Toxoplasma gondii is unique among eukaryotes in possessing a novel variant of H2B designated H2B.Z. The combination of PTMs and the use of histone variants is important for gene regulation in T. gondii, offering new targets for drug development. In this work, T. gondii parasites were generated in which the 5 N-terminal acetylatable lysines in H2B.Z were mutated to either alanine (c-Myc-A) or arginine (c-Myc-R). c-Myc-A mutant only displayed a mild effect in its ability to kill mice. c-Myc-R mutant presented an impaired ability to grow and an increase in differentiation to latent bradyzoites. This mutant line was also more sensitive to DNA damage, displayed no virulence in mice, and provided protective immunity against future infection. While nucleosome composition was unaltered, key genes were abnormally expressed during in vitro bradyzoite differentiation. Our results show that the N-terminal positive charge patch of H2B.Z is important for these procceses. Pull down assays with acetylated N-terminal H2B.Z peptide and unacetylated one retrieved common and differential interactors. Acetylated peptide pulled down proteins associated with chromosome maintenance/segregation and cell cycle, opening the question of a possible link between H2B.Z acetylation status and mitosis.Through regulation of DNA packaging, histone proteins are fundamental to a wide array of biological processes. A variety of post-translational modifications (PTMs), including acetylation, constitute a proposed histone code that is interpreted by “reader” proteins to modulate chromatin structure. Canonical histones can be replaced with variant versions that add an additional layer of regulatory complexity. The protozoan parasite  Toxoplasma gondii, offering new targets for drug development [Histones proteins function to form nucleosomes that regulate packaging of DNA, thereby affecting DNA transcription, replication, and repair. In addition to the four canonical histones , variant histones exist that can be substituted into the nucleosome, affecting its properties. Canonical and variant histones are also subject to a wide array of post-translational modifications (PTMs) that can alter the nucleosome or recruit chromatin remodeling machinery. The combination of PTMs and exchange of histone variants is believed to be important for gene regulation in the protozoan parasite elopment –3.T. gondii is a member of phylum Apicomplexa and infects between 10–90% of the population depending on the country [T. gondii converts from rapidly growing tachyzoites, which cause acute infection, to slow or non-growing bradyzoites, which cause chronic infection and typically reside in the brain [T. gondii infection is asymptomatic in healthy people, untreated clinical toxoplasmosis can be lethal in immunocompromised individuals such as transplant or HIV patients, and can produce severe disease during congenital infection, especially in early stages of development [ country , presuma country . T. gondhe brain . Althougelopment , 8. Chroelopment .T. gondii life cycle is comprised of sexual and asexual stages. While sexual replication takes place only in felids, T. gondii is able to replicate asexually as tachyzoites in any nucleated cell in any warm-blooded vertebrate. Following infection, tachyzoites convert into bradyzoites, which are house in tissue cysts that are impervious to immunity and current drug treatments. Recently, a myb-like transcription factor (BFD1) was described to be necessary and sufficient to induce bradyzoite differentiation [T. gondii remain poorly understood.The ntiation . How BFDntiation . In suppntiation . The rolT. gondii expresses histone variants that we have previously shown mark functional regions of the genome [T. gondii [e genome . H2A.Z ie genome –20, whic. gondii . Unlike . gondii –25 and t. gondii , in whicT. gondii, both H2A.Z (TGGT1_300200) and H2B.Z (TGGT1_209910) are essential during the lytic cycle with CRISPR fitness scores of −5.08 and −4.05, respectively [T. gondii H2B.Z was shown to be acetylated at 5 lysine residues in its N-terminal tail. The H2A.Z N-terminal tail has 10 acetylatable lysines, in which lysine 18 can also be methylated [T. gondii, Plasmodium or Tetrahymena termophila, this number is considerably higher (between 5 and 16). Moreover, the presence of a T. gondii double variant nucleosome that altogether carries 15 acetylatable lysines that could be regulated is highly intriguing.Histones H2A.Z and H2B.Z form a dimer that localizes with the transcriptional activation mark H3K4me3 in promoter/transcriptional start site (TSS) regions surrounding the nucleosome-free region upstream of the transcription start site . In addiectively . By massectively . T. gondthylated . H2A.Z Nthylated –32, suggthylated –37. Whilthylated . InteresT. gondii H2B.Z N-terminal tail lysines, using a mutagenesis strategy; we also pursued a gene knockout strategy for H2B.Z. In an RH strain background, we generated T. gondii that possess mutated versions of H2B.Z: c-Myc-R, in which the five N-terminal acetylatable lysines were replaced by arginines, and c-Myc-A, in which they were replaced by alanines. These lines were analyzed for their ability to grow in vitro, differentiate to bradyzoites in vitro and produce virulence in mice. In addition, expression of key genes was studied, as well as nucleosome composition and DNA damage sensitivity. Our results suggest that regulation of the N-terminal positive charge patch by lysine acetylation, rather than the histone code, is essential for the aforementioned biological processes. We have also found some differential interactors for an acetylated N-terminal H2B.Z peptide, compared to a unacetylated one, opening new questions on the function of this modification and this histone variant. The implications of these data on the putative role of H2B.Z is discussed.In the present work we studied the role of the five T. gondii and Plasmodium falciparum H2B.Z N-terminal tails contain five acetylated lysines  [H2B.Z is a variant histone of the H2B family that appears early in the phylum Apicomplexa. Previously, it was observed that and K18) , 39, 40.h2b.z gene were unsuccessful (data not shown). The failure to obtain an H2B.Z. knockout is consistent with its CRISPR fitness score of −4.05, strongly suggesting it is essential [Attempts to obtain a mutant cell line by deletion of the ssential   or a mutated form in which all the acetylatable lysines in the N-terminal tail were changed to alanine, which mimics how acetylation ablates the positive charge of the lysine residues. We also over-expressed a mutated version in which the lysines were replaced with arginines, which would generate a constitutive positive charge patch but no longer support acetylation . NeitherH2B.Z gene was deleted by using CRISPR methodology in the previously generated T. gondii lines overexpressing mutant H2B.Z versions  obtained, which could be distinguished by the c-Myc tag. Approximately equal amounts of c-Myc positive and c-Myc negative  tachyzoites were mixed and allowed to invade hTert cells, which were analyzed by IFA on different days post-infection. While the c-Myc-A parasites showed no change in growth relative to the parental line over time, the c-Myc-R line showed a marked growth deficiency compared to the parental line . As argiin vitro tachyzoite to bradyzoite differentiation using alkaline stress to trigger stage conversion and Dolichos biflorus lectin (DBL) to monitor tissue cyst wall formation. Strikingly, we found that c-Myc-R, but not c-Myc-A, showed an increase in DBL-positive vacuoles compared to the wild-type control , Bag1 (early bradyzoite), LDH2 (early bradyzoite), AP2IX-9 (mature bradyzoites) [AP2 IV-3  [BFD1 [BFD1 showed an increase in c-Myc-R, compared to RH , and late bradyzoites (96 hours post-differentiation) of the parental RH strain and c-Myc-A or c-Myc-R parasites. We analyzed the expression of yzoites) , AP2 IV-yzoites)  and BFD1s) [BFD1  mRNA by ed to RH . NotablyT.gondii effect of MMS, HU, and campothecin [In other species, H2A.Z has been linked to double strand break repair driven by the non-homologous end joining (NHEJ) pathway , 43, 44.pothecin . Topotecpothecin .50 value  and MMS (50 μM) using the competitive growth assay. We observed a significative decrease in growth compared to the parental after treatment only with MMS for c-Myc-R tachyzoites . As a result, PVs usually show 2, 4, 8, 16 and so on, tachyzoites (Tz) per PV. Treatment with MMS and Topotecan also caused a loss of synchronization, with an altered number of Tz per PV, which was also counted as “anomalous vacuole”. Anomalous vacuoles and loss of genetic material were also observed with topotecan treatment  previous to CRISPR procedure, were as lethal as the parental line (data not shown), suggesting that the randomly integrated ptub-H2B.Z-R gene is not responsible for the loss of virulence and that co-expression of H2B.Z wt and H2B.Z-R mutant histone did not generate a dominant negative T. gondii line. Infection with 1,000 and 10,000 c-Myc-R tachyzoites were also nonlethal to mice  survived until sacrifice on day 30–35 . c-Myc-W to mice .T. gondii lines infections, but not with c-Myc-R parasites (T. gondii lines) were analyzed to detect antibodies against T. gondii. An increase in total IgG in mice infected with c-Myc-R tachyzoites was observed, comparable to Me49, which indicated that an initial infection had taken place  . These rT. gondii virulence proteins that exert protection from PV establishment are the polymorphic rhoptry proteins Rop5 and Rop18, which disrupt the γIFN-inducible IGR pathway [T. gondii Rop5 (TGGT1_411430) and Rop18 (TGGT1_205250) recombinant proteins for western blot analysis. Rop5 was present in parental, c-Myc-A, and c-Myc-R parasites, whereas Rop18 only in the parental line  formation in both mutant lines by co-immunoprecipitation (co-IP) assays by using commercial anti-c-Myc agarose. Mononucleosomes of c-Myc-A and c-Myc-R lines were obtained after MNAse digestion before co-IP . In figurosstalk . Quantiflated H3 . These dSag1 (tachyzoite), tubulin (constitutive), Bag1 and LDH2 (bradyzoite) and BFD1 (master differentiation factor). Only BFD1 mRNA expression was statistically higher in c-Myc-R that in the parental or c-Myc-A  and the other unacetylated; both peptides contained a biotin tag towards the C-terminal end  to perform the pull-down assays. After resolving on SDS-PAGE, samples were analyzed by mass spectrometry. We detected a total of 48 interactors, 18 exclusive of non-acetylated oligo, 17 exclusive of acetylated oligo and 14 present in both . While mT. gondii gene expression is associated to the cell cycle, lytic cycle and differentiation [T. gondii epigenome has been studied, and it has been observed that this parasite uses a set of different histone modifications as a complex set of tools to control its gene expression [T. gondii H2BZ has not been studied. However, T. gondii H2B.Z/H2A.Z double variant nucleosomes were found in the promoter of active genes and in the gene body of inactive genes [In ntiation , 58–63. ntiation . The Apipression . Acetylapression . Until nve genes . This chin vitro. On the other hand, virulence was severely affected in vivo indicating some sort of regulation at least in those processes for this H2B.Z PTM. Those effects were not observed when lysine was replaced by alanine, an uncharged amino acid that mimics acetylated lysine, except a mild effect on virulence in vivo. For T. termophila when engineering a constitutively positive charge patch on H2A.Z, this was highly toxic, suggesting that its regulation has an essential function [in vitro, but the T. gondii c-Myc-R tachyzoites were viable except during mouse infection. This indicates that at least in vitro, if there is a toxicity due to the constitutive positive charge patch of H2B.Z, it could be partly compensated by H2A.Z, which presents 10 acetylatable lysines [T. gondii are needed to understand this.According to the results obtained in this work, the presence of a constitutive positive charge patch in the N-terminal tail of the histone (c-Myc-R mutant) causes an increase in differentiation, a decrease in growth, and an impaired DNA damage response function . Moreovefunction , 67. In  lysines . TogetheT. gondii c-Myc-R line can grow responding to DNA damage in vitro, with significant but surmountable defect. This would indicate a role in DNA damage repair for this histone variant in T.gondii, with acetylation as a central player. However, this sensitivity was only significant for c-Myc-R tachyzoites, arguing once again in favor of the positive charge patch toxicity hypothesis in this mutant. Since H2B.Z does not exist in nature beyond Apicomplexa, we can only infer the putative role of this variant histone being part of H2A.Z/H2B.Z nucleosome. The participation of H2A.Z in DNA damage repair pathways has been studied in mammals and yeast [An interesting aspect is that the nd yeast , 43, 44.in vitro are less difficult to ascertain in the in vivo model. Clearly c-Myc-R line is capable of invasion, generating an adequate immune response, but it is quickly neutralized during the infective process. This suggests that the tachyzoite is exposed in vivo to stronger environmental pressures than those observed in vitro. These pressures would include adaptation to new environments, including different cell types and organs, which may present differences in metabolism, nutrient availability, or immune responses. Under these conditions, where the tachyzoite must respond quickly, fine reprograming of gene expression and modifications in metabolic pathways would be required and the impossibility of modulating the positive charge patch of H2B.Z could hamper the ability of parasites to adapt to changing environments in vivo.The contributions of effects observed T. gondii, and interaction of H1 with H2B.Z was observed [A possible explanation for the changes provoked by the positive charges in the N-tail could be a chromatin compaction due to interaction with the negative charges in DNA. Genome size expansion in eukaryote evolution has led to an increase in histone H2A N-terminal tail arginines. Arginine-rich histones bind more tightly to DNA, compacting chromatin and, in some cases, the nuclei are also smaller . Howeverobserved . The aceT. gondii. With the results shown in this work it is only possible to correlate the decrease in one of the more virulent isoforms of Rop5 together with the absence of Rop18 protein to the loss of virulence of c-Myc-R mutant, although it cannot be ruled out that other virulence factors could be altered in these parasites. On the other hand, the absence of Rop18 protein in c-Myc-A correlates with the significative delay in mice death with c-Myc-A tachyzoites infection as reported by Resse et al [It could be expected that the phenotypic alterations are given by defects in the formation of the nucleosome with an impact on the expression of the genes. In our analysis, both situations are not altered. Although a massive analysis of gene expression is necessary to detect specific changes, part of the study shows that the expression of Rop5 and Rop18 does not occur at the transcriptional level, but rather post-transcriptionally, affecting their translation. This regulation could be given by the presence of non-coding RNAs (ncRNAs) and there is increasing evidence for a significant role for pos-transcriptional mechanisms , 71. Futse et al .BFD1 in c-Myc-R. This does not discard an association of the N-terminal tail of H2B.Z with the epigenetic modulation of gene expression in some circumstances, for example in changing environments, in an in vivo model. Regarding the significant expression of BFD1 mRNA, it is intriguing if it is related to the high differentiation rate observed in vitro for these parasites. It is important to note that the expression of the BFD1 gene is post-transcriptionally regulated [per se is not enough to induce differentiation, but the higher levels of BFD1 mRNA could favor differentiation under alkaline stress. There is also another possibility of inducing differentiation based on epigenetic regulation. HDACi FR235222 treatment was shown to favor acetylation of H4K31ac in bradyzoite gene promoters, promoting transcription of bradyzoite genes and therefore, bradyzoite development [We could not detect significant changes in most of the genes analyzed by RT-qPCR in normal conditions for tachyzoites, except for egulated . Therefoelopment . Maybe uBoth acetylated and unacetylated peptides pull-downs share 3 nuclear proteins: GCN5A, PHD and clathrin. GCN5A was shown to impact on H2A.Z acetylation via crosstalk with H4 acetylation by means of bromodomain in p300 . On the In addition, CDKs have also been linked to mitosis , 77 and ® is a live attenuated vaccine used to prevent sheep congenital toxoplasmosis [2+-dependent protein kinase (CDPK) knock-out in Me49 type II strain was evaluated in mice as a potential vaccine candidate [It is relevant that these tachyzoites were able to generate a complete immune response, which only happens when cells are invaded by tachyzoites and not with an infection with dead or lysed tachyzoites –87. In tlasmosis . Recentlandidate . The proToxoplasma gondii life cycle. The presence of 5 positive charges in the N-terminal of H2B.Z is most likely to be detrimental for the parasite. However, this toxicity is not very high, allowing the parasite to replicate, but conferring a lower fitness to the tachyzoite. Moreover, this lower fitness is traduced in a higher sensitivity to DNA damage, a mis-regulation of gene expression conducing to a higher differentiation rate in vitro, and also a complete lack of virulence when facing the immune response in mice. All these effects are not explained by an heterochromatinization of DNA due to the interaction with the positive charges, because we observed that nuclei are also smaller in c-Myc-A tachyzoites. Besides, the effects cannot be explained by a dislocalization of the histone in the genome because the nucleosome is still constituted normally. However, it is quite unlikely to find a physiologically a situation where the histone would be completely unacetylated, and c-Myc-A tachyzoites may reflect the normal condition observed in vivo. Therefore, the similarity observed between c-Myc-A and parental tachyzoites in most experiments performed in this work, would confirm that in normal conditions H2B.Z is acetylated in T. gondii.In this work, we have shown that N-terminal tail lysine acetylation of histone H2B.Z is important for the normal progression of T. gondii as well. Deficiencies in Trypanosoma cruzi H2Bv histone variant amount present similar phenotypes as observed here, and the authors also proposed a putative extra-nuclear or extra-nucleosome function [The findings in this work are not exclusively explained by regulation of gene expression. This is not surprising, as it is known that histone proteins may exert other functions, related for example to cell division. Even extra-nuclear functions are attributed to histones in many models, and research in this area could bring new insights for function .T. gondii will allow inferring about chromatin modulation in other parasites of the phylum as well as the possibility of suggesting a novel mechanism of epigenetic regulation in organisms with complex life cycles that are evolutionarily distant from fungi and vertebrates.Having a better understanding of how chromatin, chromosome maintenance, DNA integrity, DNA replication, and gene expression are modulated in hxgprt strain was used in all cases and grown in standard tachyzoite conditions in vitro: hTERT  monolayers were infected with tachyzoites and incubated with Dulbecco’s modified Eagle medium  supplemented with 1% fetal bovine serum  and penicillin -streptomycin (10 mg/ml) solution  at 37°C and 5% CO2.RHΔATGCATTCAGGGAAAGGTCCGGCACAG-3’ and R: 5’-TTAATTAACTATGCACCAGAAGTCGTG-3’. For the mutant constructs, reverse primer was the same, and forward was: ATGCATTCAGGGGCAGGTCCGGCACAGGCATCTCAGGCGGCGGCGGCGACCGCCGGGGCGTCTCTGGGAC for c-Myc-A, ATGCATTCAGGGCGCGGTCCGGCACAGCGCTCTCAGGCGGCGCGCCGCACCGCCGGGCGCTCTCTGGGAC for c-Myc-R, where lysine coding codons were replaced for alanine and arginine coding codons, respectively (in bold type). NsiI and PacI restriction sites (underlined) were engineered into the 5’ end of primers F and R, respectively, to clone into T. gondii expression vector PTUB8mycGFPPfTailTy-HX  with a strong tubulin promoter. The resulting PCR product of the expected size was removed using a Qiaex II Gel Extraction kit (Qiagen), cloned in pGEM-T vector (Promega) and sequenced (Macrogen). The cloned ORF was digested with NsiI and PacI and ligated into the plasmid. The final plasmids were electroporated into RH strain tachyzoites lacking hypoxanthine–xanthine guanine phosphoribosyl transferase (RHΔhxgprt). Transgenic parasites were selected in mycophenolic acid  and xanthine  and cloned by limiting dilution.To generate the c-Myc-H2B.Z WT construct, the open reading frame (ORF) was amplified using the primers F: 5’-h2bz gene: F: AAGTTGAGGCGGCGAAGAAGACCGCCG and R: AAAACGGCGGTCTTCTTCGCCGCCTCA. Bsa I restriction sites were included (underlined). Both oligos were annealed and cloned into the pU6 CRISPR Universal plasmid , by using the Bsa I restriction site. The plasmids were confirmed by PCR, using the gRNA as primers, were sequenced (Macrogen) and prepared for transfection in Toxoplasma. In order to obtain a clean KO, oligos were also designed to amplify the DHFR selection cassette from the pUPRT_DHFR plasmid available in the laboratory, with homology regions (20 bp underlined) to the 3′and 5′UTR of the h2bz gene . The PCR amplification product was purified from agarose gel and used to co-transfect the parasites along with the CRISPR plasmid. c-Myc-R and c-Myc-A tachyzoites (one clone for each one was selected) were transfected and Pyrimethamine  was used for selection. Cas9 was detected in the nucleus of the parasites after transfection by using α-Flag antibody . Selection was continued until three passages and cloning by limiting dilution was performed. Positive clones were selected by PCR with primers designed for that purpose: TgH2Bv-upUTR-F: GTTGTCATGCGCATTTGCATCAC; TgH2Bv-downUTR-R: GTGTGCACGCGTTATAATGAGCAC; Val-TgH2Bv-R: CATGCTCTTCTTCGACACACCAG; DHFRCX-F: GTGGCATTTCACACAGTCTCACCTC; DHFRCX-R: AGACGCAGACGCATACAACGTTAG.Single guide RNAs were designed directed to the N-terminal portion of the Intracellular tachyzoites grown in cover slips were fixed using cold methanol 100% for 8 min, washed with PBS and blocked with 1% BSA. Primary antibody αrH2B.Z (rabbit), αrSag1 (mouse), αrHSP90 (rabbit), αrROP5 (rabbit), αrROP18 (rabbit) produced in the laboratory and αc-Myc , αAcTubulin  were incubated for 1 h at room temperature. After several washes with PBS cover slips were incubated with secondary antibodies Alexa fluor goat anti-mouse 488 and anti-rabbit 594 (Invitrogen). DAPI was used to stain nuclei. Axio Imager.M2 Microscope Carl Zeiss (Germany) with objective Plan-Apochromat 63x/1.40 Oil M27 and Video digital camera Zeiss 503 monochromatic 2.8 megapixeles, was used. Image J 1.53q (Fiji) software was used to process images.7 parasites were loaded per well and resolved by 15% SDS-PAGE. Proteins were transferred to PVDF membrane for 1h at 100V. Western blot was then performed as described [Tachyzoites were collected, filtered, counted and lysed by 6 cycles of rapid freezing/defreeze in hypotonic buffer, and boiled with LB for 5 minutes. 0.5 to 1×10escribed . The priescribed , αrH2A.Zescribed , αrROP5 escribed  1/200 fohxgprt) and 50% of each clone. 3 coverslips per clone were infected with 0,05 to 0,1 tachyzoite per cell, incubated 10 minutes on ice, and 2 hours at 37°C in incubator for invasion. Slides were washed twice with PBS to remove tachyzoites that did not enter the cells, and fresh DMEM supplemented with 1% SFB was added. Slides were fixed at different times, up to 96 hours and IFA was performed with α-c-Myc and α-Sag1 antibodies to distinguish between parental and c-Myc positive vacuoles. Number of c-Myc positive and total vacuoles in at least 100 vacuoles, randomly choosing different fields of each slide were counted. In the case of competition assays with genotoxic drugs treatment, the experiment was modified as follows: 3 slides per clone mixture were fixed after 24 h, to stablish the initial percentage of c-Myc positive vacuoles. At this time point, media was changed in the rest of the slides for DMEM supplemented with DMSO or drugs at the concentrations indicated. These slides were fixed after 96 h, and IFA was performed and counted as explained before.For growth/competition assays, coverslips seeded with Htert cells were infected with a mix of 50% parental  to stain the wall of the cysts in formation and α-Sag1 antibody to stain the tachyzoites. In the case of collection of parasites for RNA extraction, differentiation assay was performed in a similar way, but in t25 dishes. After 48 or 96 h parasites were forced out of the cells by 23G, 25G and 27G syringe passages.For in vivo survival, virulence and differentiation assays, C57BL/6 female mice were used. For the use of animals, C.I.C.U.A.E-UNSAM 10/22 was approved. Mice were maintained in optimal conditions in the biotherium, with controlled temperature and free access to sterilized water and food. Infection was carried out intra-peritoneal in 10 mice per group with 100, 1000 or 10000 tachyzoites of parental lines or clones, depending on the assay. PBS was injected as negative control in five mice per experiment. Deceases were registered along 30–35 days in each experiment, and signs of illness were monitored daily. Two mice from each group (except PBS) were sacrificed at day 5 (when RH infected mice have symptoms of illness), and intraperitoneal fluids were collected in order to detect acute Toxoplasma infection. Blood samples were taken at days 0, 14, 21 and 35 and sera was frozen for ELISA assays. After sacrifice, surviving mice brains were processed for cyst observance by optical microscopy. Also, a sample was stained using DLB and checked by fluorescence microscopy.For In every phenotypical assay, three independent experiments were performed. One representative experiment is shown.Tachyzoites or parasites exposed to 48 or 96 h differentiation stress as explained before were conserved in TriZol (Invitrogen) solution at −80°C until use. All experiments were performed in three independent replicates. RNA extraction was performed according to the manufacturer’s instructions and cDNA was obtained by means of MMLV reverse transcriptase (Promega) using oligo dT with the protocol provided with the enzyme. For RT-PCR, primers used were: SAG1: Fw: TGAGAACCCGTGGCAGGGTAA; Rv: GCTTTTTGACTCGGCTGGAA; TUB: Fw: ATGTTCCGTGGTCGCATGT; Rv: TGGGAATCCACTCAACGAAGT; ACTIN: Fw: GGGCGGTTTCATGACCTAAA; Rv: ACGTATGATGCGCGAGAAAA; ROP5: Fw: CTAGCTAGCATGGCGACGAAGCTCG; Rv: CCCAAGCTTTCAAGCGACTGAGGGCGCA; ROP18: Fw: CTAGCTAGCATGTTTTCGGTACAGCGGCCA; Rv: GCCAAGCTTTTATTCTGTGTGGAGATGTTC. For RT-qPCR primers used were: SAG1: Fw: TGAGAACCCGTGGCAGGGTAA; Rv: GCTTTTTGACTCGGCTGGAA; BAG1: Fw:CAACGGAGCCATCGTTATCAAAGG; Rv: TAGAACGCCGTTGTCCATTG; LDH2: Fw: ACAATGGCCCAGGCATTCT; Rv: CAATAAACATATCGTGAAGCCCATA; AP2IX-9: Fw: GGGCGTTCTCAGCGTTCACT; Rv: GGCGCGTCTCATCTGTTTCA; AP2IV-3: Fw: GAGCCCATTGACCCCATGAA; Rv: GGCTTCGCTTCTTTCCGTGA; BFD1: Fw: ATGTCGGGAACGATGGTTTA; Rv: TCTTCACGGCATTCTCTGTG; TUB: Fw: ATGTTCCGTGGTCGCATGT; Rv: TGGGAATCCACTCAACGAAGT; ACTIN: Fw: GGGCGGTTTCATGACCTAAA; Rv: ACGTATGATGCGCGAGAAAA.The primers were first assayed for efficiency using a pooled cDNA in 1 to 0.001 dilutions, and the threshold was defined for each set of primers. Melt curves were also obtained for each set. For each set of experiments, SybrGreen master solution (Roche) was used and qPCR was run in StepOne Real time equipment (Applied Biosystems). Actin and tubulin were used as housekeeping genes and data was normalized to those genes by Infostat software. For the experiments in ™ MicroWell™ 96 well solid plates, Sigma), o.n. at 4°C. After bocking with 5% non-fat milk in PBS-T they were incubated with sera diluted 1:100 in the same solution. Total IgG (IgGt) and subtypes  HRP-conjugated anti-mouse, made in rat, were used 1:5000. Tetramethyl-bencidine  was used to reveal, and plaques were read after 20 min reaction at 655 nm in a Synergy H1. Data was analyzed in GraphPad Prism v7. Sera from mice inoculated with PBS (negative control) or infected with Me49 (positive control) were assayed.Blood samples were extracted from surviving mice at days 0 (Pi), 14, 21 and 35. Sera was frozen until processing for ELISA. TLA  in buffer Carbonate  was used to immobilize 96 well plaques  in order to obtain mononucleosomes as described [Approximately 5×10escribed  and Co-Iescribed  with minescribed . QuantifThree slides with confluent hTert cells were infected as explained before with parental or c-Myc-A/c-Myc-R/c-Myc-WT-OE clones and let replicate for 24 hours. Slides were fixed and IFA was performed as explained before using αrH2B.Z to stain nuclei. Images were processed with Image J 1.53q (Fiji) software to measure the size of the nuclei, by delimiting them with the appropriate tool in at least 50 vacuoles in fields randomly chosen, for each slide and each independent experiment. Data was graphed and statistical analyzed with GraphPad Prism 8 software.Peptides were synthesized by solid-phase multiple peptide system using tea-bags with Fmoc/tBu strategy according Guzmán , brieflyPeptides used in the pull-down assays were: Un-acetylated: GPAQKSQAAKKTAGKSLGPRK(Biotin) Acetylated: GPAQKSQAAK(Ac)K(Ac)TAGKSLGPR-K(Biotin)4)2SO4, 2% phosphoric acid) was added until covering the gel. It was stained for 1 h in gentle shaking. Then the gel was rinsed in deionized water 3 times for 5 minutes each. The background staining was removed with 30% methanol. The gel was stored in deionized water until use. Each lane of the gel was cut into individual slices. Each band was then cut into 1 mm3 cube and further treated with three washes of 50 mM NH4HCO3 in 50% CH3CN with 10 min incubations. Each group of gel cubes was then dehydrated in CH3CN for 10 min and dried in a Speed Vac. Protein samples were reduced by dithiothreitol (DTT) and alkylated by iodoacetamide [4HCO3 was used to re-swell the gel pieces completely at 4°C for 30 min, followed by a 37°C digestion overnight. A small amount of 10% formic acid was then added to stop the digestion. The sample was then centrifuged at 2,800 × g, and the supernatant was collected for LC-MS/MS.Pull-down experiments were performed as detailed in Wysocka  after nucetamide . A solutA fused silica microcapillary LC column (15-cm long × 75-μm inside diameter) packed with Halo C18 reversed-phase resin  was used with EASY-nLC 1200 system (Thermo Fisher). The nanospray ESI was fitted onto the Thermo Q-Exactive plus mass spectrometer  that was operated in a Higher-energy C-trap dissociation mode to obtain both MS and tandem MS (MS/MS) spectra. Two μL of tryptic peptide samples were loaded onto the microcapillary column and separated by applying a gradient of 3–40% acetonitrile in 0.1% formic acid at a flow rate of 300 nL/min for 80 min. Mass spectrometry data were acquired in a data-dependent top-10 acquisition mode, which uses a full MS scan from m/z 350–1700 at 70,000 resolution . Resolution for dd-MS2 spectra was set to 17,500 (AGC target: 1e5) with a maximum ion injection time of 50 ms. The normalized collision energy was 27 eV.T. gondii ToxoDB-42 _TgondiiME49 protein database using Proteome Discoverer 2.2 . The search parameters permitted a 10 ppm peptide MS tolerance and a 0.02 Da MS/MS tolerance. Carboxymethylation of cysteines was set as a fixed modification, and oxidation of methionine as a dynamic modification. Up to two missed tryptic peptide cleavages were considered. The proteins for which the False Discovery Rate was less than 1% at the peptide level were included in the following analysis. The raw data of all mass spectra had been submitted to the MassIVE .Obtained MS spectra were searched against the Supplement 11


Previous studies have shown that Mettl3 overexpression can increase the level of m6A and promote somatic cell reprogramming. Here, we demonstrate that Mettl14, another component of the methyltransferase complex, can significantly enhance the generation of induced pluripotent stem cells (iPSCs) in an m6A-independent manner. In cooperation with Oct4, Sox2, Klf4, and c-Myc, overexpressed Mettl14 transiently promoted senescence-associated secretory phenotype (SASP) gene expression in non-reprogrammed cells in the late stage of reprogramming. Subsequently, we demonstrated that interleukin-6 (IL-6), a component of the SASP, significantly enhanced somatic cell reprogramming. In contrast, blocking the SASP using a senolytic agent or a nuclear factor κB (NF-κB) inhibitor impaired the effect of Mettl14 on reprogramming. Our results highlight the m6A-independent function of Mettl14 in reprogramming and provide new insight into the interplay between senescence and reprogramming in vitro.The METTL3-METTL14 complex, the “writer” of N  •Mettl14 can facilitate somatic cell reprogramming in an m6A-independent manner•Mettl14 transcriptionally drives the senescence-associated secretory phenotype (SASP)•Mettl14-driven SASPs are mainly secreted from non-reprogramming cells•Mettl14 on reprogrammingBlocking of SASP impairs the effect of  Mettl14 can enhance somatic cell reprogramming to iPSCs in an m6A-independent manner. At the late stage of reprogramming, Mettl14 significantly increased the expression level of senescence-associated secretory phenotype (SASP). These SASPs are mainly secreted by the non-reprogramming cells and blocking of SASP impairs the effect of Mettl14 on reprogramming.Gao and colleagues found that  Depositin (FTO) . In the platform .Oct4, Sox2, Klf4, and c-Myc, known as OSKM) provides a system to study the molecular mechanisms of the cell-fate transition  by Yamanaka factors  from Rosa26-M2rtTA; Col1a1-4F2A; Oct4-GFP+ transgenic mice  accelerated Oct4-GFP+ colony formation and led to an approximately 6-fold increase in the number of Oct4-GFP+ colonies  colonies  exhibited typical embryonic stem cell (ESC) morphology with large nuclei and nucleoli, a compact appearance, and clear boundaries , cartilage (mesoderm), and cuboidal epithelium (endoderm)  E (Le et ndoderm) . Furtheron assay F.Mettl14-KD cells (OSKM+Mettl14-KD iPSCs) also exhibited an ESC-like morphology . We performed a principal-component analysis (PCA) to compare the transcriptomes of the reprogramming cells at the indicated time points. The PC1 axis was dominated by differences among reprogramming intermediate cells. Specifically, the cells showed clear stepwise transcriptome changes during the reprogramming of MEFs   and downregulation of 33 DEGs on day 15, compared with the control group  enrichment analysis showed that the DEGs increased by ractions B. To undthe SASP . Therefothe SASP  in our d heatmap C. Surpri heatmap A.Il6, C-X-C motif chemokine ligand 2 (Cxcl2), and C-C motif chemokine ligand 7 (Ccl7), were increased after day 12 and peaked on day 15  pathway, which is upstream of the SASP factors. A western blot analysis showed that OE of the gramming E.in vitro reprogramming and senescence, we examined whether both of these processes proceeded within the same time period in different cell populations. We performed double staining for AP (indicating pluripotent colonies) and SAβG (indicating senescent cells) on day 15 of reprogramming (+ colonies (Mettl14 and mutant Mettl14 triggered more cells to undergo senescence and generated more iPSCs (To explore the possible relationship between gramming . We founcolonies F. Duringre iPSCs F.Oct4-GFP+ and Oct4-GFP− cells by fluorescence-activated cell sorting (FACS) on day 15 of reprogramming and measured the expression levels of senescence and SASP genes. The expression levels of senescence genes, such as p16 (Cdkn2a) and p21 (Cdkn1a), and of SASP genes, including Il6, Cxcl1, and Ccl7, in Oct4-GFP− cells were much higher than those in Oct4-GFP+ cells    A but wer Dppa5a) A. This rNR cells B. CollecBcl-2/Bcl-xL/Bcl-w expression  to selectively reduce the viability of senescent cells by inhibiting pression  and BAY pression . Both ins mutant C. CorresP+ cells H. These Mettl14 on reprogramming mainly depended on cellular senescence and transiently upregulated expression of SASP genes in NR cells during the late phase of reprogramming in an m6A-independent manner.In conclusion, the effect of 6A-independent function of Mettl14 during in vitro reprogramming. Mettl14 significantly upregulated the expression level of SASP genes during the late phase of reprogramming. It had been previously reported that in senescent cells, Mettl14 regulated SASP genes in an m6A-independent manner  database and are accessible through GEO: C.X. and L.W. designed and performed the experiments, performed the data analysis, led discussion, and wrote the manuscript; X.X. performed the bioinformatics analyses; C.X., Y.W., X.K., Y.Z., J. Sun, Y.D., Z.S., J. Shen, D.L., W.Y., Y.L., R.Z., Y.X., H.W., L.H., L.W. and S.G. contributed to the experimental work and discussion; and S.G. and L.W. supervised the study and contributed to writing. There is no conflict of interest in this article.


Encephalomyocarditis virus into an infectious clone of YFV 17D. The resulting recombinant virus, 17D-IRES, exhibited similar replication efficiency to its parental virus (17D) in mammalian cell lines, while it was highly restricted in mosquito cells. Serial passage of 17D-IRES in BHK-21 cells showed good genetic stability. More importantly, in comparison with the parental 17D, 17D-IRES displayed significantly decreased mouse neurovirulence and viscerotropism in type I interferon (IFN)-signaling-deficient and immunocompetent mouse models. Interestingly, 17D-IRES showed enhanced sensitivity to type I IFN compared with 17D. Moreover, immunization with 17D-IRES provided solid protection for mice against a lethal challenge with YFV. These preclinical data support further development of 17D-IRES as an updated version for the approved YF vaccine. This IRES-based attenuation strategy could be also applied to the design of live attenuated vaccines against other mosquito-borne flaviviruses.The yellow fever (YF) live attenuated vaccine strain 17D (termed 17D) has been widely used for the prevention and control of YF disease. However, 17D retains significant neurovirulence and viscerotropism in mice, which is probably linked to the increased occurrences of serious adverse events following 17D vaccination. Thus, the development of an updated version of the YF vaccine with an improved safety profile is of high priority. Here, we generated a viable bicistronic YF virus (YFV) by incorporating the internal ribosome entry site (IRES) from IMPORTANCE Yellow fever (YF) continually spreads and causes epidemics around the world, posing a great threat to human health. The YF live attenuated vaccine 17D is considered the most efficient vaccine available and helps to successfully control disease epidemics. However, side effects may occur after vaccination, such as viscerotropic disease (YEL-AVD) and neurotropic adverse disease (YEL-AND). Thus, there is an urgent need for a safer YF vaccine. Here, an IRES strategy was employed, and a bicistronic YFV was successfully developed (named 17D-IRES). 17D-IRES showed effective replication and genetic stability in vitro and high attenuation in vivo. Importantly, 17D-IRES induced humoral and cellular immune responses and conferred full protection against lethal YFV challenge. Our study provides data suggesting that 17D-IRES, with its prominent advantages, could be a vaccine candidate against YF. Moreover, this IRES-based bicistronic technology platform represents a promising strategy for developing other live attenuated vaccines against emerging viruses. Yellow fever (YF) is a potentially fatal viral disease caused by infection with yellow fever virus (YFV). Infection with YFV leads to a variety of symptoms, ranging from subclinical to severe illness, including fever, nausea, vomiting, jaundice, hemorrhaging, and even death . YFV is There is no specific antiviral treatment against YFV infection; vaccination is the primary strategy for YF control. The live attenuated YF vaccine 17D  is one of the most efficient live attenuated vaccines due to the very high rates of seroconversion and long-term protection following vaccination. Since its development in the 1930s, 17D has been administered to over 850 million people worldwide and has been successfully used to control the disease in many countries of endemicity . Althoug5–Flavivirus of the family Flaviviridae, including dengue (DENV), West Nile (WNV), Zika (ZIKV), and Japanese encephalitis (JEV) viruses. YFV has a 5′-m7GpppN-capped single-stranded positive-sense RNA genome approximately 11,000 nucleotides long. The viral genome encodes a single open reading frame (ORF) flanked by 5′ and 3′ untranslated regions (UTRs), which is translated into a polyprotein co- and posttranslationally cleaved by viral and host proteases to generate 10 mature viral proteins (C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5). YFV is known to initiate its translation in a canonical cap-dependent manner. During this process, the eukaryotic initiation factor 4E (eIF4E), a component of the cap-binding complex eIF4F, recognizes an m7GpppN cap structure at the 5′ end of viral mRNAs  IRES is functional in a variety of cultured cell types , the termination codon (TAA) followed by EMCV IRES was inserted downstream of the C protein based on the infectious clone of 17D . This st3 PFU of 17D resulted in 100% lethality, with an average survival time (AST) of 5.3 days, supporting that 17D retained significant neurovirulence in mice  route. As shown in 6 PFU/g on day 9 postinoculation, when the mice began to die  receptors have been widely used for viscerotropism testing . To assen to die . In contn to die . TogetheThe IRES attenuation strategy was presumed to imbalance translation of the viral protein . In our Next, we investigated the immunogenicity of 17D-IRES. As shown in Finally, we examined the protection efficiency of the highly attenuated 17D-IRES virus against virus challenges. The lethal challenge study showed that all the 17D-IRES-immunized BALB/c and AG6 mice survived without any clinical symptoms during the observation period  and D, dThese data show that 17D-IRES is capable of effectively inducing humoral and cellular immunity, providing full protection against lethal YFV challenge.The development of new attenuated vaccines is an important aspect of YFV research. Here, we report 17D-IRES, a recombinant 17D vaccine carrying the IRES of EMCV, which displays a highly attenuated neurovirulence in mice. The 17D-IRES virus is highly sensitive to IFN-I treatment and exhibited much less viscerotropism than the 17D vaccine in AG6 mice. More importantly, immunization with the highly attenuated 17D-IRES can still provide complete protection against the virus challenge.50], >10) were developed in all mice immunized with 17D or 17D-IRES. More importantly, 17D-IRES immunization provided complete protection against i.c. challenge with 17D in mice, while 17D conferred partial protection against challenge with the same dose of 17D.The main goal of our study was to attenuate the neurovirulence and potent viscerotropism of the 17D vaccine but to maintain its immunogenicity. Using reverse genetics, we inserted the EMCV IRES into the 17D vaccine genome to make initiation of translation of the viral C protein and all other viral proteins gene 5′ cap and IRES dependent, respectively. This IRES-based rational attenuation strategy targets the level and balance of protein production and has been developed for the alphaviruses chikungunya virus and Venezuelan equine encephalitis virus and the flavivirus Murray Valley encephalitis virus , 27, 28.In summary, we generated a recombinant replication-competent 17D-IRES that carries IRES and that is notably less neurovirulent, neurotropic, and viscerotropic than the 17D vaccine. To our knowledge, this is the first report on attenuation of the 17D vaccine using the IRES-based attenuation strategy. This promising vaccine candidate is worth being tested in nonhuman primates to determine if it is suitable for evaluation in humans.Aedes albopictus C6/36 cells (ATCC CRL-1660) were cultured in RPMI 1640. Aedes aegypti cell line Aag2 (ATCC CCL-125) was cultured in Schneider’s insect medium (Gibco). All the media above were supplemented with 10% fetal bovine serum , 100 U/mL penicillin, and 100 μg/mL streptomycin. BHK-21, Vero, L929, and A549 cells were grown at 37°C in 5% CO2, whereas the C6/36 and Aag2 cell lines were grown at 28°C. The live attenuated vaccine 17D (GenBank accession no. MN072725) was recovered from the infectious plasmid pYF-17D (BHK-21 cells (ATCC CCL-10), L929 cells (ATCC CCL-1), SH-SY5Y cells (ATCC CRL-2266), and Vero cells (ATCC CCL-81) were cultured in Dulbecco’s minimal essential medium . A549 cells (ATCC CCL-185) and  pYF-17D . The virThe plasmid was constructed using the standard molecular biology protocols based on pYF-17D. To construct the IRES-based 17D infectious clone (named p17D-IRES), the EMCV IRES was amplified by PCR using plasmid pIRES-neo as a template. The EMCV IRES was inserted into the 17D genome just between the capsid and prM. The stop cordon (ATT) was added at the 3′ ends of the capsid gene to terminate the translation. The primers used here are shown in Table S1 in the supplemental material.XhoI (NEB), purified with phenol-chloroform, ethanol precipitated, and dissolved in RNase-free water. The linearized DNA was used as a template for in vitro transcription using the RiboMAX large-scale RNA production system (Promega) in the presence of the cap analog m7GpppA (Promega). RNA transcripts (5 μg) were purified using a PureLink RNA minikit (Thermo Fisher Scientific) and transfected into BHK-21 cells seeded into 6-well plates using Lipofectamine 3000 reagent (Invitrogen). Supernatants were then collected on day 4 posttransfection when a typical cytopathic effect (CPE) was observed.Plasmid p17D-IRES (10 μg) was linearized with IFAs were performed as the standard protocols. Briefly, cells were previously seeded into a 24-well plate and infected with the virus at a multiplicity of infection (MOI) of 0.01. At 36 h postinfection, the culture supernatants were removed, and the cells were fixed with ice-cold acetone and incubated with monoclonal antibody (2A10) against flavivirus E protein at 37°C for 1 h . After wGrowth curves of 17D or 17D-IRES were measured on indefinite cells. The cells were seeded into the 24-well plate and infected with 17D or 17D-IRES at an MOI of 0.01. Culture supernatants were collected at successive 1-day intervals. Progeny viral titers were tittered by plaque assay on BHK-21 cells.17D-IRES was serially passaged in BHK-21 cells at an initial MOI of 0.01. Viral RNA was extracted from the fourth and eighth passages and sequenced. The plaque phenotypes were evaluated by plaque assay in BHK-21 cells.BHK-21 cells or Vero cells were previously seeded into the 96-well plate. The monolayer cells were incubated with a medium containing various doses of IFN-αA/D  for 6 h. The supernatants were removed, and the cells were washed with PBS, followed by incubation with 17D or 17D-IRES at an MOI of 1 for 1 h at 37°C. Then, the unbound virus was removed by gentle washing with PBS. After 24 h of incubation, the supernatants were collected, and virus titers were determined by plaque assay in BHK-21 cells.50)  . Briefly50) .5 per well) were each incubated with 105 PFU of heat-inactivated 17D that was prepared in BHK cells at 37°C for 24 h, and IFN-γ spots were developed and counted . Data were normalized by subtracting the number of spots from samples incubated with noninfected BHK cell lysates.ELISpot was performed using a mouse IFN-γ ELISpot assay (Mabtech) under the manual standard. In brief, mice were sacrificed, and single-cell suspensions of splenocytes were obtained. Sham-vaccinated and vaccinated mouse splenocytes . The BALB/c mice used in this study were purchased from Beijing Vitalriver Laboratory Animal Technology Co., Ltd. 129/Sv/Ev mice deficient in IFN-α/β receptors (A129 mice) and C57BL/6 mouse deficient in types I and II interferon (IFN) receptors (AG6 mice) were maintained at the Laboratory Animal Center, AMMS.3 PFU of 17D or 17D-IRES through the intracranial (i.c.) route and monitored for clinical symptoms and mortality for 21 days. Mouse brains were collected for viral detection on days 2, 4, and 6 postinfection.To examine the attenuation of 17D-IRES in neurovirulence, groups of 4-week-old A129 mice or 3-week-old BALB/c mice were inoculated with 104 PFU of 17D or 17D-IRES and monitored for 21 days. Mouse tissues were collected for viral detection on days 3, 6, and 9 postinfection. The virus titer in each supernatant was determined by plaque assay on BHK-21 cells.To test the attenuation of 17D-IRES in viscerotropism, groups of 6-week-old AG6 mice were intraperitoneally (i.p.) inoculated with 105 PFU of 17D or 17D-IRES through the s.c. route and boosted 1 month later. Mice immunized with PBS were used as the control. All the mice were bled by tail vein puncture 4 weeks after the boost immunization. The sera were stored at –20°C for determination of neutralizing antibodies by PRNT50. To assess the cellular immunity induced by 17D-IRES, groups of 4-week-old BALB/c mice were immunized with 105 PFU of 17D or 17D-IRES through the s.c. route. On day 28 postimmunization, the mice were executed and spleens were collected for cellular immunity assay by ELISpot.To assess the immunogenicity of 17D-IRES, groups of 4-week-old BALB/c mice were immunized with 105 PFU of 17D through the s.c. route and boosted 1 month later. On day 28 postimmunization, the mice were challenged with 100 PFU of 17D by the i.c. route and monitored for clinical symptoms and mortality for 21 days.Intracranial (i.c.) challenge has been used previously to evaluate the protection conferred by YF17D and YF17D-derived vaccines in BALB/c mice . For the4 PFU of 17D-IRES. On day 28 postimmunization, the immunized AG6 mice were challenged by the i.p. route with 104 PFU of 17D; all mice were observed for clinical signs and death for 21 days.For the protection assay in AG6 mice, groups of 4-week-old AG6 mice were intraperitoneally (i.p.) inoculated with 10in vitro experiments, an unpaired t test was performed for comparison between 2 groups, and two-way ANOVA was conducted with multiple-comparison tests for multiple comparisons between groups. For survival analysis, Kaplan-Meier survival curves were analyzed by a log-rank test. Values were considered statistically significantly different at P < 0.05, P < 0.01, and P < 0.001.All data were analyzed using standard GraphPad Prism version 8.0 software. Quantitative data were represented as means ± standard deviations and obtained from at least three independent experiments. For


RAS mutations occur in approximately 20% of all cancers and given their clonality, key role as driver mutation, association with poor prognosis and undruggability, they represent attractive targets for immunotherapy. We have identified immunogenic peptides derived from codon 12 mutant RAS , which bind to HLA-A*02:01 and HLA-A*03:01 and elicit strong peptide-specific CD8+ T cell responses, indicating that there is an effective CD8+ T-cell repertoire against these mutant RAS-derived peptides that can be mobilized. Alterations in anchor residues of these peptides enhanced their binding affinity to HLA-A*02:01 molecules and allowed generation of CD8+ T cells that responded to target cells pulsed with the anchor-modified and also with the original peptide. Cytotoxic T cells generated against these peptides specifically lysed tumor cells expressing mutant RAS. Vaccination of transgenic humanized HLA-A2/DR1 mice with a long peptide encompassing an anchor-modified 9-mer G12V epitope generated CD8+ T cells reactive to the original 9-mer and to a HLA-A*02:01-positive human cancer cell line harboring the G12V mutation. Our data provide strong evidence that mutant RAS can be targeted by immunotherapy. Cancer immunotherapy is one of the most promising and rapidly advancing cancer treatment modalities –4. The pOne approach to promoting this anti-tumor immune response is cancer vaccination, which triggers specific immune responses to tumor antigens and can promote development of long-term anti-tumor memory . HistoriRecently, genomic sequencing and bioinformatics platforms have enabled rapid identification of neo-antigens expressed by individual patients that are potentially more clinically tractable vaccine targets . Neo-antThe proteins HRAS, KRAS and NRAS are the products of proto-oncogenes that appear mutated in approximately 20% of human cancers . Single ex vivo expanded spontaneous CD8+ T cells recognizing KRAS G12D in an HLA-C*08:02 colorectal cancer patient  patients –13, with patient ; and aut patient . Peptide patient –22.The limited clinical success in inducing long-lasting anti-tumor efficacy suggests a significant opportunity for improvement of mutant RAS targeting strategies. Here, we use a bioinformatics approach , 24 to pUsing the NetMHC 4.0 program that predicts HLA-peptide binding , we idenIn vitro differentiated mature DCs loaded with each individual peptide were used to stimulate autologous CD8+ T cells in four rounds of stimulation. Seven days after the last round of stimulation, CD8+ T cells were challenged with T2 cells or monocytes pulsed with the mutant RAS epitope, its wild type counterpart or left without peptide and IFN-γ production used as a measure of epitope-specific T cell response. All six RAS 10-mer epitopes induced specific CD8+ T cells in the context of HLA-A*02:01  (for 9-mers and 10-mers), position 2 with leucine (L2) (for 9-mers) and position 1 and 2 with a tyrosine and leucine (Y1L2) (for 9-mers). All modifications were predicted to enhance affinity of the peptides for the HLA-A*02:01 Table 1.in vitro differentiated DCs from eight HLA-A*02:01-positive donors were loaded with individual G12V-RAS peptides  and used to stimulate autologous CD8+ T-cells in four rounds of stimulation over four weeks. Seven days after the fourth stimulation, CD8+ T-cells were challenged with the epitopes with which they had been stimulated or with the original epitopes. The original 9-mer induced specific CD8+ T cell response in only one out of eight donors  –35. Vaccin vitro results were confirmed in an in vivo model in which transgenic humanized mice expressing HLA-A*02:01 were vaccinated with a long peptide containing the anchor-modified residue. The immunized mice generated a robust response against the L2-9-mer peptide with CD8+ T cells capable of recognizing the original 9-mer peptide and HLA-A*02:01-positive cancer cells bearing the G12V mutation. These data demonstrate that CD8+ T cells capable of reacting against this 9-mer mutant RAS peptide are present in the repertoire and can be recruited by a high affinity anchor-modified peptide. Additionally, these results strongly indicate that the MHC class I processing machinery of tumor cells is capable of generating and presenting the original G12V 9-mer on their cell surface for recognition by CTLs. Altogether, these results provide sound evidence that this anchor-modified peptide can be used in immunotherapy to treat G12V RAS-positive malignancies. To our knowledge, this is the first demonstration of this approach using a mutated antigen rather than a TAA to generate a CD8+ T cell-based response to cells presenting the original epitope.In contrast to the modifications in position 1 and 1/2, the substitution of valine with leucine in position two of the G12V 9-mer fulfilled all requirements of a viable vaccine candidate, as it enhanced its binding affinity to HLA-A*02:01 and increased its immunogenicity, generating T-cells capable of recognizing the original mutated RAS epitope. Moreover, cytotoxic T cells raised against the L2-9-mer peptide lysed tumor cells positive for HLA-A*02:01 and bearing the G12V mutation. Our To summarize, we showed that RAS peptides covering the six most common mutations were immunogenic in the context of HLA-A*02:01 and HLA-A*03:01. Moreover, modifications in anchor residues of all six RAS mutations enhanced affinity of the peptides to the HLA-A*02:01 molecule. Furthermore, immunogenicity of an HLA-A*02:01-binding 9-mer G12V peptide was greatly enhanced by substituting valine with leucine in position two, resulting in higher affinity of the peptide to the HLA and rendering it more immunogenic without losing specificity. Overall, the best HLA-A*02:01-binding G12V-derived peptides were the original 10-mer and the anchor-modified L2-9-mer. By using these two peptides, CD8+ T cell clones that recognize both 9-mer and 10-mer G12V RAS peptides can be engaged, providing the rationale for their use in a vaccine regimen or adoptive T-cell therapy targeting G12V-positive tumors. Future work is needed to assess the immunogenicity of anchor-modified peptides encompassing the other RAS mutations in the context of HLA-A*02:01, HLA-A*03:01 and HLA-A*11:01.All peptides were custom-synthesized with a purity >98% by ProImmune Ltd  or GL Biochem Ltd . Lyophilized peptides were dissolved in DMSO  at 20 mg/mL and stored at −80°C.2. For the stabilization assays, the cells were washed and incubated in serum-free RPMI medium for 4 hours at 37°C with peptide concentrations ranging from 100 µM . The peptide-loaded stable MHC class I on the cell surface was measured by flow cytometry by staining the cells with fluorochrome-labelled monoclonal antibodies to HLA-A*02:01 , HLA-A*03:01 . The mean fluorescence intensity (MFI) was taken as measure for the peptide stabilizing effect, thereby implicating peptide binding. EC50 values were calculated as the peptide concentration required for half-maximal MFI. For the cell-based competitive binding assay, lymphoblastoid CJO-A cells, homozygous for HLA-A*11:01 were incubated in serum-free RPMI medium for 4 hours at 37°C with peptide concentrations ranging from 100 µM  in the presence of 500 nM of FITC-labelled reference peptide KVFPK(FITC)ALINK . Data was analysed using WinMDi 2.9 , and EC50/IC50 calculation with Graphpad Software Prism 5.04 for Windows .Binding of HLA-A*02:01 and HLA-A*03:01 KRAS peptides was assessed in stabilization assays using TAP-deficient lymphoblastoid T2. For the assessment of HLA-A*11:01 peptides, a cell-based competitive binding assay with lymphoblastoid CJO-A cell line (European Collection of Authenticated Cell Cultures (ECACC), UK) was employed. T2 and T2 cells transfected with HLA-A*03:01 were kindly provided by Dr. Louise Boyle from University of Cambridge and by Professor Peter Cresswell from Yale University, respectively. All cell lines were grown and kept in culture in RPMI medium with 10% FCS at 37°C with 5% COTC)ALINK . The rel2 cell culture flasks in RPMI 1640 GlutaMax culture medium  with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen). For DC differentiation, on day 0 and 4 the cultures were supplemented with 50 ng/mL recombinant human GM-CSF and 50 ng/mL IL-4 (BioLegend). The cultures were maintained at 37°C in humidized atmosphere with 5% CO2. For DC maturation, on day 5 of culture, LPS  at 100 ng/mL was added to the iDCs and the culture continued. The cells were harvested on day 7 and used in T cell stimulation assays.Human DCs were differentiated from monocytic CD14+ precursors as described previously . BrieflyImmunogenicity of mutated RAS peptides was performed using HLA-matched healthy donors’ autologous PBMCs. Briefly, mature DCs were pulsed with individual mutated RAS epitopes at 25 μg/mL and co-cultured with autologous magnetic-sorted CD8+ T-cells  in RPMI with 5% human male AB serum, 10 units/mL penicillin–streptomycin, 2 mmol/L L-glutamine, 1% nonessential amino acid, IL-6 (1000 U/mL) and IL-12 (5 ng/mL). IL-2 (30 U/mL) and IL-15 (5 ng/mL) were added every 2 days. CD8+ T-cells underwent another 3 rounds of stimulation with peptide-pulsed CD14+ monocytes on day 7, 14 and 21. Seven days after the last stimulation, CD8+ T-cells were challenged by overnight incubation with peptide-loaded T2 cells or autologous monocytes. As a control, CD8+ T-cells were incubated with T2 cells or monocytes without peptide. Reactivity of the T cells was determined by intracellular IFN-γ staining measured by flow cytometry. In some experiments, cells were also stained with specific tetramers (BioLegend).For the cytotoxicity assays, the bulk of G12V RAS-stimulated CD8+ T-cells were incubated with peptide-loaded T2 cells or CFPAC-1 cell line. After overnight incubation, cells were stained with fluorochrome-conjugated anti-CD3 (BD), Calcein-AM and Ethidium Homodimer 1 (Invitrogen) to determine cell death. Target cells were gated on the CD3-negative population and cell death was determined by the percentage of cells negative for Calcein and/or positive for Ethidium Homodimer 1.The supernatants of co-cultures of tumor and CD8+ T-cells were collected after overnight incubation and stored at -80°C. IFN-γ levels from the co-cultures were measured by ELISA Max Standard kit (BioLegend) in 96-well microtiter plates according to the manufacturer’s instructions. Results are expressed as pg/mL.® Viability/Cytotoxicity Kit, for mammalian cells according to the manufacturer’s instructions (Invitrogen). Flow cytometry was done using a FACSCalibur  or BD LSRII (Becton Dickinson) flow cytometer. The data were processed with the CellQuest software  and analyzed with the WinMDi 2.9 .For cell surface labelling, cell suspensions were incubated with antibodies or tetramers for 30 min at room temperature. For intracellular staining, cells were fixed and permeabilized using Leucoperm kit (Bio-Rad) in accordance with the manufacturer’s guidelines. Cell viability was analyzed by Ethidium homodimer 1 and calcein-AM double-staining using LIVE/DEAD1-31 peptide (MTEYLKLVVGAVGVGKSALTIQLIQNHFVDE) together with 25 μg of Poly I:C (In vivogen) and 10 μg of Pan-DR peptide AKFVAAWTLKAAA. Control groups received either PBS or only poly I:C plus Pan-DR peptide. One week after the third injection the mice were culled, had their spleen harvested and used in ex vivo stimulation experiments.All mouse studies were carried out under the terms of the Home Office Project Licence PPL 70/6030 and subject to Queen Mary University of London ethical review, according to the guidelines for the welfare and use of animals in cancer research. Transgenic humanized HLA-A2/DR1 mice were obtained from Centre National de la Recherche Scientifique (CNRS), Transgénèse et Archivage d’Animaux Modèles (TAAM)  and were bred and maintained in our animal facility at the Barts Cancer Institute. To test the immunogenicity of anchor-modified G12V RAS peptide, on days 0, 14 and 21, transgenic HLA-A2/DR1 mice were injected subcutaneously in the flank with 100 μg of the 31-mer G12V RASSpleens were harvested from mice, combined with cell culture medium , and single cell suspensions were prepared by mashing the spleen against a 70 μm cell strainer. Cells were resuspended in red blood cell lysis buffer (Sigma-Aldrich), washed in PBS, and the pellet was resuspended in cell culture medium.6) were plated into each well of a U-bottom 96-well plate and restimulated with 10 μg/mL of the indicated peptides. Restimulated splenocytes were incubated overnight in the presence of brefeldin A , and intracellular IFN-γ measured by flow cytometry.Splenocytes . The results were presented as mean ± standard error of the mean (SEM). Differences between groups were analyzed using one-way ANOVA test. Differences with a P<0.05 were considered significant.via the NHS blood and transplant service and all human materials used had written informed consent.This study was reviewed and approved by the London-Westminster Research Ethics Committee (Protocol: 16/LO/1512) for access to healthy donor blood The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Queen Mary University of London ethical review. All mouse studies were carried out under the terms of the Home Office Project Licence PPL 70/6030 and subject to according to the guidelines for the welfare and use of animals in cancer research.RBB and YW conceived of and designed the study. RBB, YL and PL developed the study methodology. RBB and PL conducted experiments and acquired data. RBB, PL and LSCD analyzed and interpreted the data . RBB, PL, LSCD, NL, and YW wrote, reviewed, and/or revised the manuscript. All authors contributed to the final version of the manuscript. YL and NL provided administrative, technical, or material support. YW supervised the study.This work was supported by grants from the Pancreatic Cancer Research Fund (PCRF), grant no to RBB, PL and YW Cancer Research UK , to SL, LSCD and YW.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Despite notable clinical demand, effective treatment for TD has not been established yet. The neural mechanism involving the hyperinhibition of indirect pathway medium spiny neurons (iMSNs) in the striatum is considered one of the main causes of TD. In this study, we focused on adenosine A2A receptors (A2ARs) expressed in iMSNs and investigated whether pharmacological activation of A2ARs improves dyskinetic symptoms in a TD mouse model. A 21-day treatment with haloperidol increased the number of vacuous chewing movements (VCMs) and decreased the number of c-Fos+/ppENK+ iMSNs in the dorsal striatum. Haloperidol-induced VCMs were reduced by acute intraperitoneal administration of an A2AR agonist, CGS 21680A. Consistently, haloperidol-induced VCMs and decrease in the number of c-Fos+/ppENK+ iMSNs were also mitigated by intrastriatal injection of CGS 21680A. The effects of intrastriatal CGS 21680A were not observed when it was concomitantly administered with a β-arrestin inhibitor, barbadin. Finally, intrastriatal injection of an arrestin-biased D2R agonist, UNC9994, also inhibited haloperidol-induced VCMs. These results suggest that A2AR agonists mitigate TD symptoms by activating striatal iMSNs via β-arrestin signaling.Repeated administration of dopamine D TD emerges in about 30% of all patients treated with antipsychotics, and long-term use of D2R antagonists makes TD symptoms irreversible (Tardive dyskinesia (TD) is a neurological symptom characterized by involuntary movements of the face, torso, extremities, and sometimes, the respiratory system . It is cversible . In 2017versible . Consequ1 receptors (D1Rs) while indirect pathway medium spiny neurons (iMSNs) express D2Rs  are expressed in striatal iMSNs  were purchased from Japan SLC . All animals were housed at a constant ambient temperature (22 ± 2°C) on a 12-h light/12-h dark cycle, with free access to food and water.Haloperidol was purchased from Tokyo Chemical Industry , CGS 21680A (an A2AR agonist) from Toronto Research Chemicals , and barbadin  were performed as previously described . HaloperThe guide cannula for microinjection was implanted as previously described . On the Repetitive VCM experiments were performed as previously described . In intrWhen counting VCMs with microinjection, mice were orally administered haloperidol (2 mg/kg/day) for 21 days. On day 22, after the basal VCMs were counted once for 5 min, the mice were randomly assigned to groups receiving the vehicle or test drugs through a cannula that was pre-implanted in their dorsal striatum. After 5 min, VCMs were counted again for 5 min to determine the effect of the injected drug. During the crossover study  for permeabilization and incubated overnight at room temperature with mouse monoclonal anti-c-Fos antibody  and rabbit monoclonal anti-preproenkephalin (ppENK) antibody , followed by incubation with Alexa Fluor 594-labeled donkey anti-mouse IgG  and Alexa Fluor 488-labeled donkey anti-rabbit IgG  for 1.5 h at room temperature in the dark. Images were captured using a confocal fluorescence microscope .+/ppENK+ cells was counted in four separate fields of the dorsal striatum, 0.3 mm anterior to the bregma; each field covered 0.045 mm2. For each batch of confocal images, a uniform background value was set using Adobe Photoshop . The cells showing ppENK-mediated fluorescence signals above the background level were detected, and c-Fos signals co-localized with DAPI signals were counted.The number of c-Fost-test were performed with Prism v9.4.1 . For post hoc tests, Tukey’s multiple comparisons tests were used. P < 0.05 was considered statistically significant. Data are presented as the mean ± standard error of mean (SEM).Statistical analyses using one-way or repeated measures one-way analysis of variance (ANOVA) and two-tailed F2,26 = 13.35, P < 0.001; post hoc Tukey’s test: **P < 0.01, ***P < 0.001).We first identified the effects of repeated administration of haloperidol on spontaneous behavior and neuronal activity in the striatum of mice. The mice were administered haloperidol (1 or 2 mg/kg) daily for 21 days, and 24 h after the last treatment, their facial movements were recorded. Consistent with our previous report , haloper+ and c-Fos+/ppENK+ cells, but not of ppENK+ cells, in the dorsal striatum were significantly decreased in a dose-dependent manner .TD is related to the malfunction of iMSNs within the dorsal striatum . Therefot-test: t(11) = 2.73, P < 0.05]. These results suggest that acute activation of A2AR is effective in inhibiting orofacial dyskinetic symptoms.To determine whether pharmacological activation of A2ARs could mitigate D2R antagonist-induced dyskinesia, we intraperitoneally injected a haloperidol-induced VCM mouse model with CGS 21680A, an A2AR agonist. The number of VCMs was repeatedly measured twice on days 22 and 26 in a crossover test . CGS 216via β-arrestin, resulting in the inhibition of haloperidol-induced dyskinesia. To test this hypothesis, we examined the effects of intrastriatal injection of CGS 21680A and barbadin, a β-arrestin inhibitor . However, barbadin alone did not affect the number of VCMs in mice after repeated administration of haloperidol . Additionally, a bilateral intrastriatal infusion of an arrestin-biased D2R agonist UNC9994 (10 ng/side) suppressed haloperidol-induced VCMs , indicating that β-arrestin signaling in the dorsal striatum facilitates the antidyskinetic effect of A2AR activation.A2ARs activate iMSNs. In iMSNs, A2AR signaling contributes to synaptic plasticity . Moreovenhibitor , on halonhibitor . In the + cells after bilateral infusion with CGS 21680A or co-administration with barbadin . The number of c-Fos+/ppENK+ cells was significantly increased after bilateral infusion with CGS 21680A, whereas this effect of CGS 21680A was abolished by co-administration with barbadin . These results indicate that acute stimulation of A2ARs facilitates the expression of c-Fos protein in the dorsal striatum iMSNs of the VCM model mice, and consistent with the behavior experiments, β-arrestin signaling was required to mitigate the effect of A2AR agonists.Lastly, the neural activity of iMSNs was evaluated after the intrastriatal injection of CGS 21680A, co-administration of CGS 21680A and barbadin, or injection of vehicle in mice that received repeated haloperidol administration . In the +/ppENK+ iMSNs in the dorsal striatum. Intraperitoneal injection of the A2AR agonist, CGS 21680A, acutely decreased haloperidol-induced VCMs. Haloperidol-induced VCMs and decrease in the number of c-Fos+/ppENK+ iMSNs were inhibited after intrastriatal injection of CGS 21680A, whereas this effect was abolished by co-administration with barbadin. Additionally, the arrestin-biased D2R agonist, UNC9994, inhibited haloperidol-induced VCMs.In this study, we characterized D2R antagonist-induced TD-like symptoms and treatment responses in mice. Chronic treatment with haloperidol increased the number of VCMs and decreased the number of c-FosA2ARs are expressed at high levels in the iMSN neurons in dorsal and ventral striatum and play an important role in controlling the striatal function . Thus, t2A-D2 heterodimers positively regulates β-arrestin recruitment to D2Rs, resulting in D2R internalization (via two pathways, namely, A2AR activation followed by increased intracellular cAMP and β-arrestin-mediated decrease in the number of D2Rs. We examined the effects of intrastriatal injection of CGS 21680A and barbadin, a β-arrestin inhibitor, and observed the decrease in haloperidol-induced VCMs. These findings suggest the contribution of β-arrestin-mediated D2R internalization to the reduction of haloperidol-induced VCMs via A2AR stimulation. However, further studies investigating the direct involvement of A2AR-Gs cascade in the induction of iMSN activity are required for understanding the precise therapeutic mechanisms of A2AR stimulation in haloperidol-induced VCMs.The A2AR is a Gs-coupled receptor that activates adenylate cyclase and increases intracellular cAMP. When stimulated, A2ARs enhance the activity of striatal iMSNs by counteracting the Gi/o signaling of D2Rs , 2010. Ilization , and thelization . Thus, A+/ppENK+ iMSNs, which was successfully increased by intrastriatal activation of A2AR. Because the c-Fos gene is known as an immediate early gene that is expressed in response to neural excitation and acts as a transcriptional factor that modulates neural activity (c-Fos gene expression indicated functional changes in iMSNs. However, based on the results of the immunostaining experiments, we could not completely rule out the possibility that repeated administration of haloperidol affected the signaling pathway between neural excitation and c-Fos gene expression, instead of neural excitation itself. Future evaluation of neural excitability will be required to elucidate the underlying mechanisms.Consistent with the hypothesis that A2AR stimulation recovers the neural activity of iMSNs in haloperidol-treated mice, we showed that repeated administration of haloperidol decreased the number of c-Fosactivity , changesin vitro and in vivo studies suggest that A2AR stimulation decreases striatal D2R availability (Previous studies showed that acute administration of haloperidol, but not continuous administration, increases striatal D2R availability and thus upregulates c-Fos expression in the striatum , suggestlability . Taken t2A-D2 heterodimers in the function of D2Rs. Although further studies are needed, activation of β-arrestin signaling via A2A-D2 heterodimers will be a novel therapeutic target for the treatment of D2R antagonist-induced TD.The involvement of β-arrestin signaling was supported by the fact that intrastriatal injection of the arrestin-biased D2R agonist UNC9994 acutely mimicked the antidyskinetic effect. UNC9994 acts as a partial agonist on β-arrestin signaling without affecting conventional G protein pathway . UNC9994In conclusion, we demonstrated that pharmacological activation of A2ARs mitigates dyskinesia induced by long-term administration of D2R antagonists. This study also raises the possibility that β-arrestin recruitment may contribute to the mediation of antidyskinetic effects by stimulating A2ARs.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Kyoto University Animal Experiment Committee.KoN, NA, KaN, HS, and SK designed the research. KoN performed the research. KoN analyzed the data. KoN, NA, and SK wrote the manuscript. All authors contributed to the article and approved the submitted version.


The paucity of SARS-CoV-2-specific virulence factors has greatly hampered the therapeutic management of patients with COVID-19 disease. Although available vaccines and approved therapies have shown tremendous benefits, the continuous emergence of new variants of SARS-CoV-2 and side effects of existing treatments continue to challenge therapy, necessitating the development of a novel effective therapy. We have previously shown that our developed novel single-stranded DNA aptamers not only target the trimer S protein of SARS-CoV-2, but also block the interaction between ACE2 receptors and trimer S protein of Wuhan origin, Delta, Delta plus, Alpha, Lambda, Mu, and Omicron variants of SARS-CoV-2. We herein performed in vivo experiments that administer the aptamer to the lungs by intubation as well as in vitro studies utilizing PBMCs to prove the efficacy and safety of our most effective aptamer, AYA2012004_L.In vivo studies were conducted in transgenic mice expressing human ACE2 (K18hACE2), C57BL/6J, and Balb/cJ. Flow cytometry was used to check S-protein expressing pseudo-virus-like particles (VLP) uptake by the lung cells and test the immuogenicity of AYA2012004_L. Ames test was used to assess mutagenicity of AYA2012004_L. RT-PCR and histopathology were used to determine the biodistribution and toxicity of AYA2012004_L in vital organs of mice.We measured the in vivo uptake of VLPs by lung cells by detecting GFP signal using flow cytometry. AYA2012004_L specifically neutralized VLP uptake and also showed no inflammatory response in mice lungs. In addition, AYA2012004_L did not induce inflammatory response in the lungs of Th1 and Th2 mouse models as well as human PBMCs. AYA2012004_L was detectable in mice lungs and noticeable in insignificant amounts in other vital organs. Accumulation of AYA2012004_L in organs decreased over time. AYA2012004_L did not induce degenerative signs in tissues as seen by histopathology and did not cause changes in the body weight of mice. Ames test also certified that AYA2012004_L is non-mutagenic and proved it to be safe for in vivo studies.Our aptamer is safe, effective, and can neutralize the uptake of VLPs by lung cells when administered locally suggesting that it can be used as a potential therapeutic agent for COVID-19 management.The online version contains supplementary material available at 10.1186/s12985-022-01943-7. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported as a new human pathogen on December 31, 2019, and its human-to-human transmission was the cause for worldwide outbreak that led to the declaration of coronavirus disease (COVID-19) pandemic. SARS-CoV-2 has continued to strain the health-care system for over two years with more than one million new infections and thousands of deaths around the world each day. COVID-19 is a major public health concern and there exists a major issue of health disparity within and between countries .Moreover, SARS-CoV-2 is naturally mutating over time and producing new variants, some of which  proved to be more contagious and destructive than previous strains and are capable of therapeutic escape , ultimatNotably, emerging evidence during the COVID-19 pandemic era highlighted aptamers as promising theragnostic tools to treat COVID-19 infection during the rapid emergence of SARS-CoV-2 variants of concerns . Thus, nHere, we studied the neutralization of AYA2012004_L on the in vivo uptake of VLPs by mouse lung cells when administrated locally by intubation. We also studied efficacy, toxicity, mutagenicity, biodistribution, and inflammatory/immunogenicity characteristics of the same ssDNA aptamer against trimer SARS-CoV-2 S protein that was developed in our laboratory. Collectively, our data shows that AYA2012004_L is efficient, can mitigate the VLPs uptake in vivo, and is nontoxic. AYA2012004_L has a good biodistribution without accumulation in certain organs and is non-mutagenic. The data presented in this study shows that our aptamer has great efficacy in terms of preventing viral uptake in vivo and safety, suggesting that it might be a potential therapeutic agent for COVID-19 management.Aptamer AYA2012004_L was developed by Ayass Bioscience LLC and, together with control aptamers 1 and 2, was purchased from Integrated DNA Technologies . The aptamer sequences are as follows: AYA2012004_L (TAGGGAAGAGAAGGACATATGATTTTGGGCGGGTTGAGGTGGGGGAGGAGGAGGTAGTTAGAGTTGACTAGTACATGACCACTTGA); control 1 (TAGGGAAGAGAAGGACATATGATGCGCGGTCCCGATTTGGTGTAAAATTCCCTCAGCCCTACATTGACTAGTACATGACCACTTGA); control 2 (TAGGGAAGAGAAGGACATATGATCGGTGTAGACCCTGGAGAGGGTGTGCATATGTCCGGTTGCTTGACTAGTACATGACCACTTGA). Antibodies specific to mouse CD45 PE (BioLegend), anti-human ACE2 APC , goat IgG APC , and Fixable Viability Dye eFluor 450 (Invitrogen # 65-0863-14) were used. FluoroFix Buffer (# 422101) was purchased from BioLegend. COVID-19 S Protein/(GFP)-(6His) VLP (#VLP001) was purchased from Gentarget, Inc. RPMI 1640 (Thermo Fisher Scientific) was supplemented with 10% FCS (Gemini), 50 u/ml penicillin, 50 mg/ml streptomycin, 2 mM L-glutamate, 13 nonessential amino acids, 1 mM sodium pyruvate (SigmaAldrich), and 25 mM HEPES (Thermo Fisher Scientific). Dulbecco’s Phosphate Buffered Saline  was purchased from ThermoFisher Scientific. Cell Stimulation 500ul of DPBS buffer containing 1 mM MgClFemale B6.Cg-Tg(K18-ACE2)2Prlmn/J mice were randomly divided into the experimental groups for the following studies. First, to determine the in vivo dose of AYA2012004_L that is required to neutralize the uptake of 1y others –19. BrieMice were euthanized by using a 30–70% per minute displacement of chamber air with compressed COFemale wild-type C57BL/6J (n=3) and BALB/cJ (n=3) mice were euthanized and lung tissues were harvested and processed for single cells. Isolated lung cells cultured in cRPMI media were stimulated with or without aptamer AYA2012004_L  and LPS (200 ng/ml), ODN 1826 (10 uM), or LPS (100 ng/ml)+ODN 1826 (5 uM) as controls. Cells were incubated at 37 Human PBMCs were isolated from buffy coats (Carter BloodCare) by density-gradient centrifugation using Ficoll-Paque Plus . Human PBMCs in cRPMI media were stimulated with or without aptamer, AYA2012004_L  and LPS (200 ng/ml), ODN 1826 (20 uM), or LPS (100 ng/ml)+ODN 1826 (10 uM) as controls. After 24 and 48 hrs incubation, cell supernatant was collected by centrifugation at 200g for 3 min and cytokine levels were assessed using the LEGENDplex Human Inflammation Panel 1  according to manufacturer’s protocol. Soluble analytes were acquired using flow cytometry and analyzed with BioLegend’s LEGENDplex™.Xenometrix Ames MPF PENTA I - with S9 and Positive Controls kit was purchased from Aniara Diagnostica. The assay was performed in liquid media in 24-well plates during sample exposure and in 384-well plates for revertant growth and scoring. Growth, exposure, and indicator media, as well as S. typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli strains wp2 [pKM101] and wp2 uvrA, were included in the kit from Xenometrix. The test procedure described in the “Ames MPF Instruction for use” was followed. Briefly, bacteria were grown overnight, diluted in Exposure Medium, containing sufficient histidine (or tryptophan for E. coli) and exposed to 6 concentrations of the AYA2012004_L  as well as positive and a negative control. Bacteria were incubated with AYA2012004_L and controls in 24 well-microwell plates for 90 minutes at 37 Female wild-type C57BL/6J mice  were used for the study. All mice were randomly divided into the following groups: 1, 4, 8, 12, 24, and 48 hrs of treatment time. 200 ul of either DPBS buffer containing 1 mM MgClAptamer AYA2012004_L was quantitated by PCR using forward primer 5’-TAGGGAAGAGAAGGACAATGAT-3’, reverse primer 5’-TCAAGTGGTCATGTACTAGTCAA-3’ and probe 5’-[6FAM]CTCCTCCTCCCCCACCTCAA[BHQ1] ordered from Sigma-Aldrich. Quantitative PCR was performed on StepOnePlus real-time PCR System (Applied Biosystems) with TaqPath 1-Step Master Mix, CG . Twenty microliter reactions included 5 ul eluted total DNA, and the concentrations of primers and probe were 0.3 uM and 0.15 uM respectively. PCR was performed as follows: polymerase activation –95 Female wild-type C57BL/6J mice were randomly divided into study groups treated with 200 mentclass2pt{minimIsolated single cells from mice lungs were further stained. Briefly, As shown in the schematic representation of experimental design Fig. p values <0.05 were considered statistically significant.Significance was determined in Prism 9.3.0 (GraphPad Software) using the unpaired Student t test for two-group comparisons and the one-way ANOVA test (Dunnett’s multiple comparisons test or Tukey’s multiple comparisons test). The In this study, we used VLPs that have the full-length SARS-CoV-2 S protein expressed/presented on the surface of lentiviral particle to mimic the coronavirus. The VLPs are packaged with Green Fluorescent Protein (GFP) plasmid as a reporter signal. Our experiments show that when VLPs are uptaken by the lung (CD45-) cells that express hACE2  or the tryptophan (E. coli) operon, rendering the bacteria incapable of producing the corresponding amino acid and therefore the bacteria cannot grow in histidine- or tryptophan- deficient media. When a mutagenic event occurs, base substitutions or frameshifts within the gene may cause a reversion to amino acid prototrophy and everted bacteria will grow in selective media that is depleted of histindine and tryptophan. TA98 and TA1537 strains are used for the detection of frameshift mutations, and T100 and TA1535 for base pair substitutions . TA98 anWe explored the biodistribution of AYA2012004_L in mice tissues, including lungs, kidneys, livers, bladders, urine, spleens and hearts at 1, 4, 8, 12, 24, and 48 hours post-intubation. For that purpose, we extracted total DNA from 25 mg tissue of each organ or 25 ul of urine at the indicated time point and performed real-time PCR assay to detect AYA2012004_L aptamer  in hACE2 Transgenic mice. Lung cells were isolated from female B6.Cg-Tg(K18-ACE2)2Prlmn/J mice, female C57BL/6J and female BALB/cJ. Cells were stained with Fixable Viability Dye, anti-mouse CD45, anti-human ACE-2 and/or goat IgG. Cells were gated based on isotype staining (Goat IgG). Expression of hACE2 receptor on mice lung cells was assessed by flow cytometry analysis. Fig. S2. S protein mediated VLP uptake by lung cells of hACE2 Transgenic mice. 1x106 VLP was administered to female B6.Cg-Tg(K18-ACE2)2Prlmn/J mice in 200 of DPBS buffer containing 1 mM MgCl2 via bronchoalveolar lavage on 2 hrs intervals as 1, 3, and 6 injections. Mice were sacrificed after 24 and 96 hrs, and lung cells were analyzed by flow cytometry to detect GFP signal from VLP in FITC channel. Individual dots represent data generated with cells from different mice. Fig. S3. Expression of S protein VLP uptake by live lung cells (CD45- cells) in hACE2 Transgenic mice. Female B6.Cg-Tg(K18-ACE2)2Prlmn/J mice were administered with 1x106 S protein VLPs in 200 of DPBS buffer containing 1 mM MgCl2 via bronchoalveolar lavage on 2 hrs interval as 1, 3 and 6 injections. Mice were sacrificed after 24 and 96 hrs; and cells were stained with Fixable Viability Dye, anti-mouse CD45, anti-human ACE2 and/or goat IgG. Cells were gated based on isotype staining (Goat IgG). Expression of hACE2 receptor and S protein VLP uptake by mice lung cells were assessed by flow cytometry analysis. Fig. S4. Aptamer induced dose-dependent neutralization of S protein VLP uptake by lung cells of hACE2 transgenic mice. A Schematic experimental design of mice treatment with or without AYA2012004_L. B A representative FACS plot showing the neutralization effect of AYA2012004_L on the S protein VLP uptake by lung cells. Cells were gated based on DPBS treated group as control. C Summarized data for the frequency of GFP+ cells (uptake of S protein VLP) and D Summarized data for the percent uptake of S protein VLP by lung cells under every treatment condition. Uptake of S protein VLP by lung cells was calculated according to the formula: [(frequency of GFP+ cells of test group − frequency of GFP+ cells of control group)/(frequency of GFP+ cells of S protein VLP treated group − frequency of GFP+ cells of control group)] × 100. Individual dots represent data generated with cells from different mice (n=3). Data are expressed as mean ± SD. The p values were determined with one way ANOVA test (Dunnett’s multiple comparison test). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Fig. S5. Gating strategy for GFP-VLP signal. Lungs were processed for single-cell isolation. Single cells from the lungs were gated for GFP signal (uptake of VLP-GFP) by the CD45-Live cells. Lung cells were stained with anti-mouse CD45 PE and fixable viability dye. Stained cells were acquired for three colors flow cytometry. Cells were gated first on FS-A and SS-A and then gated for single cells that were further gated for CD45- Live cells (Lung cells). Live lung cells (CD45- Live cells) were then gated from GFP. This gating strategy is applied in Figure 1 and Figure S5. Fig. S6. Detection of AYA2012004_L in mouse vital organs and urine by RT-PCR. C57BL/6J mice were administered with 200 l of DPBS buffer containing 1 mM MgCl2 in the absence or presence of AYA2012004_L (10 M) via bronchoalveolar lavage. Mice were then sacrificed at 1, 4, 8, 12, 24 and 48 hrs post-treatment. Lungs, livers, kidneys, hearts, spleens, bladder and urine were collected and processed for nucleic acid isolation. AYA2012004_L was detected in mice tissues by real time polymerase chain reaction (RT-PCR) assays. Grey color area is the cut off ∆Ct value in the organs of AYA2012004_L that demonstrates 0.02x109 number of aptamer molecules per mg total DNA as calculated. The data are mean±SD of two independent experiments. Fig. S7. A schematic experimental design for histopathology analysis of mice tissues administered with 200 ul of DPBS buffer containing 1 mM MgCl2 in the absence or presence of AYA2012004_L (5 mM) or ODN 1826 (20 g) at day 1 via bronchoalveolar lavage. Mice body weight were measured one day before treatment (on day 0) and on day 44 (24 hrs before study termination). On day 45, mice were euthanized, and tissues were harvested and processed for Haemotoxylin and Eosin (H&E). Table S1. Assessment of the mutagenic potential of AYA2012004_L.