhodopsin (rh/RHO) (RP4) and eyes shut (eys) (RP25) (The fruit fly neration . Owing tneration . Importa) (RP25) . The flyDrosophila melanogaster model for RP13. We demonstrate that nine different RP-associated Prp8 mutant proteins delay the developmental transition when expressed in the endocrine organ specialized to produce the major insect moulting hormone. In the developing eye primordium, actively cycling cells rather than differentiated photoreceptors showed sensitivity to Prp8 malfunction. The overexpression of the two most toxic RP-Prp8 variants induced apoptosis and disturbances of the adult eye morphology. Whereas the affected tissue mounted the stress and cytoprotective response, the genetic programs underlying neuronal function were attenuated. Importantly, the expressivity and penetrance among the RP-Prp8 mutations differed and increased under prp8 heterozygosity.Here, we establish a Drosophila Prp8 protein comprises 2396 amino acids and shares 88.99% and 59.50% identity with its human and yeast counterpart, respectively  (S>F), to simplify the description -Gal4, eyeless (ey)-Gal4 and Glass multiple reporter (GMR)-Gal4 to overexpress the Prp8 transgenes in specific cells during the fly development. While phm-Gal4 expresses in the endoreplicating polyploid cells of the prothoracic gland (PG)  ey-Gal4 and GMR-Gal4 are active in the eye/antennal imaginal discs (EADs) which will give rise to the adult compound eyes, antennae and epithelia of the head capsule. Ey-Gal4 becomes active during embryogenesis in all cycling cells of the eye/antennal primordium while showing a restricted eye-specific expression in the late third instar larva . In contrast, GMR-Gal4 is a late-acting driver which targets the expression to the EAD domain posterior of the morphogenetic furrow comprised predominantly of differentiating cells arrested in G1 or G2 phase of the cell cycle, and cells within the second mitotic wave, which undergo one round of cell division . The Gal4 drivers were selected based on our previous study where we demonstrated differential response and sensitivity of the targeted cells to spliceosome deficiency (The ectively . To mimi Y2395N) . Each mucription . To studS system , which aB vector  and inteing site  to ensuring site . We seleficiency . Moreovein vivo, on the developing retina but also other tissues that fulfil highly specialized functions.Taken together, we generated nine different RP-Prp8 mutant variants, the expression of which can be easily controlled in a spatiotemporal manner. Such an approach enables rapid and parallel investigation of their impact GMR-Gal4 driver. Immunoblotting with anti-Flag and self-generated anti-Drosophila Prp8-specific antibodies  confirmed that the wild-type and RP-Prp8 proteins were stably expressed and inducible to the same level irrespective of the tag or the type of mutation  further revealed that the transgenic wild-type Prp8 (Prp8wt) as well as the RP-Prp8 mutant variants primarily localized to the cytoplasm where the endogenous Drosophila Prp8 or transgenic human Flag::PRPF8 proteins were also mostly confined. Thus, in contrast to the nuclear enrichment in mammalian cells , which causes missplicing of genes encoding key steroidogenic enzymes . In contrast, early expression of Prp8H>R and Prp8S>F using ey-Gal4 resulted in rough and irregularly shaped adult compound eyes . The Prp8H>R and Prp8S>F phenotypes sharply contrasted with undisturbed morphology of control adult eyes and those overexpressing Prp8wt and seven other RP-Prp8 mutant variants . Of note, the ey-Gal4-induced RNAi-mediated prp8 knockdown is larval lethal.To determine whether and how the RP-Prp8 mutations affect the development and differentiation of the adult compound eye, we overexpressed the wild-type and mutant variants under the control of the wt, the asymptomatic Prp8F>L and the two most pathogenic Prp8H>R and Prp8S>F variants in the heterozygous prp8 mutant animals. To this end, we used the genome editing CRISPR/Cas9 technique to generate a del14prp8 mutant allele that lacks the entire exon 14 encoding most of the C-terminal part of the protein . Consistent with a vital role of Prp8 in pre-mRNA splicing, del14prp8 homozygosity resulted in early embryonic and cell lethality  while heterozygous flies (del14/+prp8) were viable without any apparent developmental defects. Importantly, similar phenotypes have been reported for the KG03188prp8 mutant allele harboring a P-element insertion within the 5′ UTR of the prp8 gene . While ey-Gal4-driven expression of Prp8wt and Prp8F>L in del14prp8 or KG03188prp8 heterozygotes had no consequences , the expressivity of the phenotypic defects caused by Prp8H>R and Prp8S>F was higher  compared with the wild-type background , the adult eyes of H>Rey>Prp8 and S>Fey>Prp8 animals having only one wild-type prp8 allele were also significantly smaller . The reduced eye size indicated that the overexpression of the two pathogenic variants might induce cell death. Indeed, immunostaining with an antibody against the activated Drosophila Death caspase 1 (Dcp-1) revealed a marked enrichment of Dcp-1-positive cells within EADs overexpressing Prp8H>R and Prp8S>F  but also rescued the morphological abnormalities and size of the adult eyes  (up/down) in response to Prp8S>F (67/81 genes), Prp8H>R (7/2 genes) and Prp8F>L (10/10 genes) relative to EADs overexpressing Prp8wt (Supplementary Dataset 1). While several upregulated genes were shared between the transcriptional profiles of the two pathogenic variants Prp8H>R and Prp8S>F, the changes inflicted by Prp8F>L were more distant (S>F dataset highlighted over-representation of genes linked to ‘Glutathione metabolism’ including three members of the glutathione S-transferase (GST) family GstE6, GstE7, GstE5 (Supplementary Dataset 1). The induction of redox and detoxification genes positively correlated with an increased expression of several stress and damage response genes including the transcription factor ets21c  (Drosophila insulin-like peptide 8 (dilp8) , the pro-apoptotic gene reaper (rpr) was also induced (Supplementary Dataset 1). In contrast, downregulated genes were enriched for functions linked to ‘Regulation of transcription’, ‘Cell differentiation’ and GO terms describing neuronal morphogenesis and function including: ‘Dendrite morphogenesis’, ‘Synapse organization’, ‘Vesicle-mediated transport’, ‘Neurotransmitter secretion’ and ‘Visual perception’ (Supplementary Dataset 1). Importantly, the differential expression of several candidates was validated by RT-qPCR on independent samples  or complete achromatopsia, a rare, autosomal recessive inherited and congenital disorder   and a se (dilp8) . In acco samples D. Compardisorder .Fig. 6.F>R and Prp8S>F shares common features ultimately resulting in similar phenotypic outcomes.Taken together, our genome-wide transcriptome profiling revealed that the developing eye primordium induces expression of damage, stress and detoxification genes in response to the toxic RP-Prp8 protein variants, likely in an attempt to alleviate the damage and regain homeostasis at the expense of normal neurogenic development and differentiation. Despite a marked difference in the number of dysregulated genes, the genetic programme triggered by Prp8H>Rey>Prp8 and S>Fey>Prp8 samples indicated that EAD cells suffer from the breakdown of the intracellular redox homeostasis. To support the notion, we took advantage of the transgenic GstD1-GFP reporter . Indeed, we found a marked upregulation of the GstD1-GFP reporter  compared to controls . Of note, co-expression of a mock Flag tripeptide had no impact on GstD1-GFP reporter activity . These results demonstrate that although blocking apoptosis prevented alterations to the overall size and morphology of the adult eyes, it did not alleviate the redox imbalance caused by the presence of the toxic RP-Prp8 mutant proteins.GSTs are among the best-known phase II detoxifying enzymes that conjugate glutathione to harmful hydrophobic electrophiles including xenobiotics and activated metabolites. The upregulation of several of the GST genes in reporter , which hreporter  in Prp8Hcontrols A. Interereporter B–D. IntrDrosophila melanogaster model has proven successful in unravelling the disease biology of six different RP classes, including RP4, RP11, RP12, RP14, RP25 and RP26  cannot efficiently incorporate into the maturing U5 snRNP, while others (e.g. Prp8F>L Prp8Y>N) permit U5 and tri-snRNP assembly but compromise U4/U6 unwinding  and controls biogenesis of multi-subunit machines including the small nucleolar ribonucleoproteins (snoRNPs), nutrient sensing mTORC1 and RNA polymerase II (Pol II)  . In this(Pol II) . Studies(Pol II) . Whether(Pol II) . SplicinS>F and Prp8H>R mutants at the expense of genes required for neurogenesis and neuron function. With the help of the transgenic GstD1-GFP reporter, we further determined that both differentiated photoreceptors and actively cycling epithelial cells upregulated the phase II detoxifying programme. While differentiated cells survived, uncommitted eye progenitors underwent apoptosis. Interestingly, the phenomenon of acquired apoptosis resistance of terminally differentiated cells without regenerative potential in Drosophila has been attributed to the epigenetic silencing of major pro-apoptotic gene loci during development  1118w, (b) phm-Gal4 (RRID:BDSC_80577) (c) ey-Gal4 (RRID:BDSC_5534), (d) GMR-Gal4 (RRID:BDSC_1104), (e) RNAiUAS-prp8 , (f) dpp-Gal4 (RRID: BDSC_7007), (g) wtUAS-Flag::Prp8 (FRT82B (RRID: BDSC_2035), (i) del14/CyO, act-GFP JMR1prp8 (this study), (j) UAS-p35 (RRID: BDSC_5072), (k) y,v, nos-phiC31\int.NLS; attP2 (RRID:BDSC_25710), (l) UAS-myr-mRFP (RRID:BDSC_7118), (m) UAS-mCD8.ChRFP (RRID:BDSC_27391), (n) UAS-mCD8::GFP.L (RRID:BDSC_5130) (o) eyFLP, act>y+>Gal4, UAS-GFP; FRT82B tubGal80 (FRT42D (RRID:BDSC_1802), (q) eyFLP; FRT42D tub-Gal80/CyO; act>y+>Gal4, UAS-GFP/TM6B att. (this study), (r) Act5C-cas9, Lig4[169] (RRID:BDSC_54590) (GstD1-GFP (UAS-Flag (this study), (u) KG03188prp8 (RRID:BDSC_13006).The following ::Prp8wt , (h) FRTtubGal80 , (p) FRTC_54590) , (s) GststD1-GFP  (t) UAS-Gal4 driver lines crossed to 1118w and FRT42D served as controls for experiments performed in the wild-type and prp8 (del14prp8 or KG03188prp8) heterozygous background, respectively. Overexpression of the Flag tripeptide was used as a mock control to exclude saturation of the Gal4 driver. All stocks are listed in Table S1.All crosses were set up and maintained at 25°C, unless specified otherwise, on a diet consisting of 0.8% agar, 8% cornmeal, 1% soymeal, 1.8% dry yeast, 8% malt extract and 2.2% sugar-beet syrup, which was supplemented with 0.625% propionic acid and 0.15% Nipagin. Drosophila (A1Z8U0) and S. cerevisiae (P33334) Prp8 proteins was created using MUSCLE multiple sequence alignment software (Drosophila melanogaster prp8 (CG8877) was amplified from cDNA using the Phusion polymerase (New England Biolabs) and cloned into the pENTR4 vector (Invitrogen). Nine different RP mutations were introduced using the Quick Change Lightning site directed mutagenesis kit (Agilent). All primers for mutagenesis are listed in Table S3. The N-terminal Flag-tag was added by LR Clonase II-mediated recombination (Invitrogen) into the pTFW vector (DGRC). Following restriction with SphI and NotI, the UAS-Flag::Prp8 cassettes were inserted into the pattB vector backbone , software . The amisoftware . Coding ne DGRC, . The nonS>F, P2361T/Prp8P>T, F2374L/Prp8F>L, H2369P/Prp8H>P, H2369R/Prp8H>R, H2370G/Prp8H>G, H2370K/Prp8H>K, R2370S/Prp8R>S, Y2395N/Prp8Y>N) were established by PhiC31 integrase-mediated transgenesis of the respective pUAST-attB vectors into attP-9A site (99F8) (BestGene Inc.). The N-terminally-tagged RP-Prp8 constructs  were integrated into the attP2 site (68A4) (BestGene Inc.). The UAS-Flag transgenic flies expressing the Flag tripeptide were obtained by standard P-element-mediated germline transformation of pTFW plasmid into 1118 Drosophilaw embryos (BestGene Inc). All stocks are listed in Table S1.Transgenic fly lines allowing overexpression of untagged wild-type and RP-Prp8 mutant variants  technique  with eyFprp8 mutant line was obtained by CRISPR-Cas9 genome editing. Two guide RNAs (sgRNA), targeting intron 12 and 3′-UTR of Drosophila prp8 gene (Table S3) were cloned into the pCFD4-U6:1_U6:3 tandem gRNAs vector (attP2 site on the third chromosome (68A4) using PhiC31 integrase-mediated transgenesis. Upon crossing to Act5C-cas9, Lig4[169]  including the MPN/Jab1 and Tail domain were raised by immunizing two rabbits (Eurogentec) with a 6xHis-tagged Prp8 polypeptide (amino acids 2092–2341). The antigen was expressed from the Gateway pDEST17 vector (Thermo Scientific) in E. coli BL21(DE3)pLysS bacterial strain and purified under denaturing conditions using immobilized metal affinity chromatography (IMAC).Polyclonal antibodies specific to the C-terminal part of Drosophila S2 cells were cultured at 25°C in Shield and Sang M3 insect medium (Sigma-Aldrich) supplemented with 8% FBS (Merck-Biochrom). Cells were transfected in 6-well plates on glass coverslips, in serum-free medium with the desired plasmids using TransIT-Insect (Mirus Bio) according to manufacturer's instructions. Expression of UAS-driven constructs was induced by co-transfection with a pAW-GAL4 plasmid expressing Gal4 under an Actin5C promoter. Transfection efficiency was monitored by co-transfection of pIE-EGFP plasmid. Cells were processed 30 h post-transfection.https://fiji.sc/) (RRID: SCR_003070) was used to determine the intensities of Prp8, Flag and ATP5α signals using the built-in Gel analysis tools. The intensities of a loading control (ATP5α) in individual western blot replicates were compared to establish a normalization factor which was used to correct the intensities of Prp8 or Flag signals. For each biological replicate, the relative expression of Prp8 or Flag was normalized to the average expression across all samples within a replicate.Eye/antennal discs (>40 EADs/replicate/genotype) were dissected 7 days after egg-laying (AEL) in 1× PBS, and immediately lysed in 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1% Triton X-100, 0.01% Igepal and protease inhibitors (Roche Applied Science). After centrifugation, protein concentration in the supernatant was determined using Bradford assay reagent (Bio-Rad) according to the manufacturer's instructions. Samples were denatured by boiling in Laemmli buffer containing 2.5% β-mercaptoethanol for 5 min at 95°C. Equal amounts of total protein were loaded on a 10% polyacrylamide gel. After SDS-PAGE, proteins were detected by immunoblotting with mouse anti-Flag M2  (RRID: AB_262044), rabbit anti-dPrp8-CTD , and mouse anti-ATP5α  (RRID:AB_301447) followed by incubation with the corresponding HRP-conjugated secondary antibodies. Chemiluminescence was detected with ImageQuant LAS4000 reader . ImageJ (Drosophila larvae (7 days AEL) and Drosophila S2 cells were fixed for 25 min with 4% paraformaldehyde in PBS containing 0.1% Triton X-100 (PBS-T) and washed 3 times with PBS-T. After blocking in 0.3% BSA in PBS-T samples were incubated overnight at 4°C with the following primary antibodies at the indicated dilutions: guinea pig anti-Spok  (RRID: SCR_003070). Final image processing including panel assembly, brightness and contrast adjustments were performed in Adobe Photoshop CC  (RRID: SCR_014199). White outlines of the EADs and ring glands shown in figures were drawn based on DAPI staining and GFP signal, respectively. For measurements of the adult eyes, 3- to 8-day-old male flies were collected from multiple vials of the same cross and the genotypes were concealed for sample preparation and data evaluation. Z-stacks of left eyes were taken by a single person using an Olympus SZX16 fluorescent stereomicroscopes equipped with a DP72 CCD camera under the same magnification. Images were processed with cellSens 1.1 Software (Olympus) (RRID: SCR_014551). Outlines of the adult eyes (area containing ommatidia) were prepared in Adobe Photoshop CS5.1  (RRID:SCR_014199) with the magnetic or polygonal lasso tool and superimposed to represent deviations in eye morphology within and among genotypes. Statistical significance was determined by ordinary one-way ANOVA with Tukey's multiple comparisons test in GraphPad Prism (RRID:SCR_002798). For quantification of the GFP-positive clonal volume, confocal Z-stacks spanning the columnar epithelia of mosaic EADs were imported into ImageJ (https://fiji.sc/) (RRID: SCR_003070). After thresholding, the individual slices were converted to binary images and the outlines selected. The ratio of GFP and DAPI was determined using the 3D manager plug-in. The same macros were applied to all samples; all macros used are available upon request.Confocal images and stacks were acquired with Olympus FV1000 confocal microscope equipped with 20× UPlan S-Apo (NA 0.85), 40× UPlan FL (NA 1.30) and 60× UPlanApo (NA1.35) objectives. Maximum Z-projections were generated from a maximum of seven consecutive sections taken at 0.4 µm steps using Fluoview 1000 Software (Olympus) (RRID: SCR_014215) and ImageJ (phm>mCD8::GFP) crossed to the males carrying the different pUAST-attB-Prp8 transgenes were allowed to lay eggs for 24 h. Early third instar larvae (≥25) were transferred to fresh vials. Two vials for each genotype were kept at 25°C and the pupae were counted twice a day. The entire experiment was repeated twice. Statistical significance among genotypes was calculated with a Log-rank (Mantel–Cox) test. The curves prepared with GraphPad Prism represent one of two independent experiments. The number of flies per experiment and genotype (n) and P-values are specified in Supplementary Dataset 2.Female flies   of the respective phm-Gal4 genotype were collected. For RNA-seq and RT-qPCR, EADs were dissected from third instar larvae (7 days AEL)  overexpressing the RP-Prp8 variants under the ey-Gal4 driver. RNA was extracted using the standard protocol with Tri-Reagent (Sigma-Aldrich) and DNase I treatment (Ambion) (Table S3) were designed to anneal at 62°C. Data were normalized to the expression levels of rp49 transcript, and fold-changes calculated using the ΔΔCt method  . cDNA wat method . Statisteviously .n=3/genotype) was used for library preparation using the Illumina TruSeq kit according to manufacturer's instructions. The libraries were paired end sequenced on the Illumina HiSeq 2000, at 75 bp read length with >45 million reads per library. The data were processed with an in-house RNA-Seq analysis pipeline (QuickNGS). In short, initial quality check was performed using FastQC (Supplementary Dataset 1), and the reads were aligned to the Drosophila reference genome BDGP Release 6 (dm6) using Tophat2 v.2.0.10 (P<0.05 were considered as significantly up- or downregulated compared with the EADs expressing Prp8wt. The FlyBase Gene Ontology (GO) terms were used for functional annotation. The GO category enrichment analysis of differentially regulated genes was performed with DAVID considering the GO_BP_FAT ontology (http://david.abcc.ncifcrf.gov/) (http://revigo.irb.hr/) (RRID: SCR_005825)  ,b. The G_005825) .


Monogenic protein aggregation diseases, in addition to cell selectivity, exhibit clinical variation in the age of onset and progression, driven in part by inter-individual genetic variation. While natural genetic variants may pinpoint plastic networks amenable to intervention, the mechanisms by which they impact individual susceptibility to proteotoxicity are still largely unknown.C. elegans muscle cells. Here, we find that a genomic locus from C. elegans wild isolate DR1350 causes two genetically separable aggregation phenotypes, without changing the basal activity of muscle proteostasis pathways known to affect polyQ aggregation. We find that the increased aggregation phenotype was due to regulatory variants in the gene encoding a conserved autophagy protein ATG-5. The atg-5 gene itself conferred dosage-dependent enhancement of aggregation, with the DR1350-derived allele behaving as hypermorph. Surprisingly, increased aggregation in animals carrying the modifier locus was accompanied by enhanced autophagy activation in response to activating treatment. Because autophagy is expected to clear, not increase, protein aggregates, we activated autophagy in three different polyQ models and found a striking tissue-dependent effect: activation of autophagy decreased polyQ aggregation in neurons and intestine, but increased it in the muscle cells.We have previously shown that natural variation modifies polyglutamine (polyQ) aggregation phenotypes in Our data show that cryptic natural variants in genes encoding proteostasis components, although not causing detectable phenotypes in wild-type individuals, can have profound effects on aggregation-prone proteins. Clinical applications of autophagy activators for aggregation diseases may need to consider the unexpected divergent effects of autophagy in different cell types. Protein misfolding and aggregation underlie many human diseases and contribute to tissue decline during aging , 2. In fC. elegans and Drosophila models expressing polyQ-expanded Htt or ataxin-3, or isolated polyglutamine repeats, exhibit similar toxic gain-of-function behavior and the age- and polyQ-length-dependent aggregation and toxicity as those seen in patients and in mammalian models , AM140 [rmIs132(punc-54::Q35::YFP) I], CF1038 [daf-16(mu86) I], TJ356 [zIs356) IV], PS3551 [hsf-1(sy441) I], DA2123 [adIs2122(plgg-1::GFP::lgg-1 + rol-6(su1006))], KR1108 [unc-11(e47) dpy-5(e61) I], KR292 , MT1434 [egl-30(n686) I], CB1370 [daf-2(e1370) III], and AM44 [rmIs190(pF25B3.3::Q67::CFP)]. TGF205 [xzEx3(punc-54::UbG76V::Dendra2)] was made by crossing out glp-1(e2141) from AGD1033.The following stains were obtained from unc-54::GFP::UNC-54], AM583 [rmIs249], AM738 [rmIs297)], and AM930 [rmIs335(punc-54::Aβ (1-40)::CFP)] strains were kindly provided by the Morimoto lab, and the HZ1732  strain by the Colón-Ramos lab. The Q40DR1350 and recombinant inbred lines (RILs) 2, 12, 12(2), and 15 were described in , TGF130 , TGF353 , TGF208 , TGF207 , TGF088 , TGF188 , TGF086 , TGF190 , TGF187 , TGF170 , TGF036 , TGF189 , TGF203 , TGF342 , TGF261 , TGF275 , TGF089 , and TGF127 .The drxIR1;Q40 strain was made by the following scheme: Q40Bristol males were mated to RIL2 hermaphrodites, and 5–10 F1 hermaphrodite progeny, identified by the lack of RIL2-like increased head aggregation phenotype, were picked onto fresh plates. F2 generation was examined for the expected 1:3 segregation of the increased head aggregation phenotype, and 7–10 F2 hermaphrodites with this phenotype were further mated with Q40Bristol males. This mating-selection cycle was repeated 23 times. The resulting strain was named drxIR1;Q40.The drxIR1 locus by genetic crosses was confirmed by detecting the presence of the SNP 5 (WBVar00016276) . DNA was sequenced using the NextSeq 500 System  at the Wistar Institute . Unpaired short reads were analyzed using the Galaxy , F1 progeny that either did (based on segregation of unc non-dpy phenotype among their progeny) or did not inherit the sDp2 duplication were singled, and their F2 progeny scored for the increased ratio of head to body aggregation (RIL2-like) and the dumpy phenotypes. The RIL2-like phenotype behaved genetically as did the known loss-of-function dpy-5(e61) allele.Additional file 6: Table S2. Candidate genes tested by RNAi. 24 candidate genes present in the target 326 Kb of drxIR1 interval  genome assembly from WormBase release WS220 [se WS220 , availabse WS220 .


Claudin-2 expression is upregulated in multiple cancers and promotes cancer malignancy. Remarkably, the regulation of claudin-2 expression in kidney cell lines contrasts its reported regulation in other organs. However, claudin-2 role in renal clear cell carcinoma (RCC) remains unknown despite its predominant expression in the proximal tubular epithelium (PTE), the site of RCC origin.Publicly available and independent patient databases were examined for claudin-2 association with RCC. The novel protein function was validated in vitro and in vivo by gain or loss of function assays. Mechanistic results were concluded by Mass spectroscopy, immunoprecipitation and mutational studies, and functional evaluations.We show that the significant decrease in claudin-2 expression characterized PTE cells and Ex-vivo cultured mouse kidney subjected to dedifferentiation. Inhibition of claudin-2 was enough to induce mesenchymal plasticity and invasive mobility in these models. Further, a progressive loss of claudin-2 expression associated with the RCC progression and poor patient survival. Overexpression of claudin-2 in RCC-derived cancer cells inhibited tumorigenic abilities and xenograft tumor growth. These data supported a novel tumor-suppressive role of claudin-2 in RCC. Mechanistic insights further revealed that claudin-2 associates with YAP-protein and modulates its phosphorylation (S127) and nuclear expression. The tumor suppressive effects of claudin-2 expression were lost upon deletion of its PDZ-binding motif emphasizing the critical role of the PDZ-domain in claudin-2 interaction with YAP in regulating RCC malignancy.Our results demonstrate a novel kidney specific tumor suppressive role for claudin-2 protein and further demonstrate that claudin-2 co-operates with the YAP signaling in regulating the RCC malignancy.The online version contains supplementary material available at 10.1186/s13046-021-01870-5. The renal clear cell carcinoma (RCC) is the most common histological subtype, and malignant, of the renal cancers that represents 70 to 80% of all renal cancer cases . Unfortu+ and Ca++ homeostasis . Hypoxia promotes cancer progression and is reported to induce EMT in MDCK-II cells  and harvested the samples in a time dependent manner  was deleted. An HA-tag was added to the c-terminal of full-length and mutant claudin-2 constructs (termed as Cldn2FL/HA and Cldn2ΔPDZ/HA respectively) to detect the overexpressed protein. The full-length and PDZ-deleted claudin-2 expression constructs were transfected in both, MDCKII and HK-2 cells. Immunoblot analysis using anti-HA-tag and anti-claudin-2 antibodies confirmed appropriate expression of the newly constructed full-length and mutant proteins  where serine is swapped with alanine in Caki-2Cldn2 cells. Transfection of the wild type YAP protein served as control. Quantitative analysis of the cell invasion indicated that overexpression of the constitutive active YAP in Caki-2Cldn2 cells significantly increased invasive ability of these cells despite stable claudin-2 overexpression , Ankyrin Repeat Domain 1(ANKRD1), SRY-Box Transcription Factor 9 (Sox9), and Baculoviral IAP Repeat Containing 5 (Bric5). As shown in supplementary Fig. To further explore pathological significance of above findings, we overexpressed a Cldn2The concept of EMT has critical considerations in renal homeostasis and diseases, as a switch between an epithelial and mesenchymal phenotype occurs during renal diseases, albeit the extent of this switch and its reversibility are context specific . Our curhttps://www.ncbi.nlm. nih.gov/gtr/genes/9075/). Furthermore, as discussed above, renal claudin-2 expression is markedly inhibited in obese mice [Among claudin family of proteins, claudin-2 is one of the most studied protein due possibly to its role in the paracellular Na + −transport, which partly explains why its renal expression is limited to the PTE, in which there is significant paracellular reabsorption of filtrate . Howeverese mice . Obesityese mice . HoweverOur hypothesis that claudin-2 regulates YAP-activation to mediate its effects on PTE cell plasticity and EMT was based on our finding that claudin-2 expression progressively increases in PTE cells with cell density and terminal differentiation. Similar findings in a recent report using MDCK-II cells support this finding . The HipIn summary, our study highlights a novel kidney specific role of claudin-2 in regulating plasticity and differentiated epithelial phenotype of PTE cells. We believe that our finding that claudin-2/YAP signaling are mediators of PTE cell plasticity may also provide molecular explanation for potential non canonical effects of many cell-cell adhesion proteins in regulating epithelial cell homeostasis. The clinical significance of our finding is considered high in light of the unclear understanding of the molecular regulation of the renal hypoxic injury and fibrosis as an underlying cause for RCC progression.Additional file 1. Supplementary Methods and Material.Additional file 2: Fig. S1. Effects of claudin-2 expression on tumor grade and gender in patients with RCC: (a) Kaplan-Meir analysis to determine patient survival in relation to claudin-2 expression with tumor grade; (b) Kaplan-Meir analysis to determine patient survival in relation to claudin-2 expression with gender. High vs low expression of claudin-2 in tumor grade (P < 0.0001) or patient gender (P < 0.01). Moreover, P=0.028 High vs low , P= 0.013 High vs low  and P= 0.01 High vs low .Additional file 3: Fig. S2. Constitutive EGFR activation induces EMT in MDCK-II cells and specific loss of claudin-2 expression: Cells stably overexpressing the empty cloning vector (MDCKIICON) or a mutant HB-EGF construct that is constitutively secreted into the medium (MDCKIISHB) were used. Immunofluorescent analysis of the cellular expression and localization of claudin proteins and ZO-1. Arrows represent membrane tethered and disrupted expression of respective proteins.Additional file 4: Fig. S3. EGFR activation induces EMT in MDCK-II cells and specific loss of claudin-2 expression: (a) Representative phase-contrast images, and Immunofluorescent analysis of the Na+K+ ATPase expression and primary cilium (acetylated-tubulin); (b-c) Immunofluorescent co-localization of Ki-67 and claudin-2 proteins, and Quantitative analysis; (d-e) Representative images of MDCKIICON and MDCKII cells treated with EGF(100ng/ml), subjected to wound-healing , and Quantitative analysis; (f-g) Anchorage-independent growth (Colony formation assay) in soft agar, and Quantitative analysis; (h-i) Immunoblot analysis of claudin proteins and EMT marker (vimentin and fibronectin), and Densitometric analysis (C= control); (j) Immunofluorescent analysis of the cellular expression and localization of claudin proteins and ZO-1. Arrow represent membrane tethered and disrupted expression of respective proteins. CON represent Control. Data is presented as mean+sem . Statistical significance was determined by student t test. **P  <  0.01; ***P  <  0.001.Additional file 5: Fig. S4. Hypoxia Induced EMT leads to down regulation claudin-2 in MDCKII and HK2 cells: (a) Representative phase-contrast image and Immunofluorescent analysis of Na+K+ ATPase expression and primary cilium (Acylated tubulin); (b and c) Immunoblot and densitometry of claudins proteins and EMT marker from total lysate of MDCKII cell treated with hypoxic culture environment in time dependent manner. C represent control and hr. represent hours; (d) Representative immunofluorescent image of E-cadherin and vimentin of MDCKII cells exposed to hypoxia; (e) Representative phase-contrast image and immunofluorescence analysis of claudin-2 protein in HK-2 cell culture in hypoxic environment; (f and g) Immunoblot and densitometry analysis of claudins proteins and EMT marker from total lysate of HK2 cell treated with hypoxic culture environment. Data is presented as mean+sem . Statistical significance was determined by 1-way ANOVA and post hoc Tukey’s test for pairwise comparison. ***P  <  0.001.Additional file 6: Fig. S5. Claudin-2 loss promotes Mesenchymal plasticity in PTE cells and cell proliferation: (a) Phase-contrast image, and (b) immunofluorescent analysis for respective protein ; (c) Representative immunofluorescent image of E-cadherin and vimentin of MDCKII cells and MDCKIITALEN cells subjected to hypoxia; (d) Representative immunofluorescent image of Ki67 positive cell in MDCKII and MDCKIITALEN cells ; (e) Representative phase-contrast image of the wound-healing; (f and g) Representative immunoblotting and densitometry analysis using tissue lysates from 3D-cultured kidney slices from wild type and claudin-2 knockout (Claudin-2 KO) mice, unchallenged or subjected to hypoxia. CON represent Control. Data is presented as mean+sem. Statistical significance was determined student t test. ***P  <  0.001.Additional file 7: Fig. S6. Claudin-2 overexpression inhibits tumorigenic abilities of Caki-2 cells: (a) Schematic of claudin-2-m-cherry plasmid (pBR-hclaudin-2) expression system dynamics; (b) Fluorescent image of HK-2 cells transfected with pBR-hclaudin-2 plasmid for 48 hours showing GFP protein expression; (c) Fluorescent image of HK-2 cells Co-transfected with pBR-hclaudin-2 plasmid and pCMV-cre plasmid for 48 hours showing GFP and m-cherry expression protein expression; (d) Represent the immunoblotting of claudin-2 from total cell lysate of HK2 cells transfected with pBR-hclaudin-2 plasmid with/without pCMV-Cre plasmid; (e) Representative images of immunofluorescence staining of ki67/claudin-2 in HK-2, Caki-2CON and Caki-2Cldn2 cells (f). Representative images of colony formation by HK2, Caki-2CON and Caki-2Cldn2 cells. (g) Representative images from matrigel invasion assay showing invaded crystal violet-stained cells in HK-2, Caki-2 and claudin-2 over expressing Caki-2 cells (Caki-2Cldn2). Scale bar=50 μM. CON represent Control. Additional file 8: Fig. S7. Claudin-2 interacts with YAP and help maintain membrane expression of the YAP protein: (a) Immunoprecipitation was done using anti-claudin-2 antibody before mass spectrometry (MS) analysis. Lane-1 is protein ladder; Lane-2 is MDCKIITALEN (negative control) Lane-3 MDCKII cell lysate. Arrow indicate selected potential interacting protein with claudin-2; (b) Immunofluorescent analysis of YAP and ZO-1 proteins in exponentially growing MDCKII MDCKIITALEN and MDCKI cells. Arrows indicate localization of the YAP protein; (c) Representative phase-contrast image of MDCKII cells and MDCKIITALEN cells subjected to time dependent growth. Data is presented as mean+sem .Additional file 9: Fig. S8. Claudin-2 PDZ domain interact with YAP and inhibits YAP dependent invasion and regulate the YAP targeted gene expression: (a) Immunoblot analysis of HA-tagged and endogenous claudin-2 expression in HK-2 cell transfected with Full length HA-claudin-2 and PDZ motif deleted claudin-2 plasmid; (b) immunoprecipitation using anti-HA-tag antibody followed by immunoblotting using anti-HA and YAP antibodies from total cell lysate of HK2 cell transfected with Cldn2HA/Full and Cldn2ΔPDZ/HA plasmid constructs; (c and d) effects of Cldn2FL/HA and Cldn2ΔPDZ/HA expression upon cell invasion, and quantitative analysis. Here, *represents comparative analysis between control and Cldn2FL/HA expressing Caki-2 cells and $ represents comparison between Cldn2FL/HA and Cldn2ΔPDZ/HA overexpressing Caki-2 cells; (e and f) Cell invasion and quantitative analysis of Caki-2 cells expressing the Cldn2FL/HA plasmid with or without overexpression of constitutive active YAPS127A expression construct. Here, *represents comparative study between control Cldn2FL/HA overexpressing Caki-2 cells and # represents comparative analysis between cells overexpressing Cldn2FL/HA and Cldn2FL/HA+YAPS127A expression plasmid constructs. CON represent Control; (g) qPCR analysis of YAP target genes in HK2 control cell (CONsiRNA) and claudin-2 knockdown HK2 cells (Cldn2siRNA). Data is presented as mean+sem. Statistical significance was determined by student t-test. ***P <  0.001 and ##P < .01 Scale bar=50μM.Additional file 10: Supplementary Table S1. List of reagents, antibodies and kits.Additional file 11: Supplementary Table S2. Primers sequence.Additional file 12: Supplementary Table S3. Selected Potential protein Interacting with Claudin-2.


This randomized clinical trial compares the safety and efficacy of nurse practitioner–led remote intensive management with cardiologist-led standard care for low-risk patients with acute myocardial infarction. Is remote postdischarge treatment of low-risk patients with acute myocardial infarction by a centralized nurse clinician team under physician supervision feasible and safe?In this multicenter randomized clinical trial of 301 participants, there were no significant differences in safety events, medication adjustment, or left ventricular reverse remodeling outcomes in low-risk patients with acute myocardial infarction treated for 6 months after discharge by a centralized nurse practitioner–led telehealth program compared with standard in-person care by a cardiologist.Remote telehealth-enabled allied health care practitioner–led postdischarge management of low-risk patients with acute myocardial infarction is feasible and should be studied in higher-risk acute myocardial infarction cohorts. There are few data on remote postdischarge treatment of patients with acute myocardial infarction.To compare the safety and efficacy of allied health care practitioner–led remote intensive management (RIM) with cardiologist-led standard care (SC).This intention-to-treat feasibility trial randomized patients with acute myocardial infarction undergoing early revascularization and with N-terminal–pro-B-type natriuretic peptide concentration more than 300 pg/mL to RIM or SC across 3 hospitals in Singapore from July 8, 2015, to March 29, 2019. RIM participants underwent 6 months of remote consultations that included β-blocker and angiotensin-converting enzyme inhibitor/angiotensin receptor blocker (ACE-I/ARB) dose adjustment by a centralized nurse practitioner team while SC participants were treated face-to-face by their cardiologists.The primary safety end point was a composite of hypotension, bradycardia, hyperkalemia, or acute kidney injury requiring hospitalization. To assess the efficacy of RIM in dose adjustment of β-blockers and ACE-I/ARBs compared with SC, dose intensity scores were derived by converting comparable doses of different β-blockers and ACE-I/ARBs to a scale from 0 to 5. The primary efficacy end point was the 6-month indexed left ventricular end-systolic volume (LVESV) adjusted for baseline LVESV.2); 15 patients [5.9%] had a left ventricular ejection fraction <40%. The primary safety end point occurred in 0 RIM vs 2 SC participants (1.4%) (P = .50). The mean β-blocker and ACE-I/ARB dose intensity score at 6 months was 3.03 vs 2.91  and 2.96 vs 2.77 , respectively. The 6-month indexed LVESV was 28.9 vs 29.7 mL/m2 .Of 301 participants, 149 (49.5%) were randomized to RIM and 152 (50.5%) to SC. RIM and SC participants had similar mean (SD) age (55.3 [8.5] vs 54.7 [9.1] years), median (interquartile range) N-terminal–pro-B-type natriuretic peptide concentration (807 [524-1360] vs 819 [485-1320] pg/mL), mean (SD) baseline left ventricular ejection fraction (57.4% [11.1%] vs 58.1% [10.3%]), and mean (SD) indexed LVESV (32.4 [14.1] vs 30.6 [11.7] mL/mAmong low-risk patients with revascularization after myocardial infarction, RIM by allied health care professionals was feasible and safe. There were no differences in achieved medication doses or indices of left ventricular remodeling. Further studies of RIM in higher-risk cohorts are warranted.NCT02468349ClinicalTrials.gov Identifier:  Therefore, we evaluated the safety and efficacy of postdischarge telehealth-enabled, allied health care practitioner–led remote intensive management (RIM) of AMI.Telemedicine has enabled the transition from face-to-face care and is set to play a key role in the post–coronavirus disease-19 era.Improving Remodeling in Acute Myocardial Infarction Using Live and Asynchronous Telemedicine (IMMACULATE) was a multicenter randomized clinical trial of 6-month RIM compared with standard care (SC) among patients with recent AMI and who had a predischarge whole-blood N-terminal pro–b-type natriuretic peptide (NT-pro-BNP) concentration more than 300 pg/mL  at 6 months, adjusted for baseline LVESV. The secondary efficacy end points were LV ejection fraction and indexed LV mass at 6 months, reduction in NT-proBNP less than 20% from baseline to 6 months, difference in NT-proBNP concentration at 6 months, and β-blocker and ACE-I/ARB dose intensity at 1 month and 6 months  or SC  . BaselinThe primary safety end point occurred in 0 RIM participants and 2 SC participants (1.4%) . Twenty-P = .10). There was a nonsignificant increase in ACE-I/ARB dose intensity with RIM over SC at 1 and 6 months; the adjusted mean difference in ACE-I/ARB dose intensity over 6 months was 0.19 .There was no significant difference in β-blocker dose intensity at 1 and 6 months; the adjusted mean difference in β-blocker dose intensity over 6 months between RIM and SC groups was 0.12 . The adjusted mean difference in 6-month LVEF and LV mass index was 0.40%  and −2.07 g/m2 , respectively. NT-proBNP reduction was not significantly different between RIM and SC groups  of 0.67 (0-2) vs 2.70 (1-5) face-to-face visits and 17.8 (0-26) vs 0 teleconsults respectively over 6 months. The 6-month per-participant cost of RIM was 3.6-fold higher than SC ($631 vs $176), largely attributable to the high frequency of teleconsults (eMethods and eTable 4 in Among patients hospitalized for AMI with predischarge NT-proBNP concentration more than 300 pg/mL, RIM, consisting of frequent remote consultation and medication adjustment led by nurse practitioners, had similarly low safety events and achieved similar dose intensities of ACE-I/ARBs and β-blockers but did not improve the indexed LVESV at 6 months compared with face-to-face cardiologist-led SC.12 Instead, the IMMACULATE trial tested remote intensive follow-up and drug adjustment for patients in the early post-MI period. The limited window for ameliorating adverse post-MI remodeling presents itself as a unique opportunity for more cost-effective telemedicine deployment1 in contrast with chronic heart failure, which requires potentially perpetual deployment of telemedicine services to prevent recurrent hospitalization over a patient’s health span.11 Possible explanations for the present trial’s neutral primary end point were a lower-than-expected risk of participants enrolled with relatively young age, early revascularization, and preserved LVEF.Other trials have tested telemedicine strategies to follow up and adjust medications in patients after hospitalization for heart failure.Our study had several limitations. First, this trial was conducted at 3 tertiary cardiac centers, and our telemedicine unit was managed by nurse practitioners with a master’s degree in nursing and more than 10 years of nursing experience. As such, our findings may not be generalizable to allied health care professionals in other health care settings. Second, only 2 RIM participants were lost to follow-up compared with 9 SC participants, which could have biased the comparison of outcomes. Third, despite the reassuringly small number of adverse events attributable to RIM, these salutary safety signals need further validation in a larger trial of higher-risk patients with reduced LVEF or heart failure.Among patients hospitalized for AMI with elevated NT-proBNP concentration and preserved LVEF, a 6-month postdischarge multicenter RIM program by a centralized allied health care team had an equally low number of safety events and achieved similar β-blocker and ACE-I/ARB doses but did not improve LV remodeling outcomes compared with face-to-face SC by cardiologists. This feasibility study demonstrates the potential for RIM to be tested on a higher-risk AMI population with reduced LVEF or heart failure.