Here, we show that TNF70–80 binds to the TNF receptor, activating p38 MAP kinase through TNF receptor-associated factor 2. Using truncated TNFR mutants, we identify the sequence in TNFRI which enables p38 activation by TNF70–80. Peptides with this TNFRI sequence, such as TNFRI206–211 bind to TNF and inhibit TNF-induced p38 activation, respiratory burst, cytokine production and adhesion receptor expression but not F-Met-Leu-Phe-induced respiratory burst in neutrophils. TNFRI206–211 does not prevent TNF binding to TNFRI or TNF-induced stimulation of ERK, JNK and NF-κB. TNFRI206–211 inhibits bacterial lipopolysaccharide-induced peritonitis, carrageenan-induced and antigen-induced paw inflammation, and respiratory syncytial virus-induced lung inflammation in mice. Our findings suggest a way of targeting TNF-p38 pathway to treat chronic inflammatory disorders.Despite anti-TNF therapy advancements for inflammatory diseases such as rheumatoid arthritis, the burden of diseases remains high. An 11-mer TNF peptide, TNF Anti-TNF therapy has improved the treatment of inflammatory disease but can predispose to infection and malignancy. Here the authors show an anti-TNF biologic peptide that functionally and selectively targets the TNF-p38 pathway in multiple models of inflammation. However, the deleterious effects of neutralising the actions of TNF, including increased susceptibility to infection, remains a major setback for this type of therapy. Patients with rheumatoid arthritis receiving anti-TNF immunotherapy have the increased risk of fungal and bacterial infections, particularly of reactivating latent tuberculosis4, as well as non-melanoma skin cancers5. In paediatric and adult patients, combination therapy with anti-TNF biologics and other forms of immunosuppressives increased the risk of other types of malignancies6. Another approach in the treatment of chronic inflammation involves the targeting of the intracellular pathways used by the TNFR, in particular the p38 MAP kinase (MAPK). Unfortunately, this approach has encountered a number of major setbacks, including poor clinical responses and unexpected toxicity, resulting in suspension of clinical trials8. Thus, alternative approaches that target TNF and TNF-mediated signalling in chronic inflammatory conditions are needed.The pleotropic cytokine, tumour necrosis factor alpha (TNF), plays a major role in the pathogenesis of chronic inflammatory diseases. Consequently, the treatment of these diseases, in particular rheumatoid arthritis, Crohn’s disease and psoriatic arthritis, has been revolutionised by the availability of anti-TNF biologics such as monoclonal antibodies, e.g., infliximab, certolizumab, adalimumab and golimumab or soluble TNF receptors, e.g., etanercept11. Of interest was that an 11-mer TNF peptide, TNF70–80, representing the amino acid sequence 70–80 of the TNF monomer, primed neutrophils and promoted immunity to Plasmodium, as well as Mycobacterium bovis, Klebsiella pneumoniae and Aspergillus fumigatus in vitro and/or in vivo14. Here, we show TNF70–80 binds to the TNFR and initiates signalling through TNF receptor-associated factor 2 (TRAF2) and p38 MAPK, leading to activation of the neutrophil respiratory burst. Using this information, we have generated peptides from the TNFRI sequence which interact with TNF70–80 and inhibit its ability to activate p38 and the respiratory burst. The TNFRI peptides block the inflammatory response in models of autoimmunity and infection. This provides a basis for developing cytokine receptor biologics with singular therapeutic potential.It has previously been reported that short peptides of the TNF sequence promote different biological effects of the parent TNF molecule11. However, we have no understanding of how the peptide interacts with and activates cells. Here, we examined whether its stimulatory effects on the neutrophil respiratory burst is via the TNFR and p38 MAPK, both of which are required for a TNF-mediated response15. In these experiments, we used TNF and TNF70–80 at concentrations that we had previously found to enhance neutrophil responses14. The data demonstrate that in association with the activation of p38 MAPK, TNF70–80 stimulated the production of superoxide in a similar manner to TNF, measured as lucigenin-dependent chemiluminescence production . The data show that TNF70–80 enhanced the activity of p38 in cells transfected with either wild-type TRAF2 or an empty vector, but not in those transfected with ∆TRAF2  or all four (M4) cysteine-rich domains (CRD) on the extracellular portion of TNFRI  or a mutant of TNFRI. After 24 h, the cells were treated with either diluent or TNF70–80 (10 μM) and p38 activity assayed. The data showed that TNF70–80 caused ~2.5-fold increase in p38 activity in WT TNFRI- and M1 TNFR1-transfected cells and this effect was retained, albeit appearing reduced , in cells transfected with the M4 TNFRI mutant , of the extracellular portion of TNFRI  assay. The first peptide, ∆HM4 (LKPGTT)  was synthesised and shown to cause a dose-dependent suppression in the ability of TNF70–80 to stimulate CL production in human neutrophils  on TNF-induced neutrophil CL production. Treating TNF with TNFRI209–211 compared to a control peptide (TGT) caused a dose-dependent inhibition of TNF-induced CL and an IC50 of 123 μM , that contained the three residues N-terminal to TNFRI209–211 but not the corresponding scrambled peptide, GEDTST, was effective at inhibiting TNF-induced CL production with an IC50 of 171 μM  (QIENVKGTEDSGTT) increased the anti-TNF action (IC50 of 50 μM)  Fig. . The spe211 Fig.  nor TNFR211 Fig.  inhibiteely Fig. . A D-ami μM Fig. . Incubat70–80 and TNF  upregulation in neutrophils Fig.  and TNF-206–211  injection of bacterial LPS was inhibited by TNFRI211 Fig. . Similar211 Fig.  and an a211 Fig. .Fig. 6TN206–211 as an anti-inflammatory agent was further demonstrated in an acute respiratory syncytial virus (RSV)-induced inflammatory mouse model22. Mice were treated intranasally (i.n.) with TNFRI206–211 or the respective scrambled peptide, 1 day prior to an intranasal challenge with 1 × 106 plaque forming units of RSV, and then 1 and 3 days post viral challenge. Mice treated with TNFRI206–211 had significantly reduced numbers of leukocytes around the bronchi  and in the number of leukocytes around the lung blood vessels (~70%)  although this did not reach significance. The anti-inflammatory effects of TNFRI206–211 were further supported by the findings that there was a significant reduction in lymphocyte numbers . Interestingly, the anti-inflammatory effects of TNFRI206–211 were associated with a significant decrease in viral load in the lung . The data in Fig. 206–211 but not the scrambled peptide prevented TNF from inducing the activation of p38 in human neutrophils. This effect was reproduced in the MonoMac 6 cells  with TNFR206–211 did not inhibit the ability to activate p38 -induced lung inflammation. The latter is particularly useful in gauging the actions of the TNFRI peptides, as both the effects on pathogenesis and susceptibility to the virus could be monitored. Importantly, treatment with the TNFRI peptides significantly reduced lung viral load, in addition to the suppression of virus-induced inflammation. This is the first demonstration that this new form of anti-TNF therapy has the potential to be used in the treatment of infection-induced pathogenesis as well as non-infectious inflammatory conditions.206–211 to bind TNF suggests a possible mechanism by which the TNFRI peptides exert their anti-TNF effects. Such an action would prevent TNF from fully engaging TNFRI, particularly around the region whose sequence is found in the TNFRI peptides. Consistent with this idea, it was necessary to preincubate TNF with a TNFRI peptide to enable the binding of the peptide to TNF prior to addition to cells. The data also show that whereas TNFRI206–211 bound TNF, the scrambled version that was devoid of inhibitory actions, did not bind. Furthermore, hexamers of TNFRI from CRD1–CRD3, unlike TNFRI206–211, did not prevent TNF from stimulating neutrophils to produce superoxide. This also demonstrates the specificity of residues 198–211 in TNFRI in coupling TNF-p38 to the neutrophil respiratory burst function.The ability of TNFRI1. However, this form of therapy is associated with increased susceptibility to infection and cancer6. Furthermore, the FDA has issued warnings against such use in paediatric patients between the ages of 4–17 owing to increased risk of malignancy6. Another approach in the treatment of chronic inflammation has involved the targeting of the intracellular pathways used by the TNFR. Although p38 inhibitors represent one of the most heralded form of therapies for the treatment of inflammation in recent times, clinical trials with a range of unrelated pharmacological p38 inhibitors for the treatment of rheumatoid arthritis and other chronic inflammatory conditions have shown poor clinical outcomes with transient immunosuppressive effects, associated with increased risk of severe infections, liver/brain/skin toxicity and other adverse effects24. These disappointing clinical data signal that systemic inhibition of p38 is an inappropriate form of treatment.Anti-TNF biologics have revolutionised the treatment of chronic inflammatory conditions such as rheumatoid arthritis26. In addition, such blockade also resulted in a decrease in the expression of the MAP kinase phosphatase, DUSP-126 that inactivates active p38 and active JNK. Consequently, p38 activation also initiates a negative feedback mechanism that limits the strength and duration of the function of this signalling pathway. Previous studies have demonstrated that the p38-mediated regulation of IL-10 and DUSP-1 expression is mediated by the downstream mitogen- and stress-activated kinases (MSK)1 and MSK2, two of the kinases that phosphorylate the transcription factors, CREB and ATF-1, that bind to the promoters of IL-10 and DUSP-1 and enhance their transcriptional activity27. Consistent with its role in mediating an anti-inflammatory action, p38 inhibition in vivo was found to cause disease exacerbation and prolonged the duration of disease in animal models of inflammatory diseases26. This is likely to be a key reason for the failure of drugs that target p38 in clinical trials.The p38 pathway plays a dual role in regulating the inflammatory response. Thus, blockade of the p38 pathway in murine macrophages or human synovial cells not only caused a decrease in the production of proinflammatory mediators such as MMP-13, KC, TNF and IL-6, but also the anti-inflammatory IL-10206–211, which also did not affect TNF-induced ERK1/ERK2, JNK and NF-κB activation, unlike TNF-neutralising biologics. These observations demonstrate that the TNFRI peptides are a unique class of agents that have the capability to block the actions of TNF that require p38, without inhibiting the p38 pathway in general, thus enabling the normal functioning of this pathway when it is activated by other stimuli. Intriguing is the finding that TNFR206–211 selectively blocks the ability of TNF to stimulate p38 activation, most likely explaining its unique in vivo effects in experimental inflammatory models.The TNFRI peptides do not target p38 per se, unlike previously described p38 inhibitors. The peptides only targeted TNF-mediated activation of p38. Consequently, it did not affect fMLF-stimulated respiratory burst in neutrophils and p38 activation. TNF binding to the TNF receptor was not affected by TNFRI28. It is therefore likely that the peptide by inhibiting the TNF-TNFRI-p38 axis prevents the development of a cytokine ‘storm’ and its wide range of debilitating effects on leukocyte function, enabling leukocytes to kill virus-infected cells effectively. In contrast, current anti-TNF biologics may interfere with virus-clearance and promote tissue damage. We have demonstrated that Ross River virus (RRV)-infected mice treated with etanercept showed decreased weight gain, higher viral titre in muscle, joints and blood, associated with a greater degree of tissue damage and inflammatory cell recruitment than untreated RRV-infected mice29. Thus, the TNFRI peptides appear to have a number of advantages over existing anti-TNF biologics and pharmacological p38 inhibitors. Although we cannot rule out the possibility that the TNF receptor biologics would not predispose the host to infections, it is less likely that this would be an issue because of their target specificity.Our work has illustrated that the TNFR peptide while capable of binding TNF does not prevent the binding of TNF to TNFRI but it selectively causes inhibition of p38 activation and associated function. Given that the TNFR peptide sequence covers the minimal region that is required for TNF to activate p38, it is likely the peptide alters the dynamics of the TNF–TNFR interaction—allowing not only TNF binding to TNFRI, but also the activation of the other signalling molecules that the receptor normally activates. This implies that other regions of TNFRI mediate the activation of the other signalling molecules. While this interaction is interesting, the mechanism leading to the selective inhibition  of p38 remains to be fully elucidated. Nevertheless, the data from the current study illuminates a novel approach to block the TNF-p38 signalling axis and treat inflammation. Another advantage of the TNFRI peptides over current anti-TNF biologics is the ability of the peptides to promote clearance of RSV from infected mice. It has been reported that dsRNA from RSV induces the production of inflammatory cytokines in bronchial epithelial cells via a p38 and not NF-κB-dependent mechanism30. These include KAI-9803 , a selective protein kinase C δ inhibitor peptide to reduce infarct size in subjects with ST elevation myocardial infarction; KAI-4169, a peptide agonist of the calcium sensing receptor for the treatment of secondary hyperparathyroidism; XG-102 (Xigen) , a JNK-inhibiting peptide composed of D-amino acids for the treatment of stroke, inflammation, severe inner ear disorders and acute sensorineural hearing loss; the islet neogenesis-associated protein (INGAP) peptide (Exsulin Corporation) for islet beta cell neogenesis and improvement in glycemic control in type 1 diabetic patients, and AZX100 (Capstone Therapeutics), a peptide derived from HSP20 for the regulation of smooth contraction and dermal scarring. Stability can be increased by the use of D-amino acids as shown by XG-102 (AM-111). Our in vitro data also show that D-TNFRI peptides exhibited increased activity over the L-forms  and the University of Canberra Animal Ethics Committee (project number CEAE 08–13). The research was conducted according to the Animal Welfare Guidelines of the National Health and Medical Research Council of Australia.32. Blood was layered on to Hypaque-Ficoll , d = 1.114, and centrifuged at 600 × g for 20–30 min. After centrifugation, the leukocytes resolved into two bands and the neutrophils were harvested from the second band. The preparations of neutrophils were routinely of >99% viability and >98% purity. These were either used as cells in suspension or as adhered neutrophils by adding the cells to plasma-coated plates as previously described33. The 70Z/3 pre-B cell line, kindly provided by Dr. W. Langdon, University of Western Australia, Perth, Australia, are mouse pre-B lymphocytes which lack binding sites for human TNF and are non-responsive to the cytokine19. The cells were maintained in RPMI 1640 medium with 4.5 g/L glucose, 2 mM L-glutamine, 50 mM 2-mercaptoethanol and 10% FCS in an atmosphere of 95% air and 5% CO2. The monocytic cell line, MonoMac 6 was kindly provided by Dr. H.W.L. Ziegler-Heitbrock  and maintained in RPMI 1640 supplemented with 10% heat-inactivated foetal calf serum, 1 mM pyruvate, glutamine and antibiotics. A mouse fibroblastic fibrosarcoma cell line WEHI-164, clone 13 (from the ATCC) was obtained from Dr. Geeta Chauhdri . The cells were maintained in RPMI-1640 medium as above and the cells were detached with 0.25% trypsin EDTA. HEK 293T cells were maintained in DMEM, supplemented with 10% foetal calf serum. Cells, stably transfected with pRK5-TRAF2-FLAG, pRK5-TRAF287–501-FLAG , or an empty plasmid  using LipofectAMINE 2000 , were obtained from Dr. P. Xia, Centenary Institute of Cancer Medicine and Cell Biology, Sydney, Australia. The promyelotic leukaemia HL-60 cells  were cultured in RPMI-1640 supplemented with antibiotics, L-glutamine and 10% heat-inactivated foetal calf serum. Twelve-well culture plates were seeded with 1–2 × 106/ml HL-60s per well and the media supplemented with 1.25% v/v DMSO and allowed to differentiate for 4–5 days34, before treatment with TNF/peptides.The study was approved by the CYWHS Human Ethics Committee (project number HREC 595/12/10). Neutrophils were prepared from peripheral blood of healthy donors, who had given informed consent, by a rapid single-step technique1] was purchased from Prospec-Tany TechnoGene .Human recombinant TNF was a gift from Dr. G.R. Adolf  and also purchased from ProSpec-Tany TechnoGene . Recombinant mouse TNFα was purchased from Gibco . TNF stock solution was stored at −20 °C until required. Fresh dilutions of TNF were prepared in HBSS just prior to use. Human recombinant soluble TNFRI [rHusTNFRI (22–211), prepared as a chimeric protein with the 6x histidine tagged Fc portion part of human IgG70–80 and its control (GGDPGIVTH) were synthesised by Auspep Pty Ltd,  and Genscript Biotech . While it is difficult to directly equate the TNF activity to TNF70–80 because of the lack of tumour cell cytotoxicity of this peptide, comparisons on their ability to stimulate nitric oxide production in macrophage have been used. These show that 5.0 μg/ml of TNF70–80 equates to 1000 U/ml of TNF9. The sequence from the natural TNF-α was modified by substitution of isoleucine for leucine at residue 79. The modification extended the serum half-life of the peptide to >90 min and increased water solubility. Fresh dilutions of the peptide were prepared daily in HBSS or HBSS with 1% BSA. The His-tagged M4 (HM4) ∆HM4 (LKPGTT) was  purchased from Auspep Pty Ltd . TNF132–150 (LSAEINRPDYLDFAESGQV), TNFRI209–211 peptide (GTT); scrambled control (TGT) and TNFRI206–211 (EDSGTT), scrambled control (GEDTST), TNF198–211 (GIENVKGTEDSGTT) D-forms, as well as peptides of sequences in CRD of TNFRI: TNFRI77–82 from CRD1 (QDTDCR), TNFRI90–95 from CRD2 (TASENH) and TNFRI129–134 from CRD3 (KNQYRH) were purchased from GenScript Biotech . Peptides were purified by high-performance liquid chromatography (HPLC) and analysed by mass spectrometry to be >85% pure. Peptides were stored at −20 °C and solutions were made in HBSS or RPMI prior to use.TNF3VO4, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and 10 μg/ml leupeptin, aprotonin, pepstatin A and benzamidine) for 2 h at 4 °C with constant mixing35. After centrifugation , the supernatants were collected and the protein content of the lysates was determined by Lowry’s method of protein determination. Samples were stored at −70 °C until assayed. For western blot analysis, samples were mixed with Laemmli buffer and boiled before being stored for subsequent electrophoresis.The cells were lysed in 200 μl of buffer A  Nonidet P-40, 100 mM NaCl, 1 mM EDTA, 2 mM Nag for 15 s) and washed once with buffer A (4 °C), once with buffer B  and once with assay buffer 35. The assay buffer was removed following a further centrifugation in preparation for kinase activity assay.Lysates containing equal amounts of protein (0.5–1 mg) were pre-cleared with 15 μl/sample protein A sepharose   (4 °C) before being incubated with rabbit polyclonal anti-mouse p38α antibody  (3 μg/sample). After mixing for 2 h (4 °C), the immune complexes were precipitated by the addition of 20 μl/sample protein A sepharose. The immunoprecipitates were collected by centrifugation . The amounts of phosphorylated p38, ERK1/ERK2, JNK and levels of IκB-α were detected using rabbit polyclonal anti-p-p38 (Thr 180/Tyr 182)-R (sc-17852-R), mouse monoclonal anti-p-ERK1/2 (pT202/pY204.22A) (sc-136521), mouse monoclonal anti-p-JNK (G-7) (sc-6254) and rabbit polyclonal anti-IκBα (C-21)(sc-371) antibody , respectively. These antibodies were used at 1:1000 dilution. Immune complexes were visualised by enhanced chemiluminescence. Ponceau S (0.1% in 5% acetic acid) staining was performed to verify total protein loading. Some blots were stripped and re-probed with rabbit polyclonal anti-p38 (C-20)  antibody to confirm equal loading.Western blot analysis was conducted as described previously32P-ATP35. We have demonstrated that p38 immunoprecipitates do not contain ERK1/ERK235. Briefly, 30 μl of assay buffer (30 °C) containing 10 μCi of γ32P-ATP, 3.8 mM p-nitrophenyl phosphate and 15 μg of myelin basic protein was added to each sample. The samples were then incubated in a water bath at 30 °C. After 15 min, the assay was terminated by the addition of Laemmli buffer and boiling the samples (100 °C) for 5 min, Following fractionation (12% SDS polyacrylamide gels), the amount of 32P-MBP was determined using an Instant Imager .p38 activity was determined, after immunoprecipitation, as described using myelin basic protein (MPB) as a substrate for the kinase in the presence of 19 . The signal sequence from the TNFR1 cDNA was isolated by PCR and cloned, via an NheI site incorporated into the 5′ primer and a KpnI site in the 3′ primer, into the expression vector pcDNA3.1 . A double-stranded oligonucleotide encoding His 6-tag, with KpnI and BamHI sticky ends, in frame with the signal peptide, was cloned downstream of the signal sequence. The transmembrane and cytoplasmic domains of the TNF receptor were also isolated by PCR and cloned, via a 3′ BstXI site and a 5′ XhoI site incorporated into the PCR primers, 3′ of the hexahistidine sequence. This construct was designated cDNA3sigHIScTNF (WT). The mutants designated M1 and M4  was kindly provided by Dr. M. Kronke2O overnight at 4 °C and blocked with 1% BSA and 0.05% Tween-20 in PBS for 1 h at 4 °C. Aliquot of 10 μM of biotin-labelled TNF70–80  plus various concentrations of unlabelled TNF70–80 or control peptide were added to each well and incubated for 3 h at 4 °C. The wells were washed four times with wash buffer (PBS containing 0.05% Tween-20) and the plates were incubated with 100 μl/well of poly-horseradish peroxidase streptavidin conjugate (1/6000 dilution in 1% BSA and 0.05% Tween-20 in PBS) for 45 min at 4 °C. Wells were washed four times with wash buffer and bound enzyme detected by the addition of 3′,3′,5′,5′-tetramethylbenzidine (TMB) substrate . Absorbance was measured using dual filter at 570/450 nm on a Dynatech MR700 Plate Reader .Microtitre plates were coated with 100 μl/well of 3 μg/ml rHusTNFRI  in 18MQ-cm H198–211 in MilliQ water or TNFRI206–211 or the corresponding scrambled peptide in carbonate-bicarbonate buffer (pH 9.6) overnight at 4 °C and blocked with 1% BSA and 0.05% Tween-20 in PBS for 1 h at 4 °C. In the TNFRI198–211-coated plates, various concentrations of biotinylated TNF70–80 or control peptide were added to triplicate wells and incubated for 3 h at 4 °C. In the TNFRI206–211/scrambled peptide-coated plates, 50 U of TNF was added to triplicate wells, followed by washing with wash buffer (PBS/0.05% Tween-20) and then incubation with 50 μl of 1.5 μg/ml biotinylated monoclonal anti-TNF antibody . The wells from all plates were washed four times with wash buffer, then incubated with 100 μl/well of poly-horseradish peroxidase streptavidin conjugate (1/6000 dilution in 1% BSA and 0.05% Tween-20 in PBS)  for 45 min at 4 °C. Wells were washed four times with wash buffer and bound enzyme detected by the addition of TMB substrate . Absorbance was measured using dual filter at 570/450 nm on a Dynatech MR700 Plate Reader .Depending on the experiment, microtitre plates were coated with 100 μl/well of either 3 μg/ml TNFRI36 method. Using a 96-well high-binding plate, 100 μl of 0.25 μg/ml sTNFRI in 0.05 M carbonate-bicarbonate buffer (pH 9.6) was incubated overnight at room temperature. The plate was washed three times with wash solution (PBS + 1% Tween-20) for 5 min each and then blocked for 1 h at 37 °C with 100 μl of PBS + 10% BSA. Following another three washes, the plate was loaded with 50 μl of either assay solution (PBS + 4% BSA), or 500 U TNF and/or 10 mM TNFRI206–211 (pre-incubated for 10 min together or separately) in assay solution and incubated for 2 h at room temperature. After three washes, all wells were loaded with 50 μl of 1.5 µg/ml biotinylated monoclonal anti-TNF antibody  for 60 min, washed three times, then incubated with 50 μl of poly-HRP streptavidin conjugate (1/6000 in assay solution) for 30 min. After washing three times, 50 μl of TMB substrate was added and incubated for 15 min, followed by addition of 50 μl 0.5 M sulphuric acid. Absorbance was measured at 570/450 nm.TNF binding to sTNFRI was assayed by a modification of the Davis et al.206–211 or 500 µl of 100 ng/ml mouse TNF was treated with 500 µl of 8 mM of the TNFRI206–211 for 15 min at 37 °C, then added to human or mouse cells (2 × 106) in a final volume of 1 ml or 5 ml, respectively. Following incubation at 37 °C for 15 min  or 30 min (for JNK), cell lysates were prepared for western blot analyses. Adherent neutrophils were required for ensuring ERK activation by TNF. Similarly, HL-60 cell-derived neutrophils were required for inducing JNK activation by TNF.In these experiments, either 100 µl of 200 U/ml human TNF was treated with 100 µl of 4 mM of TNFRI7 cells in 15 ml culture medium. Before performing transient transfection, plasmid DNA (20 μg in 1 ml of RPMI 1640) was mixed with LipofectAMINE 2000  (60 μl in 1 ml of RPMI 1640) and the mixture was left at room temperature for 20 min. The mixture was directly added to the cells and incubated for 24 h. The cells were washed twice with HBSS before use.70Z/3 cells were plated onto 10 cm plates at 4 × 10N-methyl-acridinium nitrate)  and measuring the light output by lucigenin-dependant chemiluminescence37. Neutrophils (5 × 105) were incubated with TNF70–80, TNF or other stimuli that had previously been pre-incubated in the absence or presence of receptor peptides for 20 min. After incubation, 500 μl of lucigenin  was added and the final volume was adjusted to 1 ml. The cells were placed in luminometer  and the resulting chemiluminescence  measured. The results are expressed as maximal/peak rate of chemiluminescence produced unless specified otherwise. CR3 expression on neutrophils was measured by flow cytometry using anti-CD11b antibodies33. Neutrophils (1 × 106) were incubated for 30 min with TNF that had previously been pre-incubated in the absence or presence of receptor peptides for 20 min. After treatment, 2 μl of PE-labelled mouse IgG2a isotype control (Clone X39) or PE-labelled monoclonal anti-CD11b antibody (Clone D12)  was added to neutrophils in 200 μl of Isoton II (BD Biosciences) supplemented with 0.1% (w/v) BSA. After 30 min the cells were washed, fixed with formaldehyde (0.1%) and analysed on a FACScan . Cytokine (IL-1β and IL-8) mRNA productions were measured by quantitative RT-PCR. Briefly, total RNA was extracted from 1 × 106 neutrophils using the TRIzol reagent  according to the manufacturer’s instructions. RNA was converted to cDNA using iScript cDNA synthesis kit . The cDNA was then amplified in triplicate reactions with iQ SYBR Green supermix  and 500 nM of each primer pair for IL-1β  and IL-8  and the house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using iQ5 system with v.3.1 software.Neutrophil superoxide production was measured by an indirect assay involving the reduction of the lucigenin,  carrageenan-induced paw inflammation, (iii) antigen-induced delayed hypersensitivity38, (iv) RSV-induced lung inflammation39. Inflammation was gauged by either one or a combination of the following: tissue changes, cellular infiltration and cytokine levels. Animals were allocated randomly to the various groups. The studies were sufficiently powered based on power calculation at α of 0.05 and β of 0.2.Four models of in vivo inflammation reactions were used to assess the anti-inflammatory activity of the TNFRI peptides. Female, 6–8-week-old BALB/c mice were utilised in these models: (i) lipopolysaccharide-induced peritoneal inflammation40. At 24 h, the animals were killed and cellular infiltrates in the peritoneal exudates examined in Giemsa-stained smears. The total leukocyte and neutrophil number in the peritoneal exudates was enumerated using a hemocytometer chamber and the neutrophils from a differential count of the stained smears.Mice were injected intraperitoneally (i.p.) with 50 µg of LPS  or HBSS38. Six days later, mice were challenged in the right hind footpad with 25 μl of 40% suspension of SRBC. The DTH response was determined 24 h post challenge by measuring the footpad thickness of the unchallenged vs. SRBC-injected footpads with a caliper on a micrometre screw gauge. The inflammatory response was expressed as the difference in thickness (mm) between the unchallenged and antigen-challenged footpads.The delayed-type hypersensitivity (DTH) response was conducted by injecting mice with sheep red blood cells (SRBC) (100 µl of 10% haematocrit)  subcutaneously in the back39. The swelling/inflammation was assessed 24 h later by comparing the thickness between the unchallenged vs. carrageenan challenged footpads as described above.Carrageenan-induced paw inflammation was induced by inoculating mice with 1 ml/kg of a 1% solution of carrageenan type IV (w/v) into the right hind footpad206–211 or the control scrambled peptide administered on day −1, 1 and 3. On day 0, mice were infected i.n. with 1 × 106 plaque forming units of RSV A2. Prior to all intranasal treatments and infections mice were anaesthetised i.p. with Alfaxan. On day 5, mice were killed.BALB/c mice were housed in microisolator cages with their own food and water supply and were treated intranasally (i.n.) with three doses of 100 mg/kg of TNFRI2. After 2 h, 8 ml of DMEM with 5% foetal bovine serum was added and flasks incubated at 37 °C and 5% CO2 for ~2–3 days or until 25–50% syncytia formation was observed. At harvest time all but 2 ml of media was removed and cells were scraped from the flask, transferred to a 50 ml tube and incubated on ice for 10 min. Cells were sonicated for 5 s at 25 W, three times, with a 2 min incubation on ice between each sonication. Cell debris was removed by centrifugation at 350 × g for 7 min at 4 °C and the supernatant containing the virus stored at −80 °C. The Vero E6 cells used were VERO 1008  originally sourced from ATCC (ATCC CRL-1586). Cells were routinely tested for mycoplasma by using the LookOut Mycoplasma PCR detection kit (Sigma-Aldrich cat no. MP0035)The RSV strain A2 was propagated in Vero E6 cells. An 80% confluent T150 flask of Vero E6 cells was infected with RSV at a multiplicity of infection of 0.5 in a total volume of 6 ml of DMEM. Cells were infected for 2 h at 37 °C and 5% COg for 5 min and the supernatant collected and stored at −80 °C. The cell pellets were lysed with BD PharmLyse Lysing Buffer, washed with PBS and resuspended in a small volume of PBS. Total BAL cells were counted using a hemocytometer. Cells were centrifuged onto glass slides  using a cytospin , fixed in methanol and stained with modified Giemsa. Cell differential percentages were determined by light microscopy.After killing, the left lung was ligated at the left main branch of the trachea using surgical thread, and the right lung lavaged twice with 0.5 ml PBS. BAL fluid was centrifuged at room temperature at 350 × 41.A lobe of the non-lavaged lung was fixed in 10% formalin, embedded in paraffin and 3 μm sections stained with hematoxylin & eosin (H&E). Slides were scored by an observer blinded to the treatment groups. The severity of inflammation was quantitated on a 0–3 scale with uninfected control mice defined as 0, and 3 being the maximum inflammation scoreIL-4 and IFN-γ cytokine concentration was determined from the first BAL wash by ELISA kits from BD Biosciences and R&D systems, respectively, according to the manufacturer’s instructions.g and the supernatant collected. Twenty-four-well tissue culture plates were seeded with Vero E6 cells in DMEM containing 5% FBS incubated overnight at 37 °C and 5% CO2 to obtain 80–90% confluency. Monolayers were washed with HBSS and serial dilutions of the lung supernatants, in DMEM, were added to the cell monolayers in quadruplicate. Plates were incubated at 37 °C in 5% CO2 for 1 h. The wells were overlaid with 1 ml of 2% methylcellulose media  and incubated at 37 °C and 5% CO2. After 6 days, the overlay was removed and monolayers fixed with 1 ml of ice-cold acetone:methanol (60:40) for 10 min. Plates were air dried at room temperature and blocked in 5% skim milk powder diluted in PBS for 30 min at room temperature. Primary RSV monoclonal anti-F antibody 131–2A was added to wells for 2 h, followed by goat anti-mouse IgG-alkaline phosphatase secondary antibody for 1 h. All antibodies were diluted 1:1000 in 5% block and incubated at 37 °C. After each antibody, plates were washed three times with PBS containing 0.5% Tween-20. The assay was developed using the Alkaline Phosphatase Substrate Kit II  as per manufacturer’s protocol.RSV from a lobe of the non-lavaged lung was determined by immunostaining. The lung was homogenised in PBS  using the TissueLyser II , centrifuged at 1700 × t-test, one sample t-test or one-way ANOVA followed by Dunnett’s multiple comparison test. For non-parametric data, a Mann–Whitney analysis (one or two tailed) (comparison between two groups), a Fischer exact test (one tail) or, either a Friedman test or a Kruskal–Wallis analysis was performed, followed by Dunn’s multiple comparison test. Differences were considered statistically significant when p < 0.05. Statistical analyses were performed on GraphPad Prism 5 software.Results are presented as mean ± standard error of the mean (SEM) unless stated otherwise. The F-test or Brown–Forsythe test was used to examine homogeneity or equality of variance. For parametric data, differences were analysed by either one or two-tailed The authors declare that the data supporting the findings of this study are available within the paper and from the authors on request.


Assembly of recombinant capsid proteins into virus-like particles (VLPs) still represents an interesting challenge in virus-based nanotechnologies. The structure of VLPs has gained importance for the development and design of new adjuvants and antigen carriers. The potential of Tobacco etch virus capsid protein (TEV CP) as adjuvant has not been evaluated to date.Escherichia coli were generated: a wild-type version (TEV-CP) and a C-terminal hexahistidine (His)-tagged version (His-TEV-CP). Although both versions were expressed in the soluble fraction of E. coli lysates, only His-TEV-CP self-assembled into micrometric flexuous filamentous VLPs. In addition, the His-tag enabled high yields and facilitated purification of TEV VLPs. These TEV VLPs elicited broader IgG2-specific antibody response against a novel porcine reproductive and respiratory syndrome virus (PRRSV) protein when compared to the potent IgG1 response induced by the protein alone.Two constructs for TEV CP expression in His-TEV CP was purified by immobilized metal affinity chromatography and assembled into VLPs, some of them reaching 2-μm length. TEV VLPs administered along with PRRSV chimeric protein changed the IgG2/IgG1 ratio against the chimeric protein, suggesting that TEV CP can modulate the immune response against a soluble antigen.The online version of this article (doi:10.1186/s12985-016-0651-y) contains supplementary material, which is available to authorized users. The structural proteins of some viruses occasionally mimic the three-dimensional nature of an actual virus while lacking the virus genome packaged inside its capsid . These sTobacco etch virus (TEV) particles as an adjuvant and our findings suggested that TEV induce both humoral and cellular response without the need of any other stimulus [E. coli) the JGMV CP that assembled into virus-like particle structures. Since then, the list of VLP assembly from E. coli-expressed potyviral CPs has been extended to Potato virus Y (PVY) [E. coli cultures. We also evaluated the potential use of TEV CP VLPs as an adjuvant for a novel porcine respiratory and reproductive syndrome virus (PRRSV) chimeric protein.We previously attempted to explore the potential of stimulus . Howeverstimulus . By 1990stimulus . For thestimulus  successf Y (PVY) , Pepper  Y (PVY) , Papaya  Y (PVY)  and TEV  Y (PVY) . InteresE. coli lysates. For generation of the TEV CP expression constructs, tev cp gene (GenBank: JX512813.1) modified to contain a glycine codon at 5′- end for facilitating subsequent cloning and codon-optimized for improving E. coli expression was synthetically obtained . NcoI and XhoI recognition sites were introduced at 5′- and 3′- ends, respectively, by PCR for subsequent ligation into IPTG-inducible expression vector pET28a + . A stop codon upstream of XhoI recognition site was added in the TEV-CP version, which would result in a protein without the His-tag. The cloning procedures were performed as described previously [E. coli expression strain and IPTG-induced cultures were analyzed for TEV CP expression. Western blot revealed a band corresponding to ~ 31 kDa for each version of the TEV CP . The estimated yield of this non-tagged version was 1.2 mg/l of culture, as assessed by densitometric analysis, due to presence of several proteins belonging to the expression host that also precipitated with PEG. On the other hand, the His-tagged version was purified by Ni2+ affinity chromatography. Analysis of purification fractions showed recovery of partially purified His-TEV-CP protein  PEG 8000 for 1.5 h at 4 °C with constant shaking and left for 1 h at room temperature (22 ± 2 °C). Then, the sample was centrifuged at 4 °C, for 20 min, at 2,370 × ein Fig. . Larger ein Fig. . The yieE. coli-expressed potato virus M (PVM) VLP preparations. Nonetheless, these results must be taken carefully, because the possibility that RNA binds to the nickel-charged column used in this work to purify His-tagged TEV CP can not be ruled out [For assessing TEV CP VLPs formation, transmission electron microscopy (TEM) was performed Fig. . The miculed out .Fig. 2TEE. coli-expressed PRRSV chimeric protein, named PRRSVchim, was used as antigen. This protein comprises PRRSV GP3 and GP4 epitopes, GP5 and M ectodomains and thioredoxin as fusion partner. This chimeric protein was developed by our group for swine vaccination against this virus and all its details are included in a different manuscript that is under preparation. Twenty-five six-week-old female BALB/c mice  were divided equally into 5 groups immunized with 25 μg of His-TEV-CP (TEV VLPs25), 10 μg of PRRSVchim (PRRSV10), 10 μg of His-TEV-CP plus 10 μg of PRRSVchim (TEV VLPs10/PRRSV10), 25 μg of His-TEV-CP plus10 μg of PRRSVchim (TEV VLPs25/PRRSV10), or 10 μg of PRRSVchim plus incomplete Freund’s adjuvant (PRRSV10/IFA). Immunization protocol as well as ELISA procedure for IgG, IgG1, IgG2a and IgG2b isotype determinations against PRRSVchim were performed as described by Guerrero-Rodríguez and others [chim-specific IgG and IgG1 isotype determinations showed an increase in the antibody response elicited by PRRSVchim. However, TEV CP VLPs plus PRRSVchim formulations induced an IgG and IgG1 response against the PRRSVchim protein 5-25 times lower than PRRSVchim treatment. Conversely, PRRSVchim induced a low response of IgG2a and IgG2b isotypes, similar to the non-specific response to TEV CP VLPs. In addition, the IgG2a/IgG1 ratio against PRRSVchim protein was 125 times higher in TEV CP VLPs plus PRRSVchim formulations than PRRSVchim itself. The antibody response against TEV CP VLPs was determined as well  and the filamentous particles of Papaya mosaic virus (PapMV) . The proion Fig. , howeverchim demonstrated to elicit potent IgG antibody response without the need of any adjuvant. This high immunogenicity is perhaps due to the ability of this chimeric protein to form oligomeric structures, as demonstrated by dynamic light scattering (Additional file chim did not increase when the TEV CP VLPs were administered along with, even when different ratios of VLPs were used (Fig. chim protein alone (Fig. chim antibody response, it appears that TEV CP VLPs have a role in balancing both arms of the immune system, humoral and cellular response, against PRRSVchim. Some authors have mentioned that the success of a PRRSV vaccine relies on its ability to induce both neutralizing epitopes and cellular response [2+ affinity chromatography and self-assembles into long VLPs. Also, these particles contribute in balancing the immune response elicited by a novel chimeric protein comprising sequences from a swine virus, which in turn is a potent immunogen per se. The immune response of swine immunized with these TEV CP VLPs/PRRSVchim formulations needs to be investigated. These findings highlight the potential of TEV CP VLPs as particulate adjuvants with immune modulation properties without the need of antigen’s attachment.The novel PRRSVsed Fig. . In contone Fig. . In miceresponse . To our response . Finallyresponse . In summ


A 44-year-old African-American male was admitted to our hospital after a suicide attempt. He had depression, poor cognitive function, choreiform movements, difficulty pronouncing words, and difficulty walking. His symptoms had worsened markedly over several months. Chorea lead to genetic testing that confirmed a diagnosis of Huntington Disease (HD). A CT scan of the head showed wider ventricles than is typical of HD. The head CT and gait change suggested normal pressure hydrocephalus (NPH). Lumbar puncture (LP) led to improved neuropsychologic test scores and walking thereby supporting the diagnosis of NPH. Surprisingly, the LP also led to an 80% improvement of chorea. There are two other reports of an association between HD and NPH. NPH should be considered in HD patients with atypical symptoms, such as the inability to walk or rapid progression, as its treatment may lead to improved cognition, gait, and chorea.


Current treatments for hepatocellular carcinoma (HCC) have shown inadequate. MicroRNA-122 (miR-122) mediated RNA interference brings new prospects. A safe, efficient miRNA delivery system is an indispensable assurance. Previously, we developed an MS2 bacteriophage virus-like particle (VLP)-based microRNA delivery system crosslinked with the HIV TAT peptide, which served as an effective inhibitor in the treatments of systemic lupus erythematosus and osteoporosis. However, defects, such as low crosslinking efficiency, high cost, and potential toxicity of the crosslinking agent, needed to be confronted. Therefore, TAT peptide was designed to display on the surface of MS2 VLPs, instead of being chemically crosslinked, using the platform of phage surface display. The results reflected that MS2 VLPs displaying TAT could effectively penetrate the cytomembrane and deliver miR-122. Additionally, its inhibitory effects on HCC were significant in Hep3B, HepG2, and Huh7 cells and Hep3B related animal models. Thus, we have established a novel miR-122 delivery system based on MS2 VLPs surface displaying TAT peptide, which could effectively perform the function of penetrating cytomembrane and the inhibition of HCC. Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumor, with the third most frequent cause of cancer-related deaths –3. ConsiMicroRNAs (miRNAs), endogenous small non-coding RNAs, can interact with target messenger RNA (mRNA) 3′-untranslated regions (3′-UTR) and negatively regulate their gene expression by inhibiting translation or degrading targeted mRNAs at the post-transcriptional level . BecausemiRNA delivery depends primarily on an effective transport system , 22. HowPreviously, we developed an MS2 VLP-based microRNA-146a delivery system and conjugated human immunodeficiency virus (HIV) TAT (47-57) cell-penetrating peptide to it, using chemical cross-linkers . MS2-miRThe technique of phage surface display brings new prospects. Previously, the immunogenic epitope peptides were successfully displayed by MS2 VLP platforms, which elicited broadly cross-reactive and cross-neutralizing antibodies against diverse human papillomavirus (HPV) types –36. NeveHIV TAT was the first reported cell penetrating peptide . The regAccording to the previous report –36, the Further identifications were performed as follows. First, the assembly effects of VLPs surface displaying TAT were confirmed by transmission electron microscopy  Figure . The sizTo identify whether recombinant MS2 VLPs displaying TAT peptide could penetrate the cell membrane, the 2MS-TAT-miR122 VLPs and MS-miR122 VLPs labeled with fluorescein isothiocyanate  were respectively incubated with Hep3B and Huh7 cells. Blue fluorescence from 4′, 6-diamidino-2-phenylindole (DAPI) was observed in the nuclei; green fluorescence was emitted by FITC Figure .After incubation with 2MS-TAT-miR122 VLPs labeled with FITC for 6 h, the green fluorescence was observed in the cytoplasm of both Hep3B  of VLPs was > 100μg/mL in Hep3b, HepG2 and Huh7 Figure . AlthougP < 0.05) could be observed in 2MS-TAT-miR122 VLPs treated groups, than in MS-miR122 VLPs crosslinked with TAT groups . In invasion tests for the three kinds of cells, the decrease for 2MS-TAT-miR122 VLPs treated groups was nearly 30%, whereas that for MS-miR122 VLPs crosslinked with TAT groups was 20%  between the above two approaches.To further examine the influence of 2MS-TAT-miR122 VLPs in neoplasm invasion and migration, we performed transwell migration and invasion assays. The results for migration revealed approximately 20% decrease rate in 2MS-TAT-miR122 VLPs treated groups Figure . Neverth% Figure , which iP < 0.01).Next, we focused on cell apoptosis, which also contributes to the inhibition of cancer cells. miR-122 is known to induce hepatoma cell apoptosis , 20, 41.P < 0.05) compared to that by MS-miR122 VLPs crosslinked with TAT . Moreover, the inhibitory effects of 2MS-TAT-miR122 VLPs was more significant (P < 0.01) than MS-miR122 VLPs crosslinked with TAT group, especially during the period of administration of the last three injections.In order to investigate the inhibitory effects of 2MS-TAT-miR122 VLPs in vivo, the nude mouse models of HCC were established. After inoculation of Hep3B subcutaneously, the tumor-burdened BALB/c nude mice were randomly assigned and administrated MS2 VLPs by tail vein Figure . The sizP < 0.01).After the last injection was administered, two mice from each of the groups were selected to perform CT scan Figure . Tumor vP < 0.01). The expression levels of cyclin G1 and Igf1r significantly decreased in 2MS-TAT-miR122 VLPs group , which was in accordance with the in vitro results of the Hep3B cell line. Taken together, the results indicated that 2MS-TAT-miR122 VLPs could effective suppress tumor growth in vitro and in vivo.H&E staining revealed degeneration and necrosis of tumor tissue in the group of 2MS-TAT-miR122 VLPs Figure . Immunohp Figure   high biocompatibility and biodegradability of MS2 VLPs, (ii) feasible and simple preparation process, (iii) low cost of displaying TAT peptide without consideration of cross-linker or synthetic peptides, (iv) short preparation period, (v) stable productions without large inter-assay variations, (vi) suitable preparation conditions instead of rigorous requirements for PH value and ion concentrations, and (vii) more obvious effects for anti-tumor therapy than previously. Hence, recombinant MS2 VLPs displaying TAT are an efficient, feasible delivery strategy for miR-122 delivery and are promising in HCC treatment.2.The Hep3B, HepG2, Huh7, and HeLa cell lines were obtained from the Cell Bank of the Chinese Academy of Medical Science . The Hep3B cell line was maintained in minimum essential medium , supplemented with 15% fetal bovine serum . The HepG2, Huh7, and Hela cell lines were cultured in Dulbecco's modified Eagle's medium , supplemented with 10% FBS. All cell lines were cultured with 100 units/mL penicillin/streptomycin , and maintained in an incubator at 37°C with 5% COThe sequence of pre-miR122  (Table The plasmid p2MS-TAT-miR122 and p2MS-TAT-miRNC were transformed into competent cells of the E. coli BL21 (DE3) strain respectively. The monoclonal colony was selected, amplified, harvested and wall-cracked. The proteins were purified and the products were harvested Figure . All of The prepared MS2 VLPs were verified by TEM , SDS-PAGE and western blot analysis with anti-MS2 antibody. RT-PCR and 1% agarose gel electrophoresis were performed as well. Additionally, after digested with 1μl DNase I (10 U/mL) and 1μl RNase A (10 mg/mL) at 37°C for 2 h, the products were analyzed by 1% agarose gel electrophoresis GelRed  staining.The prepared products of pMS-miR122 and pMS-miRNC plasmids were named MS-miR122 VLPs and MS-miRNC VLPs, respectively. These VLPs were conjugated to HIV TAT (47-57) peptides with a linker . The crosslinking reagent sulfosuccinimidyl 4- cyclohexane-1-carboxylate (Sulfo-SMCC) was acquired from Thermo Fisher Scientific . SDS-PAGE was performed to verify the effect of crosslinking .The FITC  was weighed and dissolved into dimethyl sulfoxide (DMSO). The 2MS-TAT-miR122 VLPs and MS-miR122 VLPs were crosslinked with dissolved FITC at 4°C for 2 h, separately. Hep3B and Huh7 cells were used in this experiment, and the process for sample preparation was according to the reported protocols . Untreat3 cells were cultured in each well of a 24-well plate, and 2MS-TAT-miR122 VLPs or 2MS-TAT-miRNC VLPs were added to the wells with: (a) different concentrations of MS2 VLPs (8 or 40 μg/mL) for 24 h; (b) various cell lines  for 24 h; (c) final concentrations of 40 μg/mL MS2 VLPs for different time periods  in Hep3B. The processes for RNA extraction, amplification and detection were according to previous protocols [−ΔΔCt method. Primers are listed in Table 5 × 103 per well) were cultured in 96-well plates and incubated at 37°C overnight. The HCC cell lines Hep3B, HepG2, and Huh7 were used in this experiment. 2MS-TAT-miR122 VLPs (negative control: 2MS-TAT-miRNC VLPs) or MS-miR122 VLPs (negative control: MS-miRNC VLPs) (100 μL each) of different concentrations (1 μg/mL to 1000 μg/mL) were respectively administered to different cell lines in 96-well plates for 72 h. CCK-8 reagent was added, and the absorbance at 450 nm was measured. All experiments were repeated three times.Cell proliferation and cytotoxity were monitored using Cell Counting Kit-8 assay . Cells (1 × 105 cells per well), and treated with 40 μg/mL 2MS-TAT-miR122 VLPs (negative control: 2MS-TAT-miRNC VLPs) or MS-miR122 VLPs (negative control: MS-miRNC VLPs) crosslinked with TAT, individually for 36 h. The conditions of different treated cells were photographed with a light microscope .Hep3B, HepG2 and Huh7 cells were seeded in 6-well plates  coating. After incubation for 24 hours, the cells on the upper surface of the filters were removed with a cotton swab. The cells that had migrated or invaded into the lower surface of the filters were fixed and stained with a Diff-Quik Staining Kit , and were counted in five random 400× visual fields under a microscope for each group.Hep3B, HepG2 and Huh7 cells were treated with 40 μg/mL 2MS-TAT-miR122 VLPs (negative control: 2MS-TAT-miRNC VLPs) or MS-miR122 VLPs (negative control: MS-miRNC VLPs) crosslinked with TAT, individually for 48 h. Then the cells treated for apoptosis analysis were digested, washed twice with phosphate-buffered saline , double-stained with Annexin V-FITC and propidium iodide , and analyzed with a flow cytometer . The percentage of early and late apoptotic cells was distinguished, counted, and compared between different treatment groups. All experiments were performed in triplicate.Hep3B, HepG2 and Huh7 cells were treated with 40 μg/mL 2MS-TAT-miR122 VLPs (negative control: 2MS-TAT-miRNC VLPs) or MS-miR122 VLPs (negative control: MS-miRNC VLPs) crosslinked with TAT, individually for 48 h. Western blot was conducted as previously described , using p7), combined with BD Matrigel Basement Membrane Matrix , were subcutaneously injected into the posterior flank of each mouse. When the tumor reached approximately 0.5 cm3, observed with naked eye, the mice were ready for VLP administration, after being randomly grouped. Four groups were designed in this experiment: (i) treated with 2MS-TAT-miRNC VLPs (5 mice), (ii) treated with MS-miRNC VLPs crosslinked with TAT (5 mice), (iii) treated with 2MS-TAT-miR122 VLPs (7 mice), and (iv) treated with MS-miR122 VLPs crosslinked with TAT (7 mice). VLPs (100 ng per administration) were administrated twice a week via tail vein for 3 weeks. Tumor volumes were calculated using the equation,Female BALB/c nude mice  were purchased from Vital River , and fed a standard diet, under the conditions of SPF isolation in the Institute of Laboratory Animal Science . All animals used in this experiment were approved by the Ethics Committee of the National Center for Clinical Laboratories. Hep3B cells  and the tumor volume for each mouse was calculated in the way of CT scan. Subsequently, all tumor-bearing mice were euthanized. The tumors were separated and weighed, and the subcutaneous growth of each tumor was recorded.The isolated tumor tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned to 5 μm thickness. HE staining was followed using previous protocols , and immt-test. The results were considered to be statistically significant if P < 0.05. Additionally, data compared with their negative controls is represented by: * P < 0.05, ** P < 0.01, whereas data from 2MS-TAT-miR122 VLPs treated group compared with MS-miR122 VLPs crosslinked with TAT treated group is represented by: *P < 0.05, **P < 0.01.Statistical analyses were performed using SPSS 20.0 software, and presented with the GraphPad Prism 6 Software. The differences between the groups were tested using two-tailed Student's


The role of cytokines and chemokines in anti-viral defense has been demonstrated, but their relative contribution to protective anti-viral responses in vivo is not fully understood. Cytokine response modifier D (CrmD) is a secreted receptor for TNF and lymphotoxin containing the smallpox virus-encoded chemokine receptor (SECRET) domain and is expressed by ectromelia virus, the causative agent of the smallpox-like disease mousepox. Here we show that CrmD is an essential virulence factor that controls natural killer cell activation and allows progression of fatal mousepox, and demonstrate that both SECRET and TNF binding domains are required for full CrmD activity. Vaccination with recombinant CrmD protects animals from lethal mousepox. These results indicate that a specific set of chemokines enhance the inflammatory and protective anti-viral responses mediated by TNF and lymphotoxin, and illustrate how viruses optimize anti-TNF strategies with the addition of a chemokine binding domain as soluble decoy receptors. Cytokines play critical roles in the anti-viral response but their contribution in vivo remains unclear. Here the authors show that viral CrmD is a major virulence determinant and requires the SECRET and TNF binding domains to counter both the chemokine and cytokine driven inflammatory defenses. The poxvirus homologs of host tumour necrosis factor TNF (TNF) receptors (vTNFRs) block the pro-inflammatory activity of some TNF superfamily (TNFSF) ligands. Five vTNFRs have been described in poxviruses, a viral homolog of host TNFSF receptor CD30 and four TNF inhibitors named cytokine response modifiers B, C, D, and E . vTNFRs are differently expressed among viral species and show distinct binding and inhibitory properties13. While CrmE and CrmC are specific TNF inhibitors, CrmD and CrmB block TNF and lymphotoxin α (LTα). Furthermore, we have recently described that vTNFRs can inhibit membrane TNF and that CrmD and CrmB neutralize another TNFSF ligand, LTβ15. In addition to the cysteine-rich domains (CRDs), characteristic of the ligand binding region of cellular TNFRs, CrmB and CrmD have a C-terminal domain unrelated to host proteins that binds chemokines and was named SECRET  domain16. The crystal structure of the CrmD SECRET domain showed a beta-sandwich fold similar to that of the viral chemokine binding proteins (vCKBPs) 35-kDa and A41, but a different chemokine interaction region may confer its distinct narrower binding specificity19. Such variety of activities may provide poxviruses the ability to differentially block chemokines involved in distinct anti-viral responses, to inhibit chemokines at different stages of infection in the host or to simultaneously inhibit chemokines and TNF. Interestingly, the beta-sandwich fold of vCKBPs has also been observed in other unrelated poxviral proteins including CPXV203, a major histocompatibility complex I binding protein encoded by cowpox virus (CPXV), and GIF, the granulocyte-macrophage colony-stimulating factor and interleukin 2 inhibitor of the parapox Orf virus20. To reflect such diverse range of immunomodulatory activities this folding has been named as poxvirus immune evasion domain20.A unique immune evasion mechanism employed by poxviruses and herpesviruses is the production of soluble binding proteins or secreted versions of host receptors that neutralize cytokines22 genetically related to vaccinia virus (VACV), variola virus (VARV)  and monkeypox virus (MPXV)24, a human pathogen whose incidence is increasing due to the cessation of mass smallpox vaccination in Africa26. Susceptible strains of mice infected with ECTV develop mousepox, a severe disease that constitutes a good model for smallpox. ECTV infection of susceptible mouse strains via the s.c. route has been exploited as a model of generalized virus infections, genetic resistance to disease, and viral immunology27. In ECTV, CrmD is the only secreted TNFR. Similarly, both VARV and MPXV express a single vTNFR with similar characteristics, CrmB. By contrast, CPXV expresses four distinct vTNFRs13. In addition, ECTV and other poxviruses encode intracellular proteins that inhibit TNF-induced signalling, underscoring the importance of TNF in antiviral reponses28.ECTV is a mouse-specific orthopoxvirus29. A direct antiviral activity of TNF has been proposed using a recombinant VACV expressing TNF30. This direct effect was substantiated in TNF-deficient mice infected with VACV, which showed a modest (two-fold) reduction in LD50 as compared to WT mice, that was accompanied by an increased virus load but not by a diminished T cell response31. Although resistance to mousepox was associated with Th1-like cytokine expression, including TNF, blockade of TNF using monoclonal antibodies did not affect the generation of NK cell and CTL responses, virus clearance or resistance to ECTV infection32 and treatment with TNF did only reduce the mortality rate from 100% to 70% in susceptible BALB/c mice33. VACV-infected TNFR2-deficient C57BL6 mice produced higher viral titers in spleens and livers and reduced numbers of inflammatory cell foci in the liver, as compared to WT mice34.However, our knowledge of the role of TNF and LT in the control of poxviral infections in vivo is limited. Knockout mice lacking both TNFR1 and TNFR2 showed a slightly increased susceptibility to ECTV and elevated viral replication, with 60% of the infected animals succumbing to mousepox while all WT mice resisted infection35. Inactivation of a CrmB homolog (M-T2) from myxoma virus reduced clinical signs of illnesss in infeced rabbits36. However, the reported attenuation in the initial studies cannot be formally atributed solely to the absence of the vTNFR since the selection of inadvertent mutations elsewhere in the viral genome was not controlled with the construction of revertant viruses or by sequencing the complete viral genome. Additional studies with the VACV vaccine strain USSR showed that deletion of CrmC or CrmE caused no effect in virulence or a very mild attenuation not affecting mortality, respectively, after i.n. infection of mice37. Recombinant VACV strain Western Reserve expressing CrmC, CrmB or CrmE displayed increased virulence less than 10-fold in an i.n. mouse model, but high virus doses were required to cause disease because the recombinant viruses were deficient in the thymidine kinase gene38. Definitive studies addressing the role of vTNFRs in viral pathogenesis using virulent poxviruses in their natural host are lacking.A contribution of vTNFRs to pathogenesis was initially shown with a CPXV lacking CrmB, but expressing other vTNFRs, which displayed an increased LD50 in infected mice after intracranial inoculation, a route of infection not natural for poxvirusesHere we show that CrmD is an essential virulence factor as deletion of CrmD from ECTV resulted in a dramatic attenuation phenotype, generating an avirulent virus that induced strong NK cell and CD8 T cell responses but did not establish fatal mousepox. This demonstrates a critical role of TNF and a reduced set of chemokines in anti-viral defense. Moreover, this unique model of virus infection in a natural host, together with the construction of mutant viruses, allowed us to dissect the relative contribution of TNF and chemokine activities in vivo. We report that expression of the anti-TNF (CRD domain) or anti-chemokine (SECRET domain) activities of CrmD are not sufficient on their own to confer full virulence to ECTV, suggesting that the function of some chemokines complement TNF in protection against viruses. Furthermore, immunization of mice with recombinant CrmD protected from a lethal ECTV challenge.CrmD gene deleted. As a control for the selection of inadvertent mutations in other genes during the generation of ECTVΔCrmD, a revertant virus (ECTVRevCrmD) with both copies of the full length CrmD gene restored was constructed. To study the differential contribution of TNF- vs chemokine- inhibitory activities of CrmD, a virus expressing only the TNF binding domain of CrmD, composed of CRDs (ECTVRevCRD), was constructed. The complete genome sequence of ECTVRevCRD confirmed the correct incorporation of two copies of the truncated CrmD TNF binding domain and the absence of additional mutations. Replication of recombinant viruses was comparable to that of the parental ECTV  and some B cells (5%), but no CD8+ cells were detected. Sections were stained for expression of inter cellular adhesion molecule-1 (ICAM-1), an integrin ligand overexpressed on endothelial cells of the postcapillary venules in response to proinflammatory cytokines such as TNF40. More than 50% of the vessels in the footpads of ECTVΔCrmD-infected mice showed intense ICAM-1 staining, while only around 25% of the vessels expressed ICAM-1 in ECTV- or ECTVRevCrmD-infected mice  staining of sections of the footpad of ECTVΔCrmD-infected mice showed the presence of a large inflammatory infiltrate with edema in the dermis, which was not detected or much reduced in ECTV-infected mice Fig. . The infice Fig. , indicatnes Fig.  whereas The extent of viral replication and dissemination in the host in the absence of CrmD was analyzed. Viral replication was apparently not hindered at the site of inoculation, as assessed by anti-virus and anti-CrmD staining of sections from footpads of infected mice at 7 dpi Fig. . This alTo further assess the role of the CrmD SECRET domain in mousepox pathogenesis, a recombinant virus was constructed in which CrmD was replaced by a CrmD variant bearing a point mutation (N77F) that lacks TNF binding and inhibitory activity while maintaining its chemokine inhibitory activity. Figure 4 to 106 PFU per animal. With only one animal out of five succumbing to mousepox after infection with the highest dose tested  or the TNF binding domain of CrmD (ECTVRevCRD)  Fig. . ExpressRD) Fig. . Altoget22. To understand the mechanisms by which ECTV CrmD allows viral replication and whether it impairs the immune response, we studied the early NK cell response and the late T cell response against the different recombinant viruses in the DPLN at 2 dpi and in the spleen at 7 dpi, respectively.It has been shown that upon footpad inoculation of ECTV, the NK cell response during the first 4 dpi in the draining popliteal LN (DPLN) and the T cell response at late stages (peak at 7 dpi) in the secondary organs are required to survive lethal mousepox−DX5+) of all the infected groups represented around 3% of the cells in the DPLN whereas it was less than 1% in the PBS-inoculated group  of ECTV, to test the induction of an efficient protective response. Sera from CrmD-immunized mice (14 out of 15 mice), but not from control mice, neutralized the ability of CrmD to inhibit TNF activity in a cytotoxicity assay Figs. . However32. Therefore, reduced viral replication and hence necrosis in this organ may account for survival of mice infected with ECTV lacking either the full length CrmD, or the anti-TNF or anti-chemokine activities of the protein. Additionally, expression of CrmD in the spleen and liver will block anti-viral responses in these organs, consistent with an increased inflammatory response in the liver of ECTVΔCrmD- and ECTVRevCRD-infected mice.The results presented here show that ECTV CrmD is an essential factor for mousepox virulence and that both its TNF and chemokine inhibitory activities contribute to its immunomodulatory role. ECTV replication in secondary replication organs, spleen and liver, was impaired in the absence of CrmD. The most probable cause of death during mousepox is liver failure due to extensive viral replicationCrmD gene from ECTV results in one of the most profound effects on virulence described in poxviruses18. Previous reports have shown that inactivation of other vTNFRs secreted by VACV, CPXV and myxoma virus causes limited viral attenuation, and some reports are not conclusive37. Similarly, inactivation of vCKBPs, 35-kDa and A41 proteins, from poxvirus genomes causes increased leukocyte recruitment to the sites of infection without major effects on the disease progression or mortality, or a slight attenuation as a result of reduced inflammatory pathology18. Interestingly, deletion of the ECTV type I IFN binding protein, which targets a different cytokine, rendered the virus avirulent to a degree similar to that we observed after CrmD deletion44. This suggests a possible link between the type I IFN and the TNF/LT/chemokine anti-viral host responses, as the link described for the type I IFN and nuclear factor kappa B pathways45. Increased LT signalling in the absence of CrmD could contribute to the induction of the IFN response, as described for murine cytomegalovirus46, restricting virus replication through increased type I IFN signalling to immune cells or direct effects on infected cells. In accordance with this, TNF and IFN may act synergistically in anti-viral defense47. Also, a role of the LT network in controlling the type I IFN response has been proposed48.Deletion of the 29. Consistently, treatment of susceptible BALB/c mice with murine TNF hindered ECTV replication and mortality to some extent33. However, TNF had a relatively minor role during ECTV or related poxvirus infections in terms of impact on LD50. Strikingly, the lack of the secreted TNF binding protein CrmD has a profound effect, reducing virulence almost completely. While specific experimental setups may account for these differences, they indicate a role of CrmD beyond inhibition of soluble TNF, which may include inhibition of LTα, LTβ or chemokines. In addition, we have recently demonstrated that CrmD and other vTNFRs interact with membrane TNF and inhibit its cytotoxic activity14. Further, CrmD may trigger reverse signaling in membrane TNF-bearing cells, as shown for viral CD307, and this may influence the anti-viral response.Previous data suggested a role for TNF-induced signalling in mousepox pathogenesis, as transgenic resistant mice lacking functional TNFR1 and TNFR2 became susceptible to ECTV49. Despite historically considered effector cells of the innate immunity, NK cell responses are greatly modulated by dendritic cells (DC)51, being more efficient when both cells are in direct contact51, and this is important for defense against other viral infections52. Accordingly, DC-depleted C57BL/6 mice are susceptible to ECTV53. The DC-NK crosstalk is mediated in mouse and human by the engagement of DC membrane TNF, but not soluble TNF, with NK TNFR255. Therefore, CrmD or the truncated TNF binding domain may be blocking the membrane TNF-TNFR2 interaction hindering an efficient DC-NK cell crosstalk. A weak NK cell activity might explain the high viral titers of ECTV and ECTVRevCRD detected later in the spleen, whereas the dissemination of ECTVRevSECRET and the CrmD deletion mutant is curbed by a potent early NK cell response. After 4 dpi NK cells are no longer required for resistance to ECTV challenge and the T cell response takes over41. At day 7 in the spleen, we observed a depletion of CD8 lymphocytes in mice infected with ECTV expressing CrmD, however, an efficient CD8 response was mounted against all the other viruses tested here. Of note, this CD8 cell depletion did not correlate with viral loads in the spleen since ECTVRevCRD, which reaches equally high viral titers, did not cause lymphopenia. This result suggests that both CrmD immunomodulatory activities, anti-TNF and anti-chemokines, are required for complete control of the T cell response in the spleen. Consistently, the SECRET domain binds Cxcl11, one of the ligands of the chemokine receptor CXCR3 that enhances the ability of CD8 T cells to locate VACV-infected cells and to exert anti-viral effector functions56. In agreement with a role of both TNF and chemokines in T cell responses, neutralization with anti-TNF antibodies does not affect the splenic late CTL response against ECTV in C57BL/6 mice32. However, this might be an indirect effect since splenic lymphopenia has also been observed in other ECTV lethal infections where the host ability to mount an efficient TNF and chemokine response is intact42.This CrmD-membrane TNF interaction might also explain the impaired activation of NK cells in the DPLN of mice infected with ECTVRevCrmD and ECTVRevCRD. NK cells are required at early stages to curb ECTV dissemination from the lymph nodes to the spleen and liver and ultimatelly, to survive to fatal mousepox16. In the absence of CrmD, an increased inflammatory infiltrate was observed both in the footpad and in the liver, consistent with the chemokine inhibitory function of its SECRET domain. More specifically, an important fraction of the footpad infiltrate was composed of cells bearing the chemokine receptor CCR10, which supports migration of lymphocytes towards Ccl28 and Ccl27. Both chemokines are bound with high affinity by CrmD and expressed by skin keratinocytes in response to TNF and other proinflammatory stimuli57. Indeed, Ccl27 is important in T-cell mediated skin inflammation in vivo58. Thus, the potent anti-inflammatory activity of CrmD may be due to its ability to inhibit either TNF/LT as well as a set of chemokines. Consistent with this, VARV CrmB, with properties similar to CrmD, blocks cell migration induced after epicutaneous application of murine TNF59.Here we show that ECTV CrmD efficiently inhibits the establishment of a proinflammatory state at the site of virus inoculation, as evidenced by the increased ICAM-1 expression observed in its absence. The ability of CrmD to block TNF and LT activity, cytokines that induce the expression of ICAM-1 on endothelial cells, could account for thisLeishmamia major mouse model of dermal infection60 and in Listeria monocytogenes-infected mice, where Ccl2-induced recruitment of TNF and iNOS producing cells to the spleen mediates an effective innate immune response61.The infection of mice with ECTVRevCRD or ECTVRevSECRET expressing the anti-TNF/LT or anti-chemokine activity of CrmD, respectively, allowed us to address the contribution of TNF/LT vs. chemokines to inflammatory and protective responses in vivo. Interestingly, ECTVRevCRD was not able to efficiently control the inflammatory infiltrate observed in ECTVΔCrmD-infected mice, in spite of the well-documented and potent pro-inflammatory function of TNF. This suggested that the activity of the mouse chemokines targeted by the SECRET domain  was sufficient to control cell migration and to trigger inflammation when TNF was neutralized by the truncated CrmD protein. The delay in the appearance of the inflammatory infiltrate in ECTVRevCRD-infected mice as compared to that observed with ECTVΔCrmD, probably reflects TNF blockade at the inoculation site and suggests the involvement of TNF in triggering the initial response in vivo. It is important to note that the infiltrating cells are probably contributing to an increased expression of cytokines, and thus chemokine blockade by the CrmD SECRET domain expressed in ECTVRevSECRET infections may impair localized TNF expression. Limited recruitment of cytokine producing cells into the sites of infection may be a general principle in host-pathogen interactions and help pathogens escape the host response, as shown in a 18. The VACV A41 protein and its ECTV Naval orthologue E163 bind a number of chemokines including those recognized by the SECRET domain, but they do not block chemokine interaction with its receptor and are rather proposed to dissipate chemokine chemotactic gradients in vivo by targeting the glycosaminoglycan binding site of chemokines18. Thus the 35-kDa vCKBP and A41 ECTV orthologues are probably involved in controlling different aspects of the anti-viral host response. Moreover, ECTV encodes two SECRET domain-containing proteins, named E12 and E184, that show the same chemokine binding specificity16 but these proteins are not able to compensate for the loss of the CrmD SECRET domain. The phenotype of ECTVrevSECRET, expressing anti-chemokine activity but lacking TNF/LT inhibitory activity, indicates that the SECRET domain needs simultaneous TNF blockade to have a major impact on virus virulence. Additionally, the finding of the SECRET domain on the same molecule than a TNF binding domain might be relevant to ensure an efficient combined effect in the infected host. It is reasonable to propose that the coordinated blockade of cytokines and chemokines by a viral protein could serve as an excellent inhibitor of cell recruitment and the immune response in vivo, as shown here for the poxviral CrmD protein and previously for murine cytomegalovirus62.The reasons for the dramatic attenuation phenotype of the CrmD mutant could be related to the nature of the chemokines targeted and/or the inhibitory mechanism. The CrmD SECRET domain is different from the 35-kDa vCKBP in the specific set of chemokines blocked16. The VARV protein CrmB has TNF/LT and chemokine inhibitory properties similar to those of ECTV CrmD, but is better adapted to block the human immune system16. The control of TNF-induced gene expression observed in VARV-infected monkeys suggests that CrmB is expressed during infection63. Our results suggest that VARV CrmB is an important virulence factor in smallpox. The same may be true for MPXV, an orthopoxvirus causing a smallpox-like disease in humans with mortality rates of up to 10% that encodes a CrmB orthologue64. The fear of either an intentional release of VARV or the emergence of a poxviral zoonosis, caused by MPXV or other poxviruses26 in a large unvaccinated population have sparked the interest in developing safer vaccines or drugs to prevent or treat human poxviral infections. Although current vaccines have a proven efficacy record, potential risks relating to secondary effects, the increased number of immunocompromised individuals and uncertainty about the duration of protective immunity exist. Thus, alternative strategies for the development of safer vaccines are being currently pursued (reviewed in65). Recombinant protein is thought to be safer, and different combinations or single protein vaccination using components from virus particles, which induce neutralizing antibodies, protect from poxvirus infections68. Our report shows that immunization with recombinant ECTV CrmD protects mice from a lethal mousepox challenge, possibly by an antibody-mediated blockade of CrmD-ligand interaction and clearance of the CrmD protein from the infected host. Similar results have been obtained by vaccinating with the type I IFN binding protein from ECTV44, and antibodies against this secreted viral protein protect from mousepox69. Thus, secreted poxviral immunomodulators can act as effective subunit vaccines in preventing a lethal poxvirus infection in its natural host and could be used singly or in combination with other proteins. It is important to note that the smallpox VACV vaccines Dryvax and Modified VACV Ankara do not express the CrmB protein and will not induce a neutralizing response against CrmB expressed by VARV or MPXV13. However, the Dryvax vaccine induced an immune response against viral structural proteins that was sufficient to protect from smallpox.The study of the contribution of CrmD to mousepox sheds light into the mechanisms of the pathogenesis of acute viral infections. Importantly, mousepox is a model for human smallpox, a severe human disease caused by VARV and whose pathogenesis has not been extensively studied at the molecular level. VARV and ECTV share secreted immunomodulatory proteins and encode only one vTNFR70. To date soluble versions of the human TNFR2 or monoclonal anti-TNF antibodies are used, although other strategies are under development. The use of vTNFRs in this context has been proposed71, and transgenic mice expressing ECTV CrmD have shown that it can inhibit TNF driven inflammatory reactions in vivo72. The fact that CrmD acts as a potent anti-inflammatory molecule in vivo and that its SECRET domain is important for this activity suggests that addition of a chemokine inhibitory domain to the human soluble TNFRs may increase their clinical efficacy in certain settings. However, this approach should be taken with caution because an antirheumatic drug combining anti-TNF and anti-chemokine activities could be expected to further dampen the already debilitated immune response of patients under anti-TNF therapy, what might worsen the frequent infectious complications observed in these patients73.TNF and chemokines are important in the development of human pathologies unrelated to viral infection. Notably, soluble TNFRs are used to treat a variety of inflammatory conditions in the clinic such as rheumathoid arthritis, ankylosing spondylitis or psoriatic arthritisIn conclusion, the characterization of the role of CrmD in mousepox pathogenesis has demonstrated a critical role of TNF and a specific set of chemokines in defense from virulent poxvirus infections. The nature of the ligands of CrmD may point to the cytokines and chemokines important in the control of poxviral infections.24. For titration of virus in organs from infected mice, spleen and liver were aseptically removed, weighed and homogeneized and serial dilutions plated on BSC-1 cell monolayers. For infection of mice, virus stocks were semipurified by centrifugation through a 36% sucrose cushion74. Viral stocks were routinely tested for the absence of mycoplasma and the endotoxin levels detected usingToxiSensor Chromogenic LAL Endotoxin Assay kit (GenScript) were under 0.3 EU/ml.A plaque-purified and fully sequenced ECTV Naval isolate  was grown in BSC-1 cells (ATCC CCL-26)15. A N77F mutant of this CrmD-V5-6xHis protein was generated using the QuikChange II Site-Directed mutagenesis kit (Agilent Technologies) and expressed by recombinant baculoviruses15.For the generation of anti-CrmD polyclonal rabbit antibodies and immunization experiments, rabbits and mice, respectively, were injected with a recombinant CrmD fused to the Fc portion of a human IgG1 (CrmD-Fc). CrmD-Fc was expressed by recombinant baculoviruses and purified from the supernant of infected Hi5 insect cells (ThermoFisher BTI-TN-5B1-4) by affinity chromatography in a protein A coupled sepharose column. Similarly, a CrmD C-terminally tagged with V5 and 6xHis epitopes was expressed by recombinant baculoviruses as previously described16. Briefly, L929 cells (ATCC CCL-1) were incubated with TNF  which had been preincubated or not with recombinant ECTV CrmD protein or supernatants from ECTV-infected cells and cell death determined using the CellTiter OneSolution viability assay (Promega).The ability of ECTV CrmD to block TNF-induced cell death was determined as described16. Briefly, MOLT-4 cells (ATCC CRL-1582) were incubated with 70 nM of mouse Ccl25  in RPMI 0.1% FBS in the presence or absence of CrmD wild type or N77F at the indicated molar ratios. Unspecific migration in the absence of chemokine was also monitored as reference (media). Cell migration through the filter was allowed to occur during 4 h at 37 oC, and subsequently the number of cells that migrated to the bottom well was calculated by interpolation in a standard curve of number of cells using CellTiter Aqueous One Solution assay kit (Promega).The anti-chemokine activity of CrmD wild type and the N77F mutant was assessed by chemotaxis assays using a 96-well ChemoTx plate with a 3-μm pore sized filter as previously describedThe binding affinity of recombinant CrmD for mouse chemokines  and the ability of wild type CrmD and mutant N77F to interact with mouse TNF  and Ccl25  were determined by SPR using a Biacore X biosensor .For affinity determinations, recombinant CrmD was immobilized onto a flow cell of a CM4 chip at low density (900 RUs) by the amine coupling protocol. One flow cell was left empty to be used as reference. Increasing concentrations of mouse chemokines were injected in HBS-EP buffer  over the chip surface and their binding was recorded for 120 s followed by a 300 s dissociation period. Surface was regenerated with glycin-HCl pH2.0 between injections. Binding sensorgrams were analyzed by the BiaEvaluation software  and fitted to a general 1:1 Langmuir binding model.For binding assays, recombinant wild type and N77F CrmD proteins were immobilized on to a flow cell of a CM4 chip at high density (1500 RUs) as explained above. 100 nM of mouse TNF or mouse Ccl25 were injected over the chip in HBS-EP buffer and their association was monitored during 120 s followed by a 120 s dissociation. Binding sensorgrams were processed and analyzed using the BiaEvaluation software.74. The plasmid pMS30 was constructed for expression of EGFP under a VACV early-late promoter followed by an IRES cassette for expression of the puromycin acetyl transferase gene from the same transcript. For the generation of ECTVΔCrmD, the flanking regions of the CrmD gene were PCR-amplified and cloned into the EcoRI and PstI restriction sites of the polylinker region of pMS30. The 5ʹ flanking region of the CrmD gene was amplified with oligonucleotides CrmD-27 (5ʹ-GCGGAATTCCGATTTAATAACATTCGATTATATAG) and CrmD-11 (5ʹ-CGCGGATCCGGTGTATACGGAACATCTCCAC), and the 3ʹ flanking region of CrmD was amplified with oligonucleotides CrmD18 (5ʹ-CGCGGATCCTAACATGGACGTCGTCGCGTATCATAC) and CrmD28 (5ʹ-GCGCTGCAGCTCTGTAATGATGGACGTTATTTC), to generate the plasmid pMS34 (pΔCrmD). Both flanking regions and the CrmD gene were PCR-amplified with oligonucleotides CrmD27 and CrmD28 to generate the plasmid pMS37 (pRevCrmD) that was used for reinsertion of the CrmD gene into the ECTVΔCrmD genome and construction of ECTVRevCrmD. The 5´flanking region used for generation of ΔcrmD and the TNF binding domain of CrmD including a stop codon were PCR-amplified using oligonucleotides CrmD27 and CrmD30 (5´-CGCGGATCCTAACAAGAGGTCTTGTTAACAGGATAC) and pMS37 as a template. The resulting PCR product was cloned into the EcoRI and BamHI sites of pMS34 generating plasmid pAH7. This plasmid was used to generate ECTVRevCRD, which expresses a truncated version of CrmD corresponding to residues M1 to C180. For the generation of ECTVRevSECRET, the CrmD gene contained in pMS37 was mutated by directed single point mutation using the QuickChange II mutagenesis kit (Agilent Technologies) and the primers CrmD43 (AGATGACACCTTTACATCCATTCCTTTTCATAGTCCCGCGTG) and CrmD44 (CACGCGGGACTATGAAAAGGAATGGATGTAAAGGTGTCATCT). These primers introduce a N77F mutation in CrmD.Recombinant ECTVs were generated using a transient dominant selection procedure and the selection in the presence of puromycin as previously describedCrmD-specific PCR. The complete genome sequence of ECTVRevCRD and ECTVRevSECRET was determined by Illumina sequencing to confirm the genomic structure and the absence of inadvertent mutations that may affect virus virulence24. The sequences have been submited to the European Nucleotide Archive and have been assigned reference number PRJEB19928. The number of sequencing reads that aligned with the ECTV Naval genome was 1.29 × 106  for ECTVRevCRD and 1.27 × 106  for ECTVRevSECRET. ECTVRevCRD was sequenced with a 325× coverage and, including the expected introduction of a truncated version of the duplicated CrmD gene, three changes were identified: Δ5.655-5.998 (EVN006/CrmD gene); T199.552 C (EVN200P pseudogene) and Δ201.621-201.964 (EVN201/CrmD gene). ECTVRevSECRET was sequenced with a 320× coverage and, including the expected N77F mutation in the amino acid sequence of duplicated CrmD gene, three changes were identified: TT6.310-6.311AA (EVN006/CrmD gene); T199.552 C (EVN200P pseudogene) and AA201.310-201.311TT (EVN201/CrmD gene). Both recombinant viruses had the expected mutations in both copies of the CrmD gene, present at the left and right ends of the viral genome. We also identified an additional point mutation in the inactive pseudogene EVN200P that was present in both ECTVRevCRD and ECTVRevSECRET, suggesting that this mutation was introduced during the generation of ECTVΔCrmD.After transfection/infection in BSC-1 cells, the intermediate single-crossover recombinant viruses in which the complete plasmid has been inserted into the ECTV genome were selected for three to five consecutive infection rounds in the presence of puromycin and monitored for EGFP expression by fluorescence microscopy. Recombinant viruses  were finally selected by successive plaque purification of white plaques in the absence of puromycin and screening with a Q = 1%. Analyses were performed up to times post-infection at which survival rates in the corresponding groups were above 50%. ANOVA analyses with Bonferroni multiple comparison tests were performed in some experiments, as indicated, for comparisons among groups and times post-infection at which no mortalities were observed. These experiments were approved by the Biological Safety Committee of the Centro de Investigación en Sanidad Animal  and animals were housed and handled according to legal requirements.Female BALB/c OlaHsd mice (6–8 weeks old) (Harlan), housed in ventilated racks, were anesthesized with isofluorane and s.c. infected in the footpad with 10 μl of virus inoculum. Viral doses were confirmed by titrating again on the same day the virus dilutions were used for mouse infections. Mice were housed in ventilated racks (Tecniplast) under biological safety level 3 containment facilities. Monitoring of infected animals was performed daily. Animals were weighed, scored for clinical signs of illness  and footpad swelling measured. Data analysis was performed using GraphPad Prism 6 . Survival curves were compared using the Logrank (Mantel-Cox) test. Footpad swelling and % initial weight data were analyzed using multiple t tests with false discovery rate Footpad, spleen and liver samples from infected mice were removed aseptically and fixed in 10% buffered formalin solution to detect virus, ECTV CrmD protein and chemokine receptors, and in zinc fixative (BDPharmingen) to detect lymphoid cells and ICAM-1. After fixation, the samples were dehydrated through a graded series of alcohol to xylol incubations and embedded in paraffin wax. For structural and immunohistochemical analysis, sections (3 μm) were cut and stained with H&E or processed for immunohistochemical techniques. To detect virus and ECTV CrmD protein, formalin fixed serial sections were incubated with polyclonal rabbit anti-VACV antibody from a VACV-infected rabbit or a polyclonal rabbit anti-CrmD antibody against purified CrmD expressed in the baculovirus system. Both antibodies were generated in our laboratory. Secondary goat anti-rabbit IgG (Dako) was detected using an avidin-peroxidase-complex kit  and 3,3ʹ-diaminobenzidine tetrahydrochloride (Sigma) following the manufacturer´s instructions. The slides where counterstained with Mayer´s haematoxylin, dehydrated, and mounted with DPX mountant (Surgipath). Specific primary antibodies were replaced by PBS or normal goat serum in negative control sections. To detect lymphoid cells and chemokine receptors, the avidin-biotin alkaline-phosphatase staining method was used. Sections were dewaxed and immunostained with polyclonal rabbit anti-human CD3 (Dako), rat anti-mouse CD45R/B220, CD4, CD8a, and CD8b (BD Pharmingen) or goat anti-mouse CCR10 (AbCAM). Anti-mouse ICAM-1 antibody was from AbCAM. For CD3 and CCR10 immunohistochemistry, antigen retrieval was achieved by heating sections in 0.1 M citrate buffer at pH 6. Secondary goat anti-rabbit IgG, rabbit anti-rat IgG, or rabbit anti-goat IgG (Dako) were used as corresponded with the streptavidin-biotin-alkaline phosphatase kit  and “FastRed”  for detection of the immunogens, following the manufacturer’s indications. The slides were counterstained with Mayer’s haematoxylin, and mounted with Immu-mount (Thermo Shandon). Specific primary antibodies were replaced by PBS, normal rabbit serum or normal goat serum in negative control sections.n = 5), ECTVRevCrmD- (n = 5), and ECTVΔCrmD- (n = 6) infected mice were analyzed and scored as staining or non-staining. Mean percentage of staining vessels and standard deviations were calculated using the Excel spreadsheet and statistical significance was confirmed using a Student’s t-test (p < 0.01).For semi-quantitative analyses of histological sections, samples from at least 5 animals for each parameter were analyzed in every case. To establish the degree of necrosis, a minimum of 10 fields were scored per spleen and liver slice to obtain the mean value. To quantify the morphological changes, sections were graded for necrosis using an arbitrary scale: – negative findings (0%); + slight (about 25% necrosis); ++ moderate (about 50% necrosis); +++ very intense (about 90–100 % necrosis). Inflammatory infiltration was evaluated in a minimum of 10 fields per liver slice or the complete footpad section to obtain the mean value and it was scored as: – negative findings; + slight; ++ moderate; +++ very intense. For antibody staining of lymphoid cells and CCR10 chemokine receptor expressing cells, all the cells from the inflammatory infiltrate were counted for each case and mean values are presented. In the case of anti-ICAM-1 staining, all the blood vessels from sections from ECTV- . Red blood cells were lysed by hypoosmotic shock in milli-Q water and white cells were washed twice in PBS and counted manually in a haemocytometer. DPLN cells were stained with anti-DX5-FITC (eBioscience), anti-CD3e-PerCP (eBioscience) and anti-GzB-APC (R&D Biosystems). Splenocytes were stained with anti-CD3e-PerCP, anti-CD8-PE (eBioscience) and anti-GzB-APC. In parallel, cell suspensions were also stained with the appropriate isotype control antibodies. 100,000 cells were analyzed in a FACS Calibur flow cytometer (Becton Dickinson). Events were gated according to a forward and side scatter pattern compatible with healthy lymphocytes  BALB/c mice was inoculated i.p. with 10 μg of purified recombinant ECTV CrmD protein expressed in the baculovirus system per animal three times at 17 to 20 day intervals. At 18 days after the last inoculation, mice were bled and sera obtained to check for presence of anti-CrmD antibodies and challenged s.c. with 1000 PFU of ECTV as above. As a control, a group of 10 BALB/c mice was subjected to the same protocol using PBS for i.p. inoculations and infected with equal amounts of virus. Disease progression was monitored as described above.PRJEB19928.The data that support the findings of this study are available from the corresponding author upon reasonable request. The viral genomic sequences reported have been submited to the European Nucleotide Archive and have been assigned reference number Supplementary InformationPeer Review File