However, allo-HSCT leads to graft-versus-host disease (GVHD), a severe and often lethal immunological response, in the majority of transplant recipients. Current therapies for GVHD are limited and often reduce the effectiveness of allo-HSCT. Therefore, pro- and anti-inflammatory factors contributing to disease need to be explored in order to identify new treatment targets. Purinergic signalling plays important roles in haematopoiesis, inflammation and immunity, and recent evidence suggests that it can also affect haematopoietic stem cell transplantation and GVHD development. This review provides a detailed assessment of the emerging roles of purinergic receptors, most notably P2X7, P2Y Blood cancers are a highly complex group of disorders that arise from the malignant transformation of cells of the blood, bone marrow or lymphatic system. These malignancies account for approximately 10% of all cancers worldwide . Allo-HSPurinergic signalling is a form of cell-to-cell communication involving extracellular nucleosides and nucleotides, and their respective cell surface receptors, with roles in health and disease . Recent Allo-HSCTs were first performed over 50 years ago ,9 and thA major therapeutic benefit of allo-HSCT is the GVT effect. GVT was first demonstrated in a murine model where allo-HSCT eradicated residual leukaemia but also caused a fatal “wasting syndrome” characterised by diarrhoea , now rec+ T cells are the main drivers of the GVT effect [Allogeneic mouse models are commonly used to study the GVT effect and involve the transplantation of bone marrow or splenocytes from C57BL/6 mice to BALB/c mice, followed by transplantation of cancer cells such as A20 or BCL1 murine lymphoma cells ,17. ThesT effect ,20.Despite the life-saving benefits of the GVT effect, T cells that are transfused with the graft can also react against tissues of the genetically different recipient causing GVHD, which affects mainly the skin, liver, and gastrointestinal tract but may also target other organs . The maj+ and CD8+ T cells are the main mediators of GVHD [Acute GVHD, hereafter referred to as GVHD (unless stated otherwise), has a complex pathophysiology, which initially begins with damage to host tissue by the myeloablative therapy used in allo-HSCT. This causes the release of damage-associated molecular patterns, including adenosine 5′-triphosphate (ATP), which promote the maturation of host dendritic cells (DCs) through up-regulation of MHC class I and class II molecules . Host DC of GVHD ,5, there of GVHD ,30 and m of GVHD . Convers of GVHD ,33. Natu of GVHD ,35 or pa of GVHD ,37 roles of GVHD . As a reAs for the GVT studies above, allogeneic mouse models have been the main method used to investigate GVHD, providing vast insight into the pathophysiology of the disease . These mscidIl2rgnullPrkdc (NSG) and NOD.Cg-scid Il2rgtm1SugPrkdc (NOG). These strains are near-identical, except the former encodes a complete null mutation in the Il2rg gene and the latter an Il2rg gene mutation giving rise to a non-functional IL-2 receptor [+ and CD8+ T cell-mediated GVHD [This limitation is partially overcome with the use of humanised mouse models of GVHD, which involve the transfer of human immune cells, or tissue, into immunodeficient mice . This imreceptor . These mted GVHD ,42. GVHDted GVHD ,44,45. Tted GVHD ,47 or T ted GVHD ,49. Howe1, A2A, A2B and A3) [1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14) [Purinergic signalling is a form of cell-to-cell communication comprising a network of extracellular nucleotides and nucleosides, cell surface receptors, ecto-enzymes and nucleotide release pathways . Puriner and A3) . P2 rece and A3) . P2Y recd P2Y14) ,55.Cells release nucleotides and nucleosides by specific  and non-specific pathways (cell stress and cell death) . The avaThis review focuses on the role of purinergic signalling in GVHD including the use of pharmacological agents and genetic approaches to explore this role. However, purinergic signalling also has multiple roles in allo-HSCT, which will be discussed briefly first. Purinergic signalling modulates haematopoiesis and HSCs impacting cell homeostasis, differentiation, mobilisation and death . P2RX7 gene  show an increase in CD34+ HSC mobilisation [14 has been shown to modulate HSCs under stressful conditions [high Tregs in the bone marrow niche can control HSC quiescence, and both CD150high conventional CD4+ T cells and Tregs can modulate HSCs via CD73 [wt) mice [2A activation [A link between extracellular nucleotides such as ATP and mobilisation of HSCs has been shown with the P2 receptors and NLRP3 inflammasome being involved in the mobilisation of HSCs to the peripheral blood . In partlisation . Furtherlisation ,66. Uridlisation  and P2Y1nditions . CD39 annditions . Adenosivia CD73 . CD73 knwt) mice . Therefotivation ,70.Purinergic signalling also plays an important role in allo-HSCT. Prior to transplant, granulocyte-colony stimulating factor (G-CSF) mobilisation of HSCs to peripheral blood makes HSCs easily accessible and improves engraftment . HoweverATP and its analogues can impact HSC differentiation and engraftment, and play a role in trafficking of HSCs to bone marrow niches following allo-HSCT . PannexiENTPD1) or CD73 (NT5E) may establish important predictive or prognostic markers for allo-HSCT outcomes. Donor and recipient SNP variants in the NLRP3 gene have been examined in HLA-identical sibling allo-HSCT, with only the donor rs10925027 intronic TT genotype being associated with disease relapse [P2RX7 SNPs in both donor and recipients of allo-HSCT have shown limited effects on allo-HSCT outcomes. A small cohort showed that the loss-of-function (LOF) rs3751143 P2RX7 SNP (E496A) in homozygous doses was associated with poorer overall survival [P2RX7 SNPs in allo-HSCT donors and recipients [P2RX7 SNP in homozygous dosage may be associated with overall survival, while the rare LOF rs1653624 P2RX7 SNP (I568N) may be associated with reduced survival due to infection-related deaths. These studies indicate that P2RX7 (P2X7) SNPs are unlikely to be biomarkers or predictors of allo-HSCT outcome. To the best of our knowledge there are no reports examining human donor and recipient ENTPD1 (CD39), NT5E (CD73) and ADORA2A (A2A) genotypes and allo-HSCT outcomes in the clinical setting. Finally, it should be noted that purinergic receptors and molecules play emerging roles in blood cancer cells [Identifying SNPs in genes encoding the NLRP3 inflammasome complex, P2X receptors, CD39  and polyoxotungstate-1 (POM-1) used in studies of GVHD, have not been completely defined. APCP is commonly regarded as an inhibitor of CD73, with in vitro enzymatic data showing it does not impair CD39 . In contnic mice . APCP canic mice  further nic mice  can alsonic mice , again mA further limitation to past studies of purinergic signalling in GVHD has been the use of the compounds above in the absence of routine clinical GVHD therapies including corticosteroids and other drugs. However, the interaction of these clinical drugs with purinergic pathways involved in GVHD is largely unknown but remains a possibility. In this regard, the corticosteroid prednisone has been shown to reduce both P2X7 in PBMCs from a patient with the inflammatory disorder Schnitzler’s syndrome  and P2X3P2XR7 SNPs. Together the evidence indicates that the ATP/P2X7 signalling axis initiates pro-inflammatory effects by activating host DCs and causing dysfunction and/or destruction of cells with suppressor activity.There is increasing evidence that ATP and P2X7 play important roles in GVHD development. This evidence comes from studies examining the effects of ATP degradation, P2X7 expression in murine and human GVHD, P2X7 receptor blockade, and comparisons in P2X7 knockout mice or humans with Studies of allogeneic and humanised mouse models indicate a role for extracellular ATP in promoting GVHD . ATP is P2RX7 expression is increased in the PBMCs of human patients with GVHD, compared to allo-HSCT recipients without GVHD or healthy controls [P2rx7 expression is increased in the liver, spleen and thymus of allogeneic mice with GVHD [mir-188 expression, as deficiency of this microRNA results in decreased P2rx7 in murine DCs, corresponding to decreased GVHD in mir-188 knockout recipients [P2rx4 is also increased in the gut and skin of humanised mice with GVHD [The majority of studies to date indicate that P2X7 is the major P2X receptor involved in GVHD progression following ATP release. Human controls . Whilst ith GVHD ,114, andith GVHD . Moreoveith GVHD . This incipients . Murine ith GVHD , but a rPharmacological blockade of P2X7 reduces GVHD in both allogeneic and humanised mouse models of disease . The P2X−/−P2rx7 and wt mice as either the source of donor cells or recipient mice indicated that host, but not donor, P2X7 contributes to GVHD progression in an allogeneic mouse model [P2rx7−/− or wt mice demonstrated that P2X7 in the host haematopoietic system, but not non-haematopoietic tissues, influences GVHD in allogeneic mice [P2rx7−/− haematopoietic system including P2rx7−/− DCs had reduced disease compared to mice with a host P2rx7−/− haematopoietic system but wt DCs, indicating that P2X7 on host DCs is contributing to GVHD development [Genetic studies in mice confirm that P2X7 is involved in GVHD progression and provide further insight to the potential mechanism of this receptor in this disease. Comparisons of se model . Furthereic mice . Finallyelopment .P2RX7 SNPs in mice and humans. NSG mice injected with PBMCs from donors with either a LOF or gain-of-function (GOF) P2RX7 haplotype display similar GVHD development [P2RX7 genotype did not influence rates or severity of GVHD in allo-HSCT recipients [The importance of host P2X7 in GVHD is supported by studies of human elopment . Likewiscipients . Converscipients .+ T cells in an IL-2 dependant manner [+ T cells [The above studies indicate that host P2X7 is important in GVHD development however, they do not exclude the potential contribution of donor P2X7 influencing disease. P2X7 activation can promote the activation and proliferation of CD4t manner  and can  T cells ,122, thu T cells . This ma T cells  and huma T cells  mouse mo T cells , however T cells . Thus, tIn conclusion, increases in extracellular ATP during GVHD lead to P2X7 activation on host APCs, which promotes cytokine release and donor T cell activation, with additional P2X7 activation on T cells directly promoting their proliferation and survival. Further, activation of P2X7 on donor Tregs and MDSCs may lead to reduced suppression of effector cells. Therefore, P2X7 remains a potential clinical target for treatment of GVHD.2, and to a lesser extent P2Y12, in the pathogenesis of GVHD [2 expressing cells, mostly myeloid cells, are increased in the intestinal tract of allo-HSCT recipients with GVHD, with the presence of P2Y2-expressing cells associated with increased intestinal GVHD severity [P2ry2−/− recipient mice, but not P2ry2−/− donor cells, displayed reduced clinical GVHD and increased survival, which corresponded to reduced histological GVHD, serum IL-6 concentrations and other inflammatory molecules compared to wt recipient mice [2 in GVHD was found to be more dependent on the host haematopoietic system than the non-haematopoietic system. The role of P2Y2 in the host haematopoietic system was due to its expression on inflammatory monocytes but not DCs [2 in the host non-haematopoietic system was hypothesised to be due to reduced P2Y2-mediated apoptosis of enterocytes , with apoptosis of these cells reduced in P2ry2−/− host mice compared to wt host mice following allo-HSCT [In addition to P2X7 a small number of studies implicate P2Y of GVHD ,115. P2Yseverity . Notablyent mice . Using b not DCs . Whilst llo-HSCT .2 may also contribute to GVHD progression by mediating the migration of DCs to sites of inflammation and ATP release [mir-188−/− DCs, which cause less severe GVHD in an allogeneic mouse model, display reduced amounts of P2ry2 and P2ry12 compared to wt DCs [mir-188−/−, P2ry2−/− and P2ry12−/− DCs all display impaired in vitro migration compared to wt DCs [2 or P2Y12 in DC migration in GVHD in vivo is lacking.P2Y release , but as  release . mir-188o wt DCs . Whilst o wt DCs , evidenc2 can limit GVHD progression by mediating migration of Tregs to sites of inflammation and ATP release. Co-injection of donor conventional T cells with donor P2ry2−/− Tregs abrogates protection against GVHD afforded by the co-injection of donor conventional T cells with donor wt Tregs in an allogeneic mouse model [wt or P2ry2−/− Tregs reduced the expansion of donor conventional T cells to a similar extent in vivo, suggesting that the role of P2Y2 may be in directing Tregs to GVHD tissues to reduce inflammation at these sites. Thus, given the opposing roles of P2Y2 in DCs and Tregs in GVHD, systemic targeting of this receptor may be limited. To the best of our knowledge, no studies have reported the use of P2Y2 or P2Y12 antagonists to prevent GVHD in vivo.Conversely, P2Yse model . Notably2 in GVHD is in relation to dry eye disease, which is often associated with GVHD in allo-HSCT recipients [2 activation is well known to induce the shedding of various cell surface molecules including tumour necrosis factor (TNF) receptor 1 (TNFR1) [2 agonist diquafosol, an effective treatment of dry eye disease [2 within the eye may limit the inflammation associated with dry eye disease in people with GVHD.A further role for P2Ycipients . P2Y2 ac (TNFR1) . In the  (TNFR1) . Moreove disease , increas disease . Collect+ and CD8+ T cell numbers and serum IFN-γ and IL-6 concentrations and mouse mortality. As noted above, APCP also blocks CD39 [+ and CD8+ T cell numbers, serum IFNγ and IL-6 concentrations, histological damage in target organs, and mortality in allogeneic mouse models of GVHD [hi Treg cells exhibit significantly higher Entpd1 (CD39) and Nt5e (CD73) mRNA expression than CD150- Tregs and mediate their suppressive effect through adenosine production, reducing histological GVHD and mortality, and promoting HSC expansion in an allogeneic mouse model of GVHD [As introduced above, ATP released at the sites of damage in allo-HSCT recipients promotes inflammation, but its degradation by apyrase can prevent GVHD  suggesticks CD39 , therefo of GVHD ,112 supp of GVHD .scidPrkdc mice reduced histological damage, cytokine production by human T cells, and mouse mortality, however these effects were lost when these cells were incubated with the CD39 antagonist POM-1 prior to transplantation [ENTPD1 gene (encoding CD39), which results in higher proportions of CD39+ Tregs and greater suppressive capacity [Further evidence for a role of CD39/CD73 in GVHD arises from studies using humanised mice. Transfer of human gingiva-derived mesenchymal stem cells into humanised NOD.Cg-antation . Blockadantation . Finallycapacity , had worcapacity .2A to limit GVHD progression. The role of A2A in GVHD was originally shown in allogeneic mouse models of this disease  increased serum TNFα, IFN-γ and IL-6, CD4+ and CD8+ T cell numbers, and worsened mortality [2A in the host increased CD4+ and CD8+ T cell numbers [2A activation in limiting GVHD progression, treatment with the A2A agonist ATL-146e increased serum IL-10, and reduced serum IFN-γ and IL-6 [+ and CD8+ T cell numbers, decreased T cell infiltration into GVHD tissue, and reduced both weight loss and mortality in an allogeneic mouse model of GVHD. ATL-146e, as well as the other A2A agonists ATL-370 and ATL-1223, also increased donor derived Treg cells in both the skin and colon in this same model [Adenosine produced by CD39/CD73 mediates its effects largely through activation of A disease . The nonortality . Additio numbers ,112, butand IL-6 . Furtherme model .2A in GVHD has been found using humanised mouse models. Human bone marrow derived mesenchymal stromal cells reduced IFN-γ- and TNF-α-producing leukocytes and reversed clinical disease in a humanised mouse model [2A antagonist ZM241385 (from GVHD onset until endpoint), which prevented human T cell suppression, worsened disease, and reduced survival [2A agonist CGS21680 (days −2–11) had confounding effects in these mice, appearing to worsen disease by increasing weight loss and serum human IL-6 concentrations, but also potentially alleviating disease severity by reducing leukocyte infiltration into the liver and serum human TNFα concentrations [Further evidence for the role of Ase model . Howeversurvival . Contrarsurvival . While itrations .2A have pro- and anti-inflammatory effects, respectively, during GVHD, whilst CD39/CD73 exerts anti-inflammatory effects through the degradation of extracellular ATP and generation of extracellular adenosine. P2Y2 receptors play opposing roles on different cell types, and thus, may not be well-suited as a therapeutic target. Blockade of P2X7 or activation of A2A present promising therapies for future GVHD treatment, but further studies in both allogeneic and humanised mouse models of this disease, as well as patients with GVHD, are required before these molecules can be targeted clinically.The current review has briefly described how allo-HSCT is used in the treatment of haematological malignancies, reconstituting the immune system and providing a GVT effect. However, allo-HSCT also leads to GVHD, a severe immunological response against various tissues in the transplant recipient. Purinergic signalling is a modulator of HSC homeostasis, differentiation, mobilisation and death, and as such, plays a role in allo-HSCT. Furthermore, current evidence suggests that the purinergic system plays a variety of roles in GVHD progression  and as s


Influenza virus surface glycoproteins represent the main targets of the immune system during infection and vaccination. Current influenza virus vaccines rely mostly on the hemagglutinin, requiring a close match between the vaccine and circulating strains. Recently, the neuraminidase (NA) has become an attractive target; however low immunogenicity and stability in vaccine preparations remain an obstacles. Here, we took advantage of the hypervariable stalk domain of the NA to introduce cysteines at different positions and to produce more stable multimeric forms of the protein. We generated 11 N1 constructs and characterized the proteins by performing sodium dodecyl sulfate polyacrylamide gel electrophoresis and by testing their enzymatic activity and representation of antigenic epitopes. Moreover, we evaluated their potential to induce a protective immune response in vivo and characterized the polyclonal antibody responses of immunized mice. We observed that the introduction of cysteines at certain positions led to the formation of stable N1 dimers, which are capable of inducing a strong antibody response characterized by neuraminidase inhibiting activity and protection of mice from high dose viral challenge. Overall, our results provide evidence for the feasibility of introducing stalk modifications to enhance the stability and immunogenicity of NA-based recombinant antigens. Orthomyxoviridae family. They consist of enveloped particles carrying single-stranded negative sense RNA. The genome of the influenza A viruses consists of 8 segments that encode at least 11 structural and non-structural proteins [Influenza viruses belong to the proteins . These vproteins , the NA proteins . The infproteins ,6,7,8. Tproteins . A singlproteins . Throughproteins , hence mproteins . At the proteins . Consequproteins .The viral surface proteins represent the main targets of the immune system during infection and vaccination. Although the HA has been historically targeted because antibodies against it can neutralize incoming virus, recent studies point towards the NA as an attractive antigen for induction of protective immune responses ,8,16,17.Even though the stalk domain of the NA is variable and can differ significantly even within one subtype, it contains conserved structural features, including a specific cysteine residue and a potential glycosylation site ,26. ThesIn nature, the process of NA tetramerization is mediated partially by the transmembrane domain, as well as by interactions of the protomers in the head domain , but mimSpodoptera frugiperda) insect cells were grown in Trichoplusia ni medium–Fred Hink  insect cell medium supplemented with 1% penicillin–streptomycin antibiotics mix , 0.1% pluronic F-68 , and 10% fetal bovine serum . For passaging of the baculoviruses in Sf9 cells, TNM-FH insect medium  was used. BTI-TN-5B1-4  cells were passaged in serum-free Express Five insect cell medium (Gibco) containing 1% penicillin–streptomycin and 1% L-glutamine (Gibco). The challenge virus A/Singapore/GP1908/2015 (H1N1) was grown in 10-day-old embryonated chicken eggs . Viral titers were determined by performing a standard plaque assay as previously described [Sf9 (escribed . To puriThe recombinant N1 constructs (A/Michigan/45/2015 H1N1) were expressed using the baculovirus expression system . We desi® TGX™ Precast Protein Gels, BioRad). Gels were stained with Coomassie blue (ThermoFisher) for 1 h at RT and de-stained with distilled water to visualize the proteins.To determine protein integrity, 1.5 ug of the respective recombinant N1 mutant protein was mixed 1:1 with 2× Laemmli loading buffer (Bio-Rad). For reducing conditions, loading buffer was supplemented with 5% beta-mercaptoethanol. To assess the extent of protein multimerization, samples were treated with the crosslinker bis-sulfosuccinimidyl suberate  according to the manufacturer’s instructions. Bovine serum albumin (BSA) was used as a monomeric control and recombinant A/Michigan/45/2015 N1 protein containing only the globular head domain and the VASP tetramerization domain was used as control for tetramerization. Samples were heated at 95°C for 15 min prior to loading them on a sodium dodecyl sulphate polyacrylamide gel  were coated with 50 µL/well of recombinant protein (2 µg/mL) or purified virus (6 µg/mL) in phosphate-buffered saline (PBS)  overnight at 4°C. Plates were washed 3 times with PBS plus 0.1% Tween 20 (TPBS) using an automated plate washer , then blocked with 100 µL/well of TPBS containing 3% non-fat dry milk  for 1 h at room temperature (RT). The blocking solution was removed and monoclonal antibodies (mAbs) diluted in 1%-milk TPBS were added to the plates . To assess whether the recombinant N1 mutant proteins were correctly folded and properly displayed antigenic determinants, two previously described human monoclonal antibodies were used for binding assays: 1G01, a broad anti-NA antibody; and 1000-1D05, an N1-specific monoclonal antibody . As a neo-phenylenediamine dihydrochloride (OPD) developing solution (Sigma-Aldrich). The reaction was stopped by adding 50 µL/well of 3 M hydrochloric acid (HCl). The plates were read using a Synergy H1 hybrid multimode microplate reader  at an optical density of 490 nm. The data were analyzed using GraphPad Prism 7 software and values were expressed as the area under the curve (AUC).For serum ELISAs, samples were initially diluted (1:50) in 1%-milk TPBS and further serially diluted (1:3). The serum from mice vaccinated with an irrelevant protein was used as negative control and the anti-NA mouse monoclonal antibody 4A5 was used as positive control. Plates containing the mAbs or sera were incubated for 2 h at RT, followed by three washes with 100 µL/well of TPBS. The respective secondary antibody consisting of anti-mouse IgG heavy and light chain peroxidase-conjugated  or anti-human IgG Fab-specific horseradish-peroxidase (HRP) conjugated (Sigma-Aldrich) antibody was diluted 1:3000 in 1% milk/TBPS and added to the plates (100 µL/mL) for 1 h at RT. Plates were washed three times with TBPS (100 µL/well) and incubated at RT with 100 µL/well of SigmaFast The NA enzymatic activity was determined by using the NA-Star™ Influenza Neuraminidase Inhibitor Resistance Detection Kit (ThermoFisher). Briefly, the recombinant N1 mutant proteins were diluted to a starting concentration of 10 µg/mL then serially diluted (1:3). The assay was performed according to the manufacturer’s instructions. As a positive control, recombinant A/Michigan/45/2015 N1 containing the VASP tetramerization domain was used.50) to every well. Serum–virus plates were incubated at RT for 1 h 45 min while shaking. During this time, the fetuin-coated plates were blocked for 1 h at RT with 5% BSA in PBS and washed 3 times with TPBS. The serum–virus mix was added to the fetuin-coated plates (100 µL/well) and plates were incubated for 2 h at 37 °C. After 3 washes with TPBS, plates were incubated for 1 h 45 min with 100 µL/well of 5 µg/mL peanut agglutinin (PNA) conjugated to HRP (Sigma-Aldrich). Plates were washed 3 times with TPBS, then SigmaFast OPD developing solution was added to the wells (100 µL/well). After 7 min of incubation, the reaction was stopped by adding 50 µL/well of 3 M HCl. The optical density (OD) was measured at 490 nm using a Synergy 4 plate reader (BioTek). The data were analyzed by using GraphPad Prism 7 software and values were expressed as percentages of inhibition.The NI assay was performed as previously described . Brieflyn = 5 per group) with 3 µg of recombinant NA protein mixed 1:1 with AddaVax . The negative control group received 3 µg of irrelevant B/Malaysia/2506/04 HA protein via the same route. Three weeks post-prime, the mice were bled and boosted with the same antigen dose. Four weeks post-boost, the mice were bled and challenged with 25 times the 50% lethal dose (LD50) of A/Singapore/GP1908/2015 H1N1 virus diluted in PBS. For the second experiment, mice were vaccinated following the same regimen but only using the AA46-388 (T48C + N50C) and AA46-388 constructs. Subsequently, mice were challenged with 100 times the LD50 of A/Singapore/GP1908/2015 H1N1 virus. Survival and weight loss were monitored in both experiments over 14 days. Mice were euthanized if they lost 25% of their respective initial weight.Blood samples were obtained by sub-mandibular bleeding. Female 6–8 week old BALB/c mice  were administered intramuscularly  structure is needed to induce protective antibody responses . HoweverWe analyzed the integrity and conformation of all of the constructs by running SDS-PAGE gels under reducing and non-reducing conditions and after crosslinking with BS3 B–D. UndeTo further characterize the conformation and functionality of the recombinant NAs, we tested binding of two anti-NA monoclonal antibodies, which had been previously reported: 1G01, a broad anti-NA mAb; and 1000-1D05, an anti-N1 specific mAb (35). No significant differences were observed in the mAb binding profiles for the different constructs A,B. To ain vivo, we administered mice with the recombinant NAs generated in a prime boost vaccination regimen and challenged them with 25 times the LD50 of A/Singapore/GP1908/2015 (H1N1) virus. We observed different levels of protection after vaccination; mice receiving AA46-388, AA46-388 (N50C), AA46-388 (T48C + N50C), and AA46-388 (A76C) did not display significant weight loss, while all other mice experienced significant weight drops at days 6–7 post-challenge , AA46-388 (T48C + N50C), and AA46-388 (VASP); and intermediate values for AA46-388 (A76C) and AA46-388 (W81C)  and Mich15 N1 containing the VASP domain.To understand the mechanisms by which antibodies confer protection  protein A or puri protein B. Intere protein C, with t8 (W81C) D. Importin vivo, as measured by the maximum percentage of weight loss (MPWL). A negative correlation between the MPWL vs. antibody levels against purified A/Michigan/45/2015 H1N1 virus . Despite the transient weight loss that peaked at 5 days post-infection —and challenged them with 100 times the LDnfection A, mice wnfection B. OveralThe neuraminidase is a “dimer of dimers”. The first dimer is held together by a disulfide bond in the stalk domain of the protein. Dimerization of the two dimers to a tetramer is likely facilitated by both the transmembrane domains and by interactions of the protomers in the head domain . The enzHere, we aimed to connect two soluble NA dimers by introduction of different disulfide bonds to form a tetramer. While some weak tetramer formation was observed for construct AA46-388 (T48C + N50C), we failed to produce stable tetramers by introduction of an additional disulfide bond in the stalk domain. However, we showed that N1 dimers are capable of inducing strong antibody responses that show NI activity and protect mice, even from a very high dose challenge. Dimers are still outperformed by the tetramer in terms of protective immune responses; however, even responses induced by dimers may contribute to protection from disease. Another interesting observation made here is that introduction of an additional cysteine towards the head domain of the NA is counterproductive. This is likely due to interference of this free cysteine with disulfide bonds that are usually formed within the head domain of the monomer.Lessons learned here will help us to better design novel soluble recombinant NA vaccine candidates without the need for exogenous tetramerization elements such as the VASP domain. Data regarding cysteine positions in the current constructs and their impact on dimerization and tetramerization will inform next-generation cysteine-mutant-based constructs, which are already in development. Ultimately, we aim to test such constructs as trivalent  standalone vaccines or as supplement to current inactivated influenza virus vaccines in humans. Importantly, while the tetramerization mechanism is conserved across NA subtypes, additional construct designs will need to be explored for N2 and B-NA antigens.


Nicks are the most frequent form of DNA damage and a potential source of mutagenesis in human cells. By deep sequencing, we have identified factors and pathways that promote and limit mutagenic repair at a targeted nick in human cells. Mutations were distributed asymmetrically around the nick site. BRCA2 inhibited all categories of mutational events, including indels, SNVs and HDR. DNA2 and RPA promoted resection. DNA2 inhibited 1 bp deletions but contributed to longer deletions, as did REV7. POLQ stimulated SNVs. Parallel analysis of DSBs targeted to the same site identified similar roles for DNA2 and POLQ (but not REV7) in promoting deletions and for POLQ in stimulating SNVs. Insertions were infrequent at nicks, and most were 1 bp in length, as at DSBs. The translesion polymerase REV1 stimulated +1 insertions at one nick site but not another, illustrating the potential importance of sequence context in determining the outcome of mutagenic repair. These results highlight the potential for nicks to promote mutagenesis, especially in BRCA-deficient cells, and identify mutagenic signatures of DNA2, REV1, REV3, REV7 and POLQ. A nick is the simplest form of DNA damage: a discontinuity in a single strand of the DNA backbone that does not change the chemical composition. Nicks are also the most frequent form of DNA damage, as they occur naturally in the course of transcription and DNA repair, and in response to ionizing radiation. Nicks were until recently ignored as a potential source of genomic instability. Here we use deep sequencing to show that mutations do occur at nicks and to identify those mutations and the factors that cause them. Nicks are the most frequent form of DNA damage and a potential source of genomic instability in human cells. Nicks can initiate both mutagenesis and homology-directed repair . Nicks Relatively little is known about pathways that promote mutagenesis at nicks. Evidence derived largely from reporter assays has shown that nicks are normally protected by RAD51, which is loaded onto DNA by BRCA2. Mutagenesis is stimulated in response to reduction in the abundance or activity of RAD51 or BRCA2 or factors that interact with BRCA2 to load RAD51 onto DNA; and by enhancing the activity of the anti-recombinogenic helicase RECQ5, which evicts RAD51 from single-stranded DNA ,4,7. ThoTo elucidate pathways of repair at nicks, we have characterized mutagenic events at targeted nicks in human cells by deep sequencing. Building on the very detailed current understanding of DSB repair, we explicitly asked whether factors that carry out DSB repair also act at nicks, in experiments designed to compare their roles at nicks and DSBs targeted to the same site on the CD44 gene. Strikingly, deep sequencing revealed that deletions and single-nucleotide variants (SNVs) are distributed asymmetrically at nicks. This asymmetry suggested that resection occurred predominately 5’ but also 3’ of the nick site, and depletion analysis identified a critical role for DNA2/RPA, which can resect with either 5’-3’ or 3’-5’ polarity –12. FurtFig 1A). U2OS is a human osteosarcoma line that expresses wild-type P53 and that is frequently used to study DNA repair. CD44 encodes a cell-surface glycoprotein that is expressed in many cell types but not required for proliferation in cell culture. Surface CD44 is readily detected by antibody staining, making it possible to verify that observations document events at a transcribed gene by using flow cytometry to assess gene expression. DNA nicks or DSBs were targeted to exon 1 of the CD44 gene by Cas9D10A or Cas9, respectively, and CRISPR gRNA 4. Genomic DNA was isolated from populations of 10,000–30,000 transfected cells, PCR-amplified, and deletions, insertions and SNVs were scored within a 65 bp window by amplicon sequencing on an Illumina platform. Control experiments analyzing uncleaved DNA established the combined error frequencies of PCR amplification, sequencing, and computational analysis. Indels and SNVs were assessed using a computational strategy that greatly reduced PCR and sequencing errors by making a consensus call for groups of DNA molecules that contained the same unique molecular identifiers . Absolute frequencies of indels and SNVs varied among experiments, but the effects of depletion of specific factors were reproducible. Results are presented for representative sequenced libraries.We examined frequencies of mutagenic events, including deletions, insertions and SNVs, at nicks and DSBs targeted to a site in the non-transcribed strand of the endogenous CD44 gene in human U2OS cells . At nicks, most mutagenic events were deletions, with fewer SNVs and few insertions; while at DSBs most were insertions, with somewhat fewer deletions and relatively few SNVs. Depletion of BRCA2 increased the frequency of mutagenic events at nicks 7-fold (p <0.01), but had no significant effect at DSBs (p = 0.985).The frequency of mutagenic events was considerably lower at nicks than at DSBs, as illustrated by the representative analysis in Fig 1C). SNVs also exhibited asymmetric distribution around the target site of nicks, but not of DSBs . The asymmetry at nicks was especially clear in the map of SNVs in BRCA2-depleted cells . In contrast, at DSBs SNVs were clustered around the target site, in a pattern relatively unaffected by BRCA2 depletion . Insertions proved to cluster at the target site, as discussed below.Maps of the fractional decrease in the number of base calls at each position showed that, in cells depleted of BRCA2, deletions exhibited an asymmetric distribution with respect to the nick site, extending from approximately 10 bp 5’ (upstream) to 25 bp 3’ (downstream); while at DSBs, deletions were clustered within approximately 10 bp on either side of the cleavage site . If resection is predominately 5’ at both nicks, more events should be evident in the zones flanking the nick sites than the zone between the nick sites . This was tested among mutants generated within the same cell population by co-transfecting cells with RNPs composed of Cas9D10A and either gRNA 4 or gRNA 7 at RNP levels titrated to prevent dual cleavage. Sequencing showed that in >99% of molecules that bore evidence of mutation, mutagenic events were restricted to a single site. Graphs of frequencies of mutations  identified a single asymmetric peak at nicks in populations transfected with RNPs containing gRNA 4/Cas9D10A, and a single asymmetric peak at DSBs in populations transfected with RNPs containing gRNA 4/Cas9 . Two peaks were evident in populations co-transfected with RNPs composed of Cas9D10A and either gRNA 4 or gRNA 7. The mutation frequency was sufficiently high in the BRCA2-depleted sample to make it evident that asymmetry at one site was the mirror image of asymmetry at the other . A similar 2-fold reduction in a-HDR frequencies in response to depletion of DNA2 was evident at nicks targeted to either the transcribed or non-transcribed strand of the reporter construct, in a-HDR supported by ssDNA donors complementary to either the nicked (cN) or intact (cI) strand . DNA2 possesses nuclease and motor activities that enable it to expose and resect DNA with either 5’-3’ or 3’-5’ polarity in DNA replication, recombination and DSB repair [Figs 1D, 2D, and 2E), and to create a gapped substrate that can anneal with ssDNA donor complementary to the intact strand of the target in a-HDR pathways .To identify factors that might promote resection at nicks, we first used the Traffic Light (TL) reporter  to deterB repair –12. DNA2S4A Fig). We therefore pursued an alternative strategy. The S. cerevisiae mutant Rfa1-t11 (K45E) supports normal replication but causes a defect in both mitotic and meiotic recombination [S. cerevisiae K45E were previously shown to support replication but were not analyzed for function in recombination [S. cerevisiae K45 . Based on this, we generated human RPA1 expression clones bearing the R41E and R43E single mutations as well as an R41/43E double mutation, and tested their effects on replication and HDR. Ectopic expression of RPA1-R41E, RPA1-R43E, or RPA1-R41/43E did not affect cell cycle , but inhibited both a-HDR at nicks and c-HDR at DSBs . Expression of either RPA1-R43E or RPA1-R41/43E reduced both a-HDR and c-HDR more than 5-fold. Note that inhibition was evident in cells expressing endogenous RPA, so these mutants exert a dominant negative effect on recombination. The RPA1-R43E single mutant was used for further analysis.DNA2 interacts with the trimeric factor RPA, which coats single-stranded gaps and free DNA ends exposed by DNA2 to prevent annealing and structure formation and coordinate end resection –16. A hubination –24. Humabination ,26. By aFig 2C left). In BRCA2-depleted cells, deletion frequencies were high in the absence of an HDR donor and reduced by co-depletion of DNA2 or by ectopic expression of RPA1-R43E ; and provision of an HDR donor reduced deletion frequencies more than 20-fold . Sequence analysis showed that either DNA2 depletion or ectopic expression of RPA1-R43E reduced deletions  and SNVs  on both sides of the nick site. These results support the view that availability of nicks as substrates for HDR or mutagenesis depends upon DNA2 and RPA, and further suggest that pathways of mutagenesis and a-HDR compete for the same DNA substrate.The roles of DNA2 and RPA in mutagenesis and a-HDR were then analyzed by amplicon sequencing. Sequence analysis showed that depletion of BRCA2 greatly stimulated a-HDR frequencies, which were reduced by co-depletion of DNA2 or by ectopic expression of RPA1-R43E  suggested that a-HDR at nicks might also be most efficient in S phase. To test this, we assayed a-HDR and c-HDR in cells in which the nuclear activities of Cas9D10A, Cas9 or RPA were restricted to G1 or S/G2 phase of cell cycle. Expression constructs were generated bearing these factors fused to degrons derived from the CDT1 or geminin (GEM) cell cycle regulators [S5 Fig). Relative efficiencies of HDR initiated during G1 or S phase were then quantified using the TL reporter assay . Analysis of the effect of degron-tagged derivatives of Cas9D10A or Cas9 showed that, as previously reported [Fig 3B right). In contrast, G1 phase nicks initiated a-HDR more efficiently than S/G2 phase nicks .DNA2 and RPA are regulated by cell cycle and most active in S phase . c-HDR agulators , and thereported ,28, S/G2Fig 3C). Thus, RPA promotes HDR at both nicks and DSBs more effectively in S/G2 phase than in G1 phase.Cell cycle dependence on RPA function was then assessed by determining HDR frequencies in cells in which the dominant negative RPA1-R43E mutant was expressed fused to cell cycle-regulated degrons. RPA1-R43E-CDT1 will inhibit HDR in G1 phase, and RPA1-R43E-GEM will inhibit HDR in S/G2 phase, so if HDR occurs preferentially in S/G2 phase, HDR frequencies are predicted to be reduced in cells expressing RPA1-R43E-GEM relative to control cells. This pattern was evident at both nicks and DSBs . A possible explanation is that nicks generated in G1 phase undergo HDR most efficiently if they persist into S/G2 phase for repair. To address this possibility, HDR was assayed at nicks and DSBs initiated in either G1 or S/G2 phase, by Cas9D10A or Cas9 bearing CDT1 or GEM degron tags, in cells expressing dominant negative RPA1-R43E or its derivatives bearing CDT1 or GEM degron tags . In all cases, HDR frequencies were higher in cells expressing RPA1-R43E-CDT1, which inhibits HDR in G1 phase, than in cells expressing RPA1-R43E-GEM, which inhibits HDR in S/G2 phase. Thus G1 phase nicks may persist to undergo repair later in cell cycle.At nicks, a-HDR frequencies were unexpectedly higher in cells expressing RPA1-R43E-CDT1 than in control cells, an effect not evident at DSBs . In control cells (siNT2), the frequency of deletions was 20-fold lower at nicks than at DSBs . At nicks, depletion of BRCA2 stimulated deletion frequencies more than 10-fold, and most deletions were >20 bp in length; while at DSBs, depletion of BRCA2 had little effect on deletion frequencies, and most deletions were <10 bp. At nicks, co-depletion of DNA2+BRCA2 reduced the overall deletion frequency nearly 2-fold relative to depletion of BRCA2 alone, reflecting reduced frequencies of longer deletions . At DSBs, the overall deletion frequency was also reduced nearly 2-fold by depletion of DNA2, but due primarily to a reduction in short deletions . Separate analysis of deletions 1–6 bp in length revealed that, at nicks, co-depletion of DNA2+BRCA2 caused a 6-fold increase in the frequency of 1 bp deletions but reduced the frequency of deletions of 2–6 bp; while at DSBs, DNA2 depletion reduced the frequencies of deletions of each length .Amplicon sequencing was used to examine the frequencies and lengths of deletions and insertions at nicks and DSBs targeted by gRNA 4 in cells depleted for BRCA2 and DNA2 . At nicks, insertion frequencies increased 3-fold in response to depletion of BRCA2 alone, and 17-fold in response to co-depletion of DNA2+BRCA2, reaching 5%; while at DSBs, depletion of BRCA2 or DNA2 had little effect on insertion frequencies. Surprisingly, the great majority of insertions at both nicks and DSBs were 1 bp in length, in all conditions tested.Insertions were rare (<1%) at nicks in control cells (siNT2), and far more frequent at DSBs . DNA2 promoted deletions at nicks, as it does at DSBs; however, at nicks this effect was restricted to deletions longer than 1 bp, and DNA2 specifically inhibited 1 bp deletions. DNA2 also inhibited the 1 bp insertions that constituted the majority of insertion events at nicks. At DSBs, 1 bp insertions frequently result from fill-in reactions at 5’ overhangs generated as a result of staggered cleavage by Cas9 [Taken together, the results above identify an unexpectedly complex role for DNA2 at nicks targeted by gRNA 4 . At DSBs at the same site, reductions in deletion frequencies in the range of 10%-40% resulted from depletion of REV1, REV3, REV7 or POLQ .Analysis of deletion frequencies showed that there was no effect of co-depletion of REV1 or REV3 and BRCA2 relative to depletion of BRCA2 alone, while co-depletion of REV7 or POLQ and BRCA2 reduced deletion frequencies  showed that at nicks, co-depletion of BRCA2 and REV1, REV7 or POLQ reduced the overall frequencies of deletions in this range, but with contrasting effects on 1 bp deletions, which were reduced 2-fold by REV1 depletion, unaffected by REV7 depletion, and increased nearly 2-fold by POLQ depletion. Depletion of these same factors, most notably POLQ, also reduced the frequencies of 1–6 bp deletions at DSBs. These results and the analysis of the effects of co-depletion of BRCA2 and DNA2 at nicks .Separate examination of short deletions . Depletion of BRCA2 increased insertion frequencies at nicks several-fold ; and co-depletion of DNA2+BRCA2 increased the overall insertion frequency and the frequency of 1 bp insertions . Co-depletion of REV1+BRCA2 caused a 3-fold reduction in the frequency of all insertions relative to BRCA2-depleted cells (0.29% vs 0.87% respectively) and eliminated 1 bp insertions. This immediately implicated REV1 as the source of 1 bp insertions at nicks targeted by gRNA 4. Insertion frequencies increased nearly 3-fold in response to depletion of REV3+BRCA2 relative to depletion of BRCA2 alone , suggesting that REV3 inhibits these 1 bp insertions. Sequence analysis further identified unexpected roles for REV3 and DNA2 in suppressing 1 bp insertions. Co-depletion of REV7+BRCA2 had little effect on insertion frequencies; and co-depletion of POLQ+BRCA2 caused a modest increase in the frequency of 1 bp insertions, and a modest reduction in longer insertions.Frequencies of insertions were nearly two orders of magnitude lower at nicks than at DSBs targeted by gRNA 4 . At DSBs, depletion of either REV3 or REV7 caused a reduction in insertion frequencies of similar magnitude (15%), which may reflect function of these factors in concert as components of POLζ. This contrasts with the increase in insertion frequencies at nicks in REV3- but not REV7-depleted cells , and suggests that REV3 and REV7 may function independently at nicks but not DSBs targeted by gRNA 4. At DSBs, depletion of POLQ caused a modest increase in 1 bp insertions and a decrease in longer insertions.Frequencies of insertions at DSBs targeted by gRNA 4 were relatively unaffected by depletion of BRCA2 and modestly increased by depletion of REV1 . These +1G insertions could be mapped to the site of the nick . However, we note that this mapping is not definitive as we cannot formally distinguish between insertion of G at the nick site  or 1 bp 3’ (downstream) of it (to generate TGG). The +1 insertions frequently evident among repair products of DSBs generated by Cas9 result from filling-in of cleavage products that carry 1 bp 5’ overhangs rather than blunt ends [Figs 6B left, S7B).At nicks targeted by gRNA 4, insertion of a single G (+1G) on the nicked strand accounted for almost all insertion events in control (siNT2) cells and in other samples tested, except cells depleted for REV1 . The frequency of insertions was comparable at nicks and DSBs targeted by either gRNA 4 or 7 . At nicks targeted by gRNA 7, insertion frequencies were not increased in response to co-depletion of DNA2 or REV3 with BRCA2, and only modestly reduced in response to co-depletion of REV1 and BRCA2; and the vast majority of insertions were +1T . We cannot distinguish between +1T insertions that occurred at the nick site and at other positions in the mononucleotide run. Sequence analysis further showed that repair of DSBs targeted by gRNA 7 resulted in insertions of +1T on the top strand of the amplicon . This is consistent with the view that +1 insertions at DSBs result from filling-in 5’ overhangs of some cleavage products.Sequence analysis was also carried out on repair products of nicks and DSBs targeted by gRNA 7 to a site located 35 bp upstream of the gRNA 4 site in CD44 exon 1, and on the opposite (transcribed) DNA strand . This could be coincidental. However, presuming that insertions at nicks occur at the nick site, the identity of insertions at nicks and DSBs can be explained mechanistically if the Cas9D10A nuclease occasionally cleaves not just one but both strands to generate DSBs bearing 1 nt 5’ overhangs rather than the predicted nicked substrates. If these aberrant cleavage products undergo repair by fill-in followed by ligation, sequencing will score them as 1 bp insertions.It is potentially significant that the sequences of +1 insertions at both nicks and DSBs targeted by gRNA 4 were identical, +1G; and that sequences of +1 insertions at both nicks and DSBs targeted by gRNA 7 were identical, +1T . At nicks targeted by gRNA 4, the majority of insertions were +1G and the insertion frequency was considerably reduced (>10-fold) in response to depletion of REV1, a translesion polymerase with dCMP transferase activity. These +1C insertions could occur if REV1 bridged the discontinuity caused by the nick by adding a single C on the opposite strand, which would then template incorporation of a G on the opposite strand . In contrast, at the site targeted by gRNA 7, the length of a mononucleotide T-tract adjacent to the target site increased from 4 to 5 nt, raising the possibility that sequence expansion might occur as a result of slippage during replication .Alternatively, the sequences of 1 bp insertions at nicks targeted by gRNAs 4 and 7 could reflect distinct mechanisms of repair active in either sequence context . At nicks targeted by gRNA 4, frequencies of SNVs increased 4-fold in response to depletion of BRCA2, accompanied by a switch to a predominance of transversion mutations (from 25% to 70%). Co-depletion of REV1, REV3 or REV7 along with BRCA2 reduced SNV frequencies 15–30% relative to cells depleted of BRCA2 alone, but had relatively little effect on the fraction of transversion mutations. At DSBs, depletion of BRCA2 had little effect on SNV frequency or the fraction of transversions relative to control (siNT2) cells, and depletion of REV1, REV3 or REV7 reduced SNV frequencies with little effect on the fraction of transversions. Strikingly, SNV frequencies at both nicks and DSBs targeted by gRNA 4 were considerably reduced in response to depletion of POLQ. POLQ depletion also caused the fraction of transversions to diminish at nicks, though not at DSBs. These results identify an unexpected role for POLQ in generating SNVs at both DSBs and nicks.SNV frequencies at nicks and DSBs were determined as described in Methods and normalized to siBRCA2- or siNT2-treated cells, respectively, to facilitate comparisons of the effects of each factor . The pattern of distribution of SNVs at nicks was unaffected by co-depletion of REV1, REV3 or REV7 along with BRCA2 ; or by limiting the activity of DNA2 or RPA, although the latter two treatments did reduce SNV frequency . Taken together, those observations suggest that resection by DNA2/RPA creates a gap that is filled in by POLQ to generate SNVs, as diagrammed in Fig 7C.Sequence analysis identified an asymmetric distribution of SNVs around the cut site at nicks but not at DSBs .Nicks are normally protected from mutation by BRCA2, which limits the threat they pose to genomic instability. To permit sequence analysis to be carried out on a dataset that was not limited by a very low frequency of mutations, the pathways of nick repair were defined in cells depleted for BRCA2, which increased the frequencies of mutations and of a-HDR at nicks . Asymmetry was evident at nicks targeted to either strand , so it does not reflect the direction of transcription. Asymmetry may at least in part reflect resection by DNA2/RPA. DNA2 possesses both 3’ and 5’ exonucleolytic activities, and interaction with BLM or WRN helicase can drive its 5’ resection activity [Fig 2). RPA exhibits some sequence-specificity of DNA binding [At nicks, both deletions and SNVs were distributed asymmetrically, extending approximately 10 bp 5’ (upstream) and 25 bp 3’ (downstream) of the nick site, while no corresponding asymmetry was evident at DSBs . The evidence that depletion of DNA2 caused a 5-fold increase in 1 bp deletions at nicks targeted by gRNA 4 .DNA2 and RPA are activated in S phase. Analysis of cell cycle dependence of repair leads to a model in which nicks generated in G1 phase may persist until S/G2 phase to undergo resection by DNA2/RPA . REV7 is the non-catalytic subunit of the TLS polymerase POLζ (REV3/REV7), but depletion of REV3 did not affect the deletion frequency, making it unlikely that repair synthesis by POLζ limits deletions. The role of REV7 may instead be architectural, protecting nicks to enable their repair by other factors, as it does at DSBs. The evidence that DNA2 inhibited insertions at nicks while stimulating long deletions  raises the possibility that resection by DNA2/RPA generates a gap that becomes a source of deletions, and that POLQ may limit deletion by extending DNA 3’ ends at these gaps, as it does at DSBs [Fig 7). POLQ is highly error-prone, generating single base changes at a rate 10- to 100-fold higher than other family A polymerases on undamaged DNA templates in vitro [Sequence analysis showed that both REV7 and POLQ limit deletions at nicks targeted by gRNA 4 . The frequency of insertions was comparable at nicks and DSBs targeted by either gRNA , but the sequences inserted were different. Insertions were uniformly +1G at both nicks and DSBs targeted by gRNA 4, and at nicks and DSBs targeted by gRNA 7, they were uniformly +1T. This may be coincidental, but these results also raise the possibility that Cas9D10A "nickase" occasionally cleave both strands to generate DSBs with 5’ overhangs that are repaired by the same fill-in process active at cleavage products of Cas 9 that carry 5’ overhangs [Fig 6C). Systematic characterization of repair products of nicks targeted to a number of different sites can distinguish between occasional generation of DSBs by Cas9D10A and more physiological repair mechanisms.Insertions were analyzed at two different sites, targeted by gRNAs 4 and 7 to positions 35 bp apart and on opposite strands of the CD44 gene . About 2 in DMEM (Gibco) containing 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco # 15140–122). For each siRNA transfection, 3x105 U2OS cells were reverse transfected with 5 pmol of each siRNA (QIAGEN) using lipofectamine RNAiMAX (Thermo Fisher) in a 12-well tissue culture plate. At 24 hr post-transfection, cells were trypsinized, washed once with PBS, resuspended in 100 μl of Ingenio electroporation solution (Mirusbio), mixed with 10 pmol premade complex of CRISPR gRNA and Cas9 (DSBs) or Cas9D10A (nicks) recombinant protein (IDT), transferred to a cuvette and transfected with a 4D-Nucleofector (Lonza), using program CM-104. CRISPR gRNA 4 targeted exon 1 of the CD44 gene at the sequence 5’-cctCGTGGCCGCTGAGCCTGGCAC-3’ . Parallel transfection with an eGFP expression plasmid  was used to determine nucleofection frequencies (>90%). The frequency of indels was about 40% among molecules recovered from transfected control populations (siNT2) in which DSBs were targeted by gRNA 4/Cas9  in a 12-well tissue culture plate for 2 hr. Cells were the trypsinized, washed once with PBS, and resuspended in 100 μl of Ingenio electroporation solution (Mirusbio). After addition of 10 pmol premade complex of CRISPR gRNA and Cas9 or Cas9D10A recombinant protein (IDT) to each well, contents were transferred to a Lonza cuvette and transfection was carried out with the 4D-Nucleofector (Lonza), using program CM-104. Cells were then transferred to a 6-well plate for further culture and harvested at 72 hr post-transfection. Transfection frequencies were determined by parallel transfection with 240 ng of a control eGFP expression plasmid (IDT). Genomic DNA was isolated with the DNeasy Blood and Tissue Kit (QIAGEN) at 72 hr post-transfection. Parallel analysis of DSBs targeted to the same site provided a physiological control for depletion.For siRNA depletion, 3x10 and siMRE11  from Thermo Fisher Scientific; and siDNA2 , siPOLQ ; siREV1 ; siREV3  and siREV7  from Qiagen.siRNAs used were siNT2 (4390847), siBRCA2 (s2085), siEXO1 6 cells were used for genomic DNA preparation. Sequencing libraries were prepared followed a modified version of Safe-seq [Genomic DNA for sequencing was isolated 6–7 days post-transfection and prepared as previously described . GenomicSafe-seq  in whichPrimers containing UMIs (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNNNNACTTCGGTCCGCCATCCTCGTC-3’ and 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNNNNNGCAAATCCCAGCCCTGCTTTC-3’) were used to amplify a 323 bp region of exon 1 of the CD44 gene spanning the cut site for 4 cycles using Phusion Hot Start II DNA Polymerase (Thermo Fisher Scientific). PCR products were purified with Ampure XP beads (Beckman Coulter) and further amplified using a pair of primers containing Illumina Nextera P5 and P7 adapter sites for a total of 25 cycles. Primers for both amplification steps were obtained from IDT. PCR products (480 bp) were purified with 0.7 volume of Ampure XP beads (Beckman Coulter), and reversely cleaned up by 7% Polyethylene Glycol 8000 (PEG) in 875 mM NaCl solution  followed by a 0.7 volume of Ampure XP beads purification. Purified PCR products were then pooled in equal molar ratio, and sequenced on Illumina MiSeq  at the Fred Hutchinson Cancer Research Center.Reads were first converted to unaligned SAM files using FastqToSam command from PICARD tools. Then the UMI sequences were extracted and converted to "ab/ba" format where ’a’ and ’b’ are the tag sequences from Read 1 and Read 2 using an inhouse script. Remaining reads were then assembled to BAM files in which reads having the same UMI were assembled to consensus sequences using single strand consensus sequence (SSCS) assembly in FASTQ format. Remaining reads were aligned with the reference sequence and analyzed using two established methods. (1) In an approach designed to accurately call SNVs by scoring only mutations found at complementary positions on both DNA strands, SSCS read 1 and read 2 were aligned by Burrows-Wheeler Aligner (BWA) MEM and realigned around INDELs by GATK. Variants were called using Samtools mpileup. INDELs were normalized using bcftools. Output of mutation frequencies, SNVs, insertions, deletions at each position was generated by a python program derived from . (2) SSCINDELs and SNVs from the resulting Variant Call Format (VCF) sequence data files  were theCCTAcgg-3’ . Donors for HDR were either the duplex plasmid pCS14GFP [Cell culture, transfection, depletion and reporter assays using the Traffic Light (TL) construct in 293T TL cells were carried out as previously described in detail . In brieSSO-2: 5’-TGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCgagggtgagggcgatgcCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCG-3’Frequencies of GFP+ cells (HDR) were determined three days post-transfection by flow cytometry, and normalized for transfection efficiency as determined by parallel transfection of a GFP expression plasmid. Data were collected using a BD Biosciences LSR II Flow Cytometer. Each set of assays was performed in triplicate, and a mean frequency of HDR was determined. The values presented represent the mean ± SEM from a representative experiment. Two-tailed T-tests were performed using Microsoft Excel (2015) to determine if the differences between HDR and mutEJ frequencies at different stages of cell cycle were statistically significant. Results are presented as frequencies of HDR among transfected cells, as measured in parallel by transfection with a GFP expression construct.GAATATCGACTGCTCATGAGTGATGGATTGAACACTCTATCC-3’ and 5’-AGCAGTCGATATTCCGGCGGACTATTCCCCGTAGTAATGGGACGGATGTTG-3; for RPA1-R43E 5’-CCGCCGCGTTATGAACTGCTCATGAGTGATGGATTGAACACTCTATCC-3’ and for RPA1-R41/43E, 5’-AGCAGTTCATAACGCGGCGGACTATTCCCCGTAGTAATGGGACGGATGTTG-3’; 5’-CCGCCGGAATATGAACTGCTCATGAGTGATGGATTGAACACTCTATCC-3’ and 5’-AGCAGTTCATATTCCGGCGGACTATTCCCCGTAGTAATGGGACGGATGTTG-3’.RPA1 expression constructs were generated as derivatives of a RPA1-WT expression construct pLX304-hRPA1 bearing an N-ter V5 tag (Addgene Plasmid #25890). The R41E, R43E, and R41/43E mutations were made by site-directed mutagenesis  using thS2 Fig).C-terminal CDT1 and GEM tags were added using a modified overlap extension PCR cloning method . The CDTS3 Fig).To generate the pCDNA-Cas9-CDT1, pCDNA-Cas9-GEM, pCDNA-Cas9D10A-CDT1 and pCDNA-Cas9D10A-GEM expression constructs, we replaced the T2A-BFP tag in both pCDNA-Cas9-T2A-BFP and pcDNA-Cas9D10A-T2A-BFP  with the6 U2OS or HEK293T cells were transfected with 750 ng of plasmid, and at 36 hr post-transfection cells were harvested by trypsinization (0.05%) and washed twice with cold PBS. Cells were then fixed and permeabilized by incubation with 500 μl of 1x Foxp3 /Transcription Factor Fixation/Permeabilization solution (Invitrogen) for 30 min at room temperature; washed twice with the same buffer; and resuspended in 500 μl of the same buffer. Samples were then divided into two 250 μl aliquots and stained at room temperature for 1 hr with anti-RPA1 antibodies  to detect endogenous RPA1 or anti-V5 tag antibodies  to detect ectopically expressed RPA1. Cells were then washed once in 1 ml PBS and stained with DAPI  in 10 mM Tris, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 22 mM MgCl2, 0.1% NP-40, 0.05 mg/ml BSA and 10% DMSO, and then resuspended in 200 μl PBS containing 1% FBS and analyzed by flow cytometry on a BD LSR II instrument, set to record 50,000 cells per sample. Data were analyzed using FlowJo software (version 9.6).To analyze ectopic expression of CDT1- or GEM-tagged RPA1-R43E, 1x105 U2OS or HEK293T cells were transfected with 200 ng of expression plasmid, and at 36 hr post-transfection cells were harvested by trypsinization (0.05%), washed twice with cold PBS, then stained with DAPI and further analyzed as above.To analyze cell cycle dependence of expression of CDT1- or GEM-tagged Cas9D10A, 3x10S1 Fig(A) Diagram of nicks targeted to the non-transcribed or transcribed strands by gRNAs 4 and 7, respectively. P, promoter; arrowheads, nick target sites. (B) Sequence of the region of the CD44 gene targeted by gRNAs 4 and 7. Target sites, red font; regions of DNA that form hybrids with the CRISPR gRNAs, underlined.(PDF)Click here for additional data file.S2 Fig(A) Diagram of predominately 5’ asymmetric resection at nick sites on opposite DNA strands. (B-D) Maps of mutations  flanking nicks or DSBs targeted to sites 35 bp apart by gRNA 4 or gRNAs 4 and 7. Cells were depleted with siNT2 (control) or siBRCA2, as indicated. Each panel maps the 240 bp region centered on the gRNA 4 target site, with site(s) cleaved indicated by arrowhead. Frequencies of mutations at the gRNA 4 site shown at the top of the y-axis.(PDF)Click here for additional data file.S3 Fig(A) Effects of DNA2, EXO1 or MRE11 depletion on frequencies of a-HDR or c-HDR at nicks or DSBs, respectively. Frequencies were normalized relative to frequencies in 293T TL cells treated with siBRCA2 (nicks) or siNT2 (DSBs). Cleavage was targeted by gRNA9 (see panel B) and supported by a cN ssDNA donor for a-HDR (nicks) or a plasmid donor for c-HDR (DSBs). (B) Left, diagram of the cleavage sites for gRNA 2 and gRNA 9 in the TL construct (arrowheads), with the promoter (P) upstream. Right, effects of depletion of DNA on frequencies of HDR at nicks or DSBs targeted to the TL reporter construct in 293T TL cells by the indicated gRNA. HDR was supported at nicks by a donor complementary to the nicked or intact strand , in cells treated with siNT2, siBRCA2 or siDNA2+siBRCA2, as indicated; or at DSBs by a dsDNA donor, in cells treated with siNT2 or siDNA2. Frequency values represent the mean ± SEM from a representative experiment; and * and *** indicate p<0.05 and p<0.001, respectively, for the frequency difference between indicated sample and sample treated with siBRCA2 (nicks) or siNT2 (DSBs). (C) Working model for the role of DNA2 resection in a-HDR at nicks supported by a cN or cI donor. Results in panel B show that DNA2 promotes a-HDR by both pathways, and the first step shown in resection 3’ of the nick. The cN or cI donors anneal as shown, and processing then generates a heteroduplex which is resolved by replication.(PDF)Click here for additional data file.S4 Fig(A) Cell cycle profiles of U2OS control cells (siNT2) or cells treated with siDNA2 or siRPA1. (B) Cell cycle profiles of U2OS control cells (mock) or cells expressing RPA1-WT, RPA1-R41E, RPA1-R43E or the RPA1-R41/R43E double mutant.(PDF)Click here for additional data file.S5 Fig(A) Cell cycle profiles of 293T cells transfected with Cas9D10A-mKO2-CDT1 or Cas9D10A-mAGGEM expression constructs, showing either the entire population or the population gated for PE+ (mKO2) or GFP+ (mAG) cells. (B) Cell cycle profiles of cells mock-transfected or transfected with CDT1- or GEM-tagged derivatives of RPA1-R43E bearing a C-terminal V5 tag, and stained with anti-RPA antibody, which detects endogenous and ectopically expressed RPA; or with anti-V5 antibody, which detects ectopically expressed RPA1-R43E.(PDF)Click here for additional data file.S6 Fig(A) Tabulated effects of depletion of indicated factors on frequencies of 1 bp insertions of A, C, G or T or all nucleotides at nicks targeted by gRNA 4. These and other sequences shown illustrate the top DNA strand, which is the strand targeted for nicks by gRNA 4/Cas9D10A . (B) Graph of effects of depletion of indicated factors on frequency of molecules bearing +1bp insertions of A, C, G or T at the gRNA 4 target site. (C) Sequence of predominant 1 bp insertions at the gRNA 4 target site. Nick site, underlined; PAM, blue font; insertion, red font. (D) Tables show nucleotides identified at indicated positions in products of repair containing a 1 bp insertion within the 4 bp region spanning nick site, in U2OS cells treated as indicated Positions are numbered -2, -1, +1, +2, relative to the nick site in the reference sequence GT/GC, where the slash marks the site at which the nick targeted to the CD44 gene by gRNA 4 cleaves the phosphodiester backbone.(PDF)Click here for additional data file.S7 Fig(A) Left, effects of depletion of indicated factors on frequencies of 1 bp insertions of A, C, G or T at DSBs targeted by gRNA 4. Right, the 12 bp region surrounding the gRNA 4 target site (underlined) at DSBs bearing 5’ overhangs; and +1G insertion at that site (red). PAM, blue font. The postulated repair intermediate is shown for clarity. (B) Left, effects of depletion of indicated factors on frequencies of 1 bp insertions of A, C, G or T at nicks targeted by gRNA 7. Right, the 12 bp region surrounding the gRNA 7 target site (underlined in nicked strand) and +1T insertion at that site (red). PAM, blue font. (C) Left, effects of depletion of indicated factors on frequencies of 1 bp insertions of A, C, G or T at nicks targeted by gRNA 7. Right, the 12 bp region surrounding the gRNA 7 target site (underlined) at DSBs bearing 5’ overhangs and +1T insertion at that site (red). PAM, blue font. The postulated repair intermediate is shown for clarity.(PDF)Click here for additional data file.S1 Data(ZIP)Click here for additional data file.


Different aminonaphetalenesulphonic acid derivatives like 5-aminonaphthalene-1-sulphonic acid (5AN1SA), 2-aminonaphthalene-1-sulphonic acid (2AN1SA), 8-aminonaphthalene-2-sulphonic acid (8AN2SA) and 4-amino-3-hydroxynaphthalene-1-sulphonic acid (4A3HN1SA) were used to construct polymer/bismuth film modified electrode for simultaneous determination of Pb(II) and Cd(II) ions with the aim of developing a cheaper and sensitive electrode that could possibly replace Nafion. Among the different modified electrodes, poly (8AN2SA)/bismuth film modified electrodes showed the highest electrochemical response for both ions. These electrochemical results were also supported by density functional theory (DFT) calculations. Based on these experimental and theoretical results, poly (8AN2SA)/bismuth film glassy carbon modified electrode was further investigated to develop a simple and sensitive electrochemical method for the simultaneous determination of Pb(II) and Cd(II) ions. After optimizing the different experimental parameters, the proposed method gave a linear range of 1–40 μg/L with the detection limit of 0.38 and 0.08 μg/L for the simultaneous determination of Pb(II) and Cd(II) ions, respectively. 8-Aminonaphthalene-2-sulphonic acid, Square wave anodic stripping voltammetry, Pb(II), Cd(II). AmonMajor sources of pollution of the environment by lead are anthropogenic including emissions of automobiles; lead paints, pipes and batteries; lead-glazed ceramics and containers; fertilizers, pesticides, cosmetics and toys . Lead poCadmium which is released mainly from batteries, coatings and platings, stabilizers for plastics, fossil fuel combustion, phosphate fertilizer, and waste incinerationis classified as a Group 1 human carcinogen , 10. CadHence, a cheap, sensitive, selective, and environmentally friendly analytical methods and materials affordable for developing countries are of utmost importance to regularly monitor the amounts of these toxic metals in different matrices. Several methods have been developed to monitor toxic metals in various sample matrices such as atomic absorption spectroscopy , 17, graNafion polymer was added to bismuth film electrode the modified electrodes gave better results for the simultaneous determination of lead and cadmium , poly (8-poly (8AN2SA)] and poly (4A3HN1SA) were electrodeposited on GCE following previously reported procedures . Briefly2.3The experimental conditions were first optimized by varying the deposition potential (−0.6 – 1.6 V), deposition time (60–240 s), pH of the supporting electrolyte (ABS) (3.0–6.0), film thickness of the polymer electrodes (5–25 cycle) and the concentration of Bi(III) (0.1–2.5 mg/L). Then the determination of Pb(II) and Cd(II) in water samples was performed using Square Wave Anodic Stripping Voltammetry (SWASV) under the optimized conditions by scanning the potential range from −1.2 to 0.2 V. The potential step, square wave amplitude, and frequency were set to 4 mV, 0.025 mV, and 15 Hz, respectively. The calibration curves were used to determine the concentrations of Pb(II) and Cd(II) in wastewater samples. The interference study was carried out by measuring 30 μg/L of Pb(II) and Cd(II) in the presence of potential interfering ions with three fold higher concentration than the analyte amount. Recovery study was also performed by spiking 10, 20, and 30 μg/L of Pb(II) and Cd(II) in to the wastewater samples.2.4Eint(M@D) is the interaction energy for the M@D complex (Edeform(M) and ΔEdeform(D) are the deformation energies for M and D, respectively (note that the deformation energy for M is zero). The deformation energies were calculated as the difference between the energy of M in the complex M@D orbital basis set and both M and D in their isolated states. The stabilization energies of the complexes, the change in zero point energy was added to the binding energy ΔEsta33.13. Typical cyclic voltammograms for the electrosynthesis of the polymer film are shown in The polymer film electrodes were prepared on glassy carbon electrode by cycling the potential between -0.8 to +2.0 V for 15 cycles at 0.1 V/s from 2.0 mM monomer solutions prepared in 0.1 M HNO2SO4. All polymer modified GCEs showed distinct peaks at 0.32 V and 0.34 V which was not shown in bare GCE. Thus, the results suggested an electroactive polymer film is deposited on the GCE. The highest peak current was observed for poly , indicating that the reaction involves surface-controlled redox process [The mechanism of electron transfer at poly (8AN2SA)/Bi/GCE in monomer free of 0.5 M H process . Surface process .(4)ip=n22), among other polymer films. In addition, it exhibits higher peak current response compared to other voltammograms as shown in The calculated values were put in 3.2The structures of the monomers used for the preparation of the polymer electrodes and computational calculations are shown in Fig. S-2 and bind) with better stabilization energy compared with the other Pb(II) complexes. Moreover, the analysis of the total atomic charges listed in bind) and showed comparable stabilization energy with the other complexes listed in The results indicate that the dimers  interestingly interact with Cd(II) and Pb(II) ions and form complex, as evidenced by the less exothermic energy. In particular, the results presented in the table show that the total energy for Pb(II) complexes are more exothermic than the Cd(II) complexes in general. The Cd(II)@dimer-8AN2SA complex showed relatively less binding energy  and Cd(II) was studied as shown in The effect of deposition potential on the peak current was investigated by changing the potential from -1.6 to -1.0 as shown in When the accumulation time was varied from 60 to 270 s, the peak current increased almost linearly with the increase of pre-concentration time C. There Fig.4Dshows the effect of Bi(III) ion concentration on the peak currents of Pb(II) and Cd(II). Bi(III) ion is known to form fussed alloy with heavy metals and facilitate the nucleation process during the accumulation step. The result at low Bi(III) concentration is not favorable for efficient formation of Bi alloy . GradualThe effect of poly (8AN2SA) film thickness on the peak current was also optimized by potentiodynamic electrodeposition  of the polymer films as shown in 3.5p (μA) = 0.2677c + 0.079, correlation coefficient of R2 = 0.995 (p (μA) = 0.3619c - 0.804 with a correlation coefficient R2 = 0.992 was observed in a concentration range of 1.0–40.0 μg/L of Cd(II) ion (The poly (8AN2SA)/Bi/GCE was used for the determination of Pb(II) and Cd(II) by applying SWASV and as shown in  = 0.995 A. Simila(II) ion B. The el(II) ion C. The se3.63+, Cr3+, Al3+, Cu2+, Zn2+, Fe2+, and K+) into 0.1 M ABS containing 30 μg/L of Pb(II) and Cd(II). The anodic peak currents of both Pb(II) and Cd(II) ions decrease by less than 10% for most potentially interfering ions except for Cu(II) and Zn(II) as shown in Under the optimized condition, the stability of the poly (8AN2SA)/Bi/GCE was investigated by measuring anodic stripping responses of 30 μg/L of Pb(II) and Cd(II) ions for nine replicate measurements. The relative standard deviation (RSD) were found to be1.2% and 2.3% for Pb(II) and of Cd(II), respectively. The interference study was carried out by adding three fold higher concentration of potentially interfering ions /Bi modified electrode for the determination of Pb(II) and Cd(II) were tested in the industrial wastewater sample collected . The electrochemical result of Pb(II) in the wastewater was found to be 657.1 μg/L, which is comparable with the result from AAS (625.0 μg/L) with p-value 0.002. Similarly, the anodic stripping result in the wastewater for Cd(II) was found to be 93.8 μg/L, close to the result of AAS (96.6 μg/L) with p-value 0.04. The p-values in both cases are less than 0.05 which tells no significant difference between the two methods, indicating that the simple and cheap electrochemical method, anodic stripping voltammetry applied for the determination of Pb(II) and Cd(II) at poly (8AN2SA)/Bi modified glassy carbon electrode can be useful for the analysis of heavy metal ions.Furthermore, in order to evaluate the reliability of the poly (8AN2SA)/Bi modified electrode, recovery studies were carried out by spiking standard sample to the industrial wastewater samples. It can be seen that the recoveries obtained were found to be 83.9–90.9% for Cd(II) and 92.0–101.0% for Pb(II) see . The res4Among the several different aminonaphetalenesulphonic acid derivatives, poly  was found to be the best polymer for in-situ modification bismuth film on glassy carbon electrode in agreement with the prediction from computational studies (DFT). For the simultaneous determination of Pb(II) and Cd(II), poly (8AN2SA)/bismuth film glassy carbon modified electrode exhibited a linear range of 1.0–40 μg/L with detection limits of 0.38 and 0.08 μg/L, respectively. Hence, the proposed method provides an alternative and cheaper modifier that can replace the expensive Nafion polymer.Alemayehu Yifru: Performed the experiments; Wrote the paper.Gossa Dare: Performed the experiments.Taye B. Demissie: Analyzed and interpreted the data; Wrote the paper.Solomon Mehretie; Shimelis Admassie: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Data will be made available on request.The authors declare no conflict of interest.No additional information is available for this paper.


NS1 also prevented the generation of protective immunity against a heterologous virus challenge. These data indicate that shutoff active NS1 plays a major role in suppressing host immune responses against IAV infection.Influenza A virus (IAV) is a significant human pathogen that causes seasonal epidemics. Although various types of vaccines are available, IAVs still circulate among human populations, possibly due to their ability to circumvent host immune responses. IAV expresses two host shutoff proteins, PA-X and NS1, which antagonize the host innate immune response. By transcriptomic analysis, we previously showed that PA-X is a major contributor for general shutoff, while shutoff active NS1 specifically inhibits the expression of host cytokines, MHC molecules, and genes involved in innate immunity in cultured human cells. So far, the impact of these shutoff proteins in the acquired immune response in vivo has not been determined in detail. In this study, we analyzed the effects of PA-X and NS1 shutoff activities on immune response using recombinant influenza A/California/04/2009 viruses containing mutations affecting the expression of shutoff active PA-X and NS1 in a mouse model. Our data indicate that the virus without shutoff activities induced the strongest T and B cell responses. Both PA-X and NS1 reduced host immune responses, but shutoff active NS1 most effectively suppressed lymphocyte migration to the lungs, antibody production, and the generation of IAV specific CD4 Seasonal influenza A virus (IAV) infection is a major public health problem causing 3–5 million hospitalizations and 50,000 deaths annually in the USA . In addiNS1 is a multifunctional protein expressed from the NS gene segment with a strain-specific size of 230–237 amino acids ,9. NS1 iAnother IAV host shutoff protein is PA-X, which is expressed by a ribosomal frameshift during translation of the viral polymerase subunit PA mRNA . PA-X shTo analyze the target specificity and functional role of IAV shutoff proteins, we created mutant viruses using a 2009pH1N1 virus, A/California/04/2009 , as a backbone . We mutaInfluenza A virus A/California/04/2009 (H1N1) was provided by R. Webster and R. Webby . Stock virus was propagated in 10-day-old embryonated chicken eggs. A/Puerto Rico/8/34 (H1N1) was provided by S. Dewhurst  and grown in MDCK cells. Rescue of the mutant viruses were reported previously  outgrew the other viruses, expressing shutoff protein(s) at 1 dpi, similar to what we observed in cultured MDCK cells [50 and weight loss values (50 by 5–7 fold when compared to the corresponding viruses (PA-X low or high)  D. Body wigh)  and spleen at 7 and 21 dpi. IAV-specific CD4 peptide ,27. + and CD8+ T cells in the MLN and spleen at 7 dpi  resulted in significantly fewer IAV-specific CD4at 7 dpi A. The efe spleen B. The sut 21 dpi C. The EL+ and CD8+ T cell levels were significantly reduced by either NS1 or PA-X shutoff activities, suggesting that both proteins can suppress the generation of memory T cells  of wt Cal or A/Puerto Rico/8/34 (H1N1) (PR8). Body weight loss and survival rate were monitored for 15 days. Initial infection with any of the four different viruses produced sufficient immunity to protect the mice from a homologous virus challenge  has not been determined in detail, but considering the strong impact on the outcome of immunity found in this study, it is possible that modification of NS1 shutoff activity strongly improves the potential of the LAIV and generates robust, long lasting, cross-reactive immunity.Not surprisingly, infection with the virus lacking shutoff activity induced strong early cytokine responses, long-lived increases in IAV-specific CD4The functional differences of PA-X and NS1 shutoff activities in generating protective immunity could be an important factor for IAV adaptation to human hosts. Most of the circulating human IAV strains express shutoff active NS1 and weakly active PA-X. The key NS1 residues responsible for CPSF30 interaction and shutoff activity are less conserved among animal isolates. Comparison of PA-X shutoff activity of human and animal isolates also indicate species-specific differences. PA-X of avian viruses and 2009 pH1N1 containing the PA segment originated from avian viruses is highly active compared to those from human viruses . The preIn conclusion, shutoff active NS1, which is conserved among human IAVs except 2009pH1N1, efficiently limits the generation of IAV-specific B and T cells and long-lasting immunity to protect against subsequent infection. While PA-X has stronger shutoff activity on general gene expression, it is less effective in regulating the acquired immune response.