Immune-mediated inflammatory diseases (IMIDs), such as spondyloarthritis (SpA), psoriasis, Crohn's disease (CD), and rheumatoid arthritis (RA) remain challenging illnesses. They often strike at a young age and cause lifelong morbidity, representing a considerable burden for the affected individuals and society. Pioneering studies have revealed the presence of a TNF-dependent proinflammatory cytokine cascade in several IMIDs, and the introduction of anti-TNF therapy 20 years ago has proven effective to reduce inflammation and clinical symptoms in RA, SpA, and other IMID, providing unprecedented clinical benefits and a valid alternative in case of failure or intolerable adverse effects of conventional disease-modifying antirheumatic drugs  or non-steroidal anti-inflammatory drugs . However, our understanding of how TNF inhibitors (TNFi) affect the immune system in patients is limited. This question is relevant because anti-TNF therapy has been associated with infectious complications. Furthermore, clinical efficacy of TNFi is limited by a high rate of non-responsiveness (30–40%) in RA, SpA, and other IMID, exposing a substantial fraction of patients to side-effects without clinical benefit. Despite the extensive use of TNFi, it is still not possible to determine which patients will respond to TNFi before treatment initiation. The recent introduction of antibodies blocking IL-17 has expanded the therapeutic options for SpA, as well as psoriasis and psoriatic arthritis. It is therefore essential to develop tools to guide treatment decisions for patients affected by SpA and other IMID, both to optimize clinical care and contain health care costs. After a brief overview of the biology of TNF, its receptors and currently used TNFi in the clinics, we summarize the progress that has been made to increase our understanding of the action of TNFi on the immune system in patients. We then summarize efforts dedicated to identify biomarkers that can predict treatment responses to TNFi and we conclude with a section dedicated to the recently introduced inhibitors of IL-17A and IL-23 in SpA and related diseases. The focus of this review is on SpA, however, we also refer to RA on topics for which only limited information is available on SpA in the literature. Immune-mediated inflammatory diseases (IMID) is a term used to define a group of clinically heterogeneous, unrelated conditions that share common inflammatory pathways and derive from aberrant immune responses of the human adaptive or innate immune system. Overall, the estimated incidence of IMIDs in Western populations approximates 5–7%  and encoRecent genome-wide association studies (GWAS) performed with thousands of patients and controls from different populations have provided detailed information about the genetic variants associated with immune-mediated inflammatory diseases . These sFrequently, multiple immune-mediated inflammatory disorders co-exist within the same patient. This was observed in a large study involving 3,287 AS patients, of which the 39% also developed uveitis, 16% psoriasis, and 8% inflammatory bowel disease . In addiTNF mRNA is induced by the cooperation of AP-1 transcription factors with nuclear factor associated with activated T cells (NFAT) and nuclear factor-kB (NF-kB), which can bind directly to the promoter of the TNF gene  in vitro , which is subsequently cleaved in the extracellular domain by the metalloprotease TNF-converting enzyme , resulting in the release of the mature soluble TNF monomer (sTNF), a protein of 17 kDa , 28. TheTNF-α exerts its activity by binding to two different receptors that differ in cellular localization and signaling mechanisms . Like thTNFRSF1A) is ubiquitously expressed (except for erythrocytes), and is characterized by the presence in its intracellular portion of a “death domain” motif. Binding of TNF-α to TNFR1 initiates a complex network of downstream events that may result in both the induction of apoptosis and of acute inflammation. Upon activation, TNFR1 recruits TNFR1-associated death domain protein (TRADD) to the plasma membrane, followed by the assembly of a scaffolding signaling complex (complex I) that includes TNF receptor associated factor 2 (TRAF2), and that results in the activation of AP-1 and NF-kB transcription factors (https://www.bu.edu/nf-kb/gene-resources/target-genes/)  . Among t-genes/) , that su-genes/) , 37, 38.-genes/) .TNFRSF1A locus with AS, primary biliary cirrhosis and MS (https://www.immunobase.org/). A genetic variant identified selectively in multiple sclerosis has been demonstrated to affect splicing and induce expression of a soluble form of the receptor, which can block TNF. Of note, this variant has not been identified in diseases that are ameliorated by inhibition of TNF, such RA, psoriasis, and Crohn's disease (The relevance of TNFR1 for immune-mediate diseases is supported by the association of genetic variations at the  disease .TNFRSF1B) is mainly expressed on lymphocytes, endothelial cells and astrocytes or oligodendrocytes  in the membrane proximal region of TNFR2 . This vaThe important pathogenetic role of TNF-α in chronic inflammatory diseases is supported by the therapeutic efficacy of anti-TNF agents.As mentioned above, TNF-α was initially considered as a potential onco-therapeutic agent. However, despite the name, administration of recombinant TNF-α to patients with malignant diseases resulted in disease progression and severe side effects, rather than disease improvement . Phase IInitially, treatment options for IMID were limited to non-steroidal anti-inflammatory drugs (NSAIDs) and physical therapy. At present, NSAIDs such as ibuprofen, naproxen, diclofenac, celecoxib, highly effective in reducing stiffness and back pain in axial SpA, are recommended by the ASAS/EULAR guidelines as first-line treatment . When papneumonia, influenza, and adenovirus infections, latent viral infections , skin and soft tissue infections and urinary tract infections. Cases of exacerbated legionella were also reported. More rare side effects include severe hepatic reactions, nervous system disorders, hypersensitivity reactions and leucopenia. Some meta-analyses have been performed to assess the increased risk of malignancies following anti-TNF therapy and there is no proven increase of any malignancy so far. An increased risk of non-melanoma skin cancer during therapy with TNF-blockers has been reported  remains indeed the major concern . Other ireported , 60. Increported .H and VL), that recognizes the target and a C-terminal constant region (Fc). The engineered monoclonal antibody monomer comprises a variable region that can be either murine or human, and a constant region (Fc), usually human to preserve favorable pharmacokinetic properties.Natural and engineered anti-TNF antibodies share a similar structure , constitAt present, there are five TNF-inhibitors approved for the treatment of spondyloarthritis in the UE, the USA and many other countries . Three aThe first TNF blocker introduced in the clinic for the treatment of RA was the monoclonal antibody infliximab (trade name Remicade) . InflixiAdalimumab  and golimumab (Simponi) , are fulA different type of anti-TNF compound is etanercept , an engineered dimer composed of 2 extra-cellular portions of the human p75 TNF-α receptor (TNFR2) fused to a C-terminal human IgG1 Fc domain . EtanercH and VL domains. The hinge region of certolizumab is modified by polyethylene glycol, which reduces immunogenicity, improves solubility and the in vivo half-life  or complement-dependent (CDC), although these effects have never been demonstrated in patients , 72, 82.Given the complexity of the cellular pathways involved, the molecular mechanism of action of anti-TNF agents is still not fully elucidated. In general, the biological effects of anti-TNF agents can be summarized by these main functions: (i) binding of soluble TNF-α and LT-α, reducing the signaling cascades downstream of these cytokines; (ii) binding to FcR-expressing cells, inducing antibody-mediated cellular cytotoxicity; (iii) binding to membrane-bound TNF (tmTNF), triggering reverse signaling.Given the pleiotropic functions of TNFα, its blockade has long ranging effects on a variety of immune cells. However, the effects of TNF inhibition on immune cell populations are not always consistent in different studies, and may vary depending on the disease setting, the treatment (often studies group patients treated with different inhibitors) and whether the patients respond to therapy.+ T cells into functionally distinct T helper (Th) subsets, characterized by the secretion of specific “cytokine signatures.” Th1 cells secrete IFNγ and are important for host defense against intracellular pathogens, while Th2 cells produce IL-4, IL-5, IL-13, and are involved in the protection against parasitic infections. The more recently identified Th17 cells secrete IL-17, IL-21, IL-22, and IL-26 in humans, and contribute to immune responses against extracellular bacteria and fungi. Both Th1 and Th17 responses have been associated with autoimmune disease  frequencies in patients vs. healthy controls see , 2. HighThe increase of Th17 cells after TNF-blockade has also been correlated with lack of response to treatment in AS  or RA 8, althougIn several studies, the decrease in Th subsets was mirrored by an increase in regulatory T cells (Treg), indicating that the balance between effector T cells and Treg is important for the re-establishment of immune homeostasis. Altered Treg functions have been described in IMID, and Treg frequency is often decreased in patients, compared to healthy donors. These defects can be reversed by anti-TNF therapy, depending on the agent used , 158 Ta. AdalimuAnti-TNF treatment downregulates the production of a large range of inflammatory cytokines/chemokine, including IL-6, IL-1β, IL-8, RANTES, and MCP-1 .More recently, granulocyte macrophage-colony stimulating factor (GM-CSF), a myelopoietic cytokine that induces myeloid cells activation and differentiation, has emerged as a potential target in the treatment of rheumatic diseases , 171.GM-CSF gene expression tends to occur locally in inflamed tissues and can be induced by inflammatory cytokines such as IL-6, IL-1β, TNF-α in many different cell types, both immune  and not  . GM-CSF +CD4+ T cells  or anti-inflammatory (IL-10) functions .B cells exit the bone marrow as “transitional” cells that give rise to mature/naïve B cells, memory (CD27+) B cells and antibody-secreting plasmacells. In secondary lymphoid tissue follicles B cells can be activated to class-switch the B Cell Receptor from IgM/IgD to IgG, IgA, or IgE .+CD24highCD38high markers contains the largest fraction of IL-10-producing B cells upon CD40 ligation in human peripheral blood  and poor response to therapy, in particular for infliximab treated patients . In a laThe clinical characteristics described so far may help clinicians to guide treatment decisions in daily practice, however none of them has strong specificity.Different strategies have been proposed in the past 15 years with the aim to identify markers predicting clinical responsiveness to anti-TNF therapy. However, results reported in the literature have not always been validated in subsequent studies. Many different strategies have been pursued to identify biomarkers, such as measuring soluble molecules in serum or applying a variety of molecular and genomic techniques. More recently, alternative approaches including molecular imaging with fluorescent antibodies, analysis composition of the gut microbiota and the hunt for biomarkers in tissues have been proposed. Since there are only limited numbers of studies performed in SpA patients, we will extend our discussion to other chronic inflammatory diseases, the results of which might also be relevant for SpA.A report from Pedersen and colleagues investigated circulating biomarkers of inflammation including CRP, IL-6 and YKL-40, vascular endothelial growth factor , C-terminal crosslinking telopeptide of type II collagen (CTX-II), matrix metalloproteinase 3 (MMP-3), total aggrecan, cartilage oligomeric matrix protein and bone turnover in axSpA patients undergoing anti-TNF treatment. Patients that reached a major improvement in ASDAS at week 22 had higher baseline CRP, IL-6, CTX-II, and MMP-3 and lower YLK-40 levels as compared to partial or non-responders. BASDAI responders had higher baseline CRP and VEGF, and lower YLK-40 as compared to BASDAI non-responders .+ T cells  in PBMCs after TNF-α blockade was associated with poor long-term clinical outcome  was significantly higher in responders at baseline and was able to predict the therapeutic outcome with 100% sensitivity and 91.7% specificity. Clinical responders revealed an almost complete reset to normal levels of inflammatory monocyte markers whereas in non-responders the levels of those genes remained elevated  and the chemokine receptor 2 (CCR2) were upregulated in biopsies obtained from non-responders, whereas no significant difference for these two genes was observed in blood. Increased levels of genes in the CCL7-CCR2 pathway suggest that in non-responders there is an increased recruitment of inflammatory TNF-secreting macrophages to the inflamed tissuers, which contributes to increased plasmacell abundance ultimately impacting clinical outcome. Interestingly, one of the upstream regulators of the CCL7-CCR2 axis, TREM-1, the expression of which was measured in the blood of 22 CD patients prior to anti-TNF therapy, was significantly downregulated in non-responders, and showed a high prediction accuracy.Still in the context of IBD, a recent publication from the Powrie lab has demonstrated that oncostatin M (OSM) levels are higher in inflamed intestinal tissue of IBD patients with deep mucosal ulcerations (severe disease). Of particular interest was the observation that OSM gene expression levels were substantially higher in colonic biopsies of IBD patients refractory to anti-TNF therapy. These findings identified OSM as a potential biomarker of disease activity and therapeutic response to anti-TNF therapy. In addition, OSM could be an interesting novel therapeutic target for patients not adequately responding to TNF-blockers . Howeverin vivo molecular imaging of the intestinal mucosa of Crohns' disease patients to identify mTNF-expressing cells. After topical application of labeled adalimumab through a standard spray catheter onto the most inflamed region of the bowel during colonoscopy, in vivo imaging of the intestinal mucosa showed a specific fluorescence signal of mTNF+ cells. Interestingly, cells expressing mTNF were mainly lamina propria CD14+ macrophages and some CD4+ T lymphocytes. The authors correlated the number of immune cells expressing mTNF with clinical outcome to adalimumab, and demonstrated that responders had a significantly higher mean number of mTNF-expressing cells than patients refractory to the therapy. This study was based on molecular imaging with fluorescent antibodies, which constitutes a new approach for the identification of patients responding to therapy, both in chronic inflammatory and autoimmune disorders, and in cancer  was also associated with response to TNFi, but none of the other RA-associated risk alleles had an effect on treatment responses (PTPRC with response to anti-TNF therapy (p < 5 × 10−8). Cui et al. have performed a GWAS meta-analysis on 2,706 RA patients. 733 of these patients were treated with the soluble TNFR-Fc fusion etanercept, while 1,071 or 894 patients were treated with the monoclonal anti-TNF antibodies adalimumab or infliximab, respectively. They identified in the etanercept group, but not in the two other treatment groups a SNP in the 3′-untranslated region of the CD84 gene, which may disrupt a transcription factor binding site. The allele associated with better treatment response was associated with higher gene expression levels of CD84 and explained 2.6% variance in response to treatment with etanercept . Genotyping data from 2,706 RA patients treated with anti-TNF were given to challenge participants to develop predictive models of treatment responses to TNFi. This challenge ran for 8 months in 2014 and 73 research teams submitted computer code covering a wide range of state-of-the-art modeling methodologies. In the validation phase, these models were evaluated with 591 anti-TNF-treated RA patients from an independent cohort. Despite the remarkable assembly of expertise in this crowdsourcing approach, no significant genetic predictors of treatment responses could be identified. The authors concluded that genetic information does not significantly enhance prediction of therapeutic responses over standard clinical assessments and suggested to embark on other research strategies to identify biomarkers   . Taken t+ T cells from SpA patients , did not correlate with expression of Th17 and Th1 genes, suggesting that these SNPs may contribute to SpA pathogenesis through distinct cellular mechanisms. These data showed that genetic variation at multiple loci within the IL-23/Th17 pathway, such as IL23R, IL12B, and CCR6, affects CD4+ effector functions in SpA patients. Of note, the effect of genetic variation on CD4+ T cell function could be detected in activated, but not in resting T cells, consistent with the context-dependent action of expression quantitative trait loci (eQTL) observed in several studies  have been identified in several tissues as potent producers of proinflamatory cytokines, including IL-17A and IL-22. In collaboration with the team of D. Baeten we have recently characterized the presence and composition of ILCs and investigated if these cells are an important source of IL-17A in the synovial tissue of patients with peripheral SpA. We analyzed matched synovial tissue (ST), synovial fluid, and peripheral blood from SpA patients with actively inflamed knee joints. We found that ILCs, and in particular NKp44athology . Thus, fWith respect to IL-23, a phase 2 clinical study tested the safety and efficacy of a fully human monoclonal antibody (ustekinumab) targeting the p40 subunit shared by IL-12 and IL-23 in psoriasis . This stIL23R have been associated with AS (Blocking the activity of IL-12/23 and of IL-23 has also been tested in moderately to severely active Crohn's disease and patients receiving either of these monoclonal antibodies had significantly higher response rates than did those receiving placebo , 282. In with AS  and this with AS , 286. Se with AS , 264 and with AS . Finally with AS . The rea with AS , or that with AS . ClearlyThe clinical relevance of a “personalized” approach in medicine is well-accepted. However, despite the well-known individual variability in the susceptibility to infections and inflammatory diseases, disease progression and response to therapy, medical practice and public health policies typically take a “one size fits all” approach to disease management. This is due to a lack of understanding of what determines the individual predisposition to disease and the mechanisms associated with the response to a specific therapy. Biological strategies that block specific immune mediators such as TNF, IL-6, IL-17A, IL-23, or JAKs are effective only in a subpopulation of patients and can be associated with serious side effects. To improve clinical outcome, tools that allow prediction of treatment responses are needed. In addition, a better understanding of the pathogenic mechanisms will permit a more efficient use of existing therapies, as well as the development of novel targeted therapies. In a context in which new treatments for chronic inflammatory diseases have recently been introduced in the clinics, we highlight the importance of establishing a path toward personalized medicine by defining immunological correlates associated with therapeutic responses to anti-TNF therapy in SpA and related diseases.The limitations of developing reliable biomarkers that can be used in daily practice derive from several factors. Many of the studies reviewed here have been performed in different centers and in relatively small cohorts of patients, limiting the unbiased identification of robust and validated biomarkers. In this sense, increased collaboration between centers and “merging” of cohorts of patients affected by IMIDs to increase sample size and power may help to define more reliably define biomarkers. “Consortium science” has been key to the success of large GWA studies aimed at identifying risk alleles for various diseases and should be reinforced as a valuable strategy in translational research.Due to limited patient numbers, several studies have grouped patients not homogeneous for disease status, treatment agent, response to therapy, and other patient characteristics. Most of these variables have significant effects on the measurable parameters of patient immune functions, and are important confounders for the analysis of the impact of TNF-blockade on the immune system. This may be the cause underlining the many controversial findings reported in the literature about the modifications in immune function imparted by TNFi. Accurate patient stratification may allow a better understanding of the molecular and functional consequences of anti-TNF therapy, and improve the chances to identify strong biomarkers of response.With respect to axial SpA, an additional challenge is provided by the difficult access to inflamed axial tissues. Sequential sampling during treatment in large cohorts of patients is almost impossible to perform, except for SpA patients with a predominant peripheral inflammation. In this particular case, peripheral blood still remains the only easily accessible material to study pathophysiologic processes and mechanisms contributing to the response to therapies.Finally, recent clinical trials testing the safety and efficacy of monoclonal antibodies targeting IL-17A and IL-23 in SpA, psoriasis, psoriatic arthritis and Crohn's disease have been very encouraging and have increased the therapeutic options for these diseases. At the same time, their introduction into the clinics has increased the urgency to implement personalized treatment strategies. A trial and error approach, as it is currently applied in most cases, is simply not compatible with optimal patient care and efficient use of resources. However, defining objective criteria to guide treatment decisions for each individual patient remains a major challenge. Furthermore, clinical studies testing blockade of IL-17A and IL-23 have not been without surprises. The unexpected failures of anti-IL-17A in Crohn's disease and of anti-IL-23 in AS are stark reminders of our limited understanding of the pathogenic mechanisms of these diseases and are a call to action to all of us in the biomedical research community.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TNF-α-neutralizing antibodies, such as infliximab (IFX) and adalimumab (ADA), are effective in the treatment of inflammatory bowel diseases (IBD), but they are expensive and become ineffective when patients develop anti-IFX or anti-ADA antibodies . Second-generation anti-TNF-α antibodies, such as Golimumab, Etanercept, Certolizumab-pegol and IFX biosimilars, may solve these issues.To determine the neutralizing capacity of first- and second generation anti-TNF-α antibodies and to determine whether ATI show cross-reactivity with the IFX biosimilar CT-P13 (Inflectra).2-fragment, biosimilar CT-P13 (Inflectra) and ADA.TNF-α neutralization was measured using a quantitative TNF-α sensor assay consisting of HeLa 8D8 cells that express the Green Fluorescence Protein (GFP) under control of a NF-кB response element. All available anti-TNF-α drugs and the IFX biosimilar CT-P13 (Inflectra) were tested for their TNF-α-neutralizing capacity. In addition, patient sera with ATI were tested for their potential to block the activity of IFX, IFX (F)abTNF-α strongly induced GFP expression in Hela 8D8 cells. Higher concentrations of first-generation anti-TNF-α drugs were required to neutralize TNF-α compared to the second-generation anti-TNF-α drugs. Serum of IBD patients with proven ATI blocked TNF-α-neutralizing properties of IFX biosimilar CT-P13 (Inflectra), whereas such sera did not block the effect of ADA.The second-generation anti-TNF-α drugs show increased TNF-α-neutralizing potential compared to first-generation variants. ATI show cross-reactivity toward IFX biosimilar CT-P13 (Inflectra), consequently patients with ATI are unlikely to benefit from treatment with this IFX biosimilar. TNF-α blocking agents, such as infliximab (IFX) and adalimumab (ADA), are highly effective in the induction and maintenance of remission in Crohn’s disease (CD) and ulcerative colitis (UC) and have led to remarkable improvements in the therapy of CD and UC. –8 HoweveBesides the cost of these drugs, another problem in the treatment with TNF-α blocking agents is that approximately 10%-21% of the patients annually lose their response to the treatment partially due to formation of antibodies against the drug (antigenicity), causing low trough levels.–16 AntibCurrently second generation TNF-α blocking agents as Certolizumab-PEGOL (Cimzia) and Golimumab are shown to be effective in the treatment of CD and UC. –22It is unclear whether ATI always neutralize the anti-TNF-α drug and whether they show cross-reactivity towards other available anti-TNF-α therapeutics or IFX biosimilar.Here, we compared the TNF-α-neutralizing capacity of all commercially available anti-TNF-α drugs. Furthermore, we tested the neutralizing capacity of ATI, as well as their cross-reactivity with IFX, ATI, ADA and ATA. Finally, we tested if ATI towards the original IFX cross react with the biosimilar of infliximab (CT-P13 (Inflectra)).th, 2009, protocol number METc 2008.338. Written, informed consent was obtained from each patient included in the study. Within this informed consent patient agrees to link the patient’s biobank ID to the electronic patient’s dossier. The authors had full access to the electronic patient dossier. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a prior approval by the institution’s human research committee.In a retrospective cohort, 23 IFX (Remicade) treated IBD patients were identified with anti-IFX antibodies (ATI) and their clinical parameters were collected from their electronic patient dossier. This is a retrospective study on biobank material. This biobank was approved by the ethical review board of the UMCG approved August 52-fragment, ADA, Etanercept, Certolizumab and Golimumab, we used HeLa 8D8 cells that express the Green Fluorescence Protein (GFP) under control of the NF-kB response element [To investigate the difference between the neutralizing effect of IFX, IFX (F)ab element .2 at 37°C. The peak of TNF-α (1 ng/ml)-induced GFP expression was observed after 16 h of incubation, which was chosen for all further experiments. Reagents were added directly to the cells after seeding them and incubated for 16 hours. After incubation, cells were suspended in 350 μl of ice-cold phosphate-buffered saline (PBS) supplemented with 1% bovine serum albumin (BSA) and kept on ice until analysis. The number of GFP-positive cells was quantified by flow cytometry using a FACSCalibur™ flow cytometer. The fluorescent signal was measured in at least 10,000 cells. Different concentrations of IFX, IFX biosimilar CT-P13 (Inflectra), ADA, Certolizumab, Etanercept and Golimumab (0-5-10-20-40-100 ng/mL) were analyzed for their neutralizing capacity on 1 ng/mL TNF-α. All experiments were conducted four times.Cultures of HeLa 8D8 cells were maintained in plates at a density of 250,000 cells/well in 1 mL Dulbecco’s-modified Eagle medium (DMEM), supplemented with 10% heat-inactivated fetal calf serum (FCS) and incubated in an atmosphere of 5% COin vitro blocking effect of sera of patients with proven ATI. Serum samples that contained ATI were incubated with 1 ng/mL of TNF-α and 10–40 ng/mL of IFX were added to the Hela 8D8 cells. The quantity of serum was adjusted to a concentration of 7 AU/mL of ATI. Different amounts of normal human serum were used as negative controls.Blood samples of 23 IBD patients with ATI were collected. The concentrations of antibodies against IFX and the serum levels of free IFX were determined by Sanquin, Amsterdam, The Netherlands ,25. We eCross-reactivity between antibodies against IFX and ADA was tested using sera of 2 CD patients with ATI (7 AE/ml) and sera of 1 AS and 1 PsA patient with ATA (30 AE/mL).Sera with ATI of 6 different IBD patients were used to show antigenic cross-reactivity between IFX and the IFX biosimilar CT-P13 (Inflectra).t- test and otherwise the Mann-Whitney U test, depending on normality of the data, for continuous data. All tests of significance were two-sided, with p-values of <0.05 assumed to indicate significance. Statistical analyses were performed using IBM SPSS Statistics version 25.0  unless otherwise mentioned.Continuous variables were given as mean and range and categorical data as numbers with percentages. Differences between groups, at baseline, different follow-up times, were evaluated by the Student–Blood samples of 23 IBD patients with ATI were used to test the blocking effect of the neutralizing effect of IFX. The characteristics of these patients are shown in A 16 h treatment with 1 ng/ml TNF-α resulted in a strong induction of GFP-positive HeLa 8D8 cells (approximately 50%), validating their use as TNF-α reporter cells . Next, ISera of 17 different IBD patients with different concentrations of ATI were added together with 1 ng/mL of TNF-α and 10–40 ng/mL of IFX to the HeLa 8D8 cells.We descriptively show that there is a blocking effect of ATI of the 17 different IBD-patient’s serum samples on the neutralizing effect of IFX as shown in Sera of patient 15 and 17 had a low blocking effect possible due to that they had lower levels of ATI, 46 AE/ml and 59 AE/ml, respectively. We show in the Serum samples of 2 IBD patients with ATI and serum samples of 2 RA patients with ATA were used to evaluate the cross-reactivity between ATI and ADA, and ATA and IFX, respectively. The sera with ATI inhibited the effect of the IFX F(ab)2-fragment, but did not block the neutralizing effect of ADA . Therefoin vitro reporter assay, we show in this study that Etanercept (ETA), Certolizumab and Golimumab are more effective in neutralizing TNF-α compared to the first generation anti-TNF-α drugs IFX and ADA. We also show that serum with proven ATI blocked the TNF-α-neutralizing properties of IFX, the F(ab) 2 of IFX, and the IFX biosimilar CT-P13 (Inflectra), whereas ATI did not block the effect of ADA.Using an et al reported in a retrospective review that there was no difference in clinical remission rates between IFX, ADA and Certolizumab pegol.[IFX was the first anti-TNF-α biological for the treatment of IBD patients. The ACCENT1 trial showed that CD patients treated with IFX therapy were 3 times more likely to have clinical remission in the maintenance phase.,26 ADA aab pegol.Recently, biosimilars of IFX, such as CT-P13 (Remsima/Inflectra), manufactured by Celltrion and Flixabi manufactured by Biogen came available for the treatment of IBD.,13 The tin vitro. The ATI seems to be directed against the F(ab)2-fragment of the anti-body and since an antibody reaction is very specific this proves similarity. These findings correspond to the findings with Remsima.[In this study we show that there is no difference in either the anti-TNF-α effect or antigenicity of IFX and its biosimilar CT-P13 (Inflectra)  Remsima. This is  Remsima. As a res Remsima. In accorAs shown in clinical studies, IFX-treated patients with loss of response due to the development with ATI can be switched safe and effective to ADA and vice versa.As the expiration date for IFX was considered to be only 24 hours we tested fresh and 4 year-old IFX from different batches in solubilized and dry conditions and found no difference in the TNF-α neutralizing capacity. Therefore, spill of this expensive biological can probably be reduced by compounding and storing IFX in sterile and cool conditions, according to normal practice for antibody storage. Our study has some limitations. First, the sample size is relatively small and therefore data is restricted to observations and hypotheses and cannot reveal  mechanisms. Any additional subgroup analysis have not been performed due to size of the study, therefore potential clinical implications as patient tailored therapy remains hypothetical. Strengths include our well-characterized cohort, and uniform collection of laboratory data.In conclusion, we show that the second generation anti-TNF-α drugs show increased TNF-α neutralizing potential compared to first generation anti-TNF-α drugs. We also show that IFX and the IFX biosimilar CT-P13 (Inflectra) have the same neutralizing capacity as the original IFX. Furthermore, we show that ATI show cross-reactivity toward the IFX biosimilar CT-P13 (Inflectra) proving that the CT-P13 (Inflectra) IFX biosimilar also has the same antigenic properties and therefore the clinical implication is that patients with ATI should not be switched to IFX biosimilar. However, patients treated with IFX without IFX antibodies can be switched safely and effectively to this cheaper IFX biosimilar in order to reduce costs of anti-TNF-α treatment in IBD. Patients with ATI could benefit from a switch to adalimumab as there is no cross-reactivity.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


Anti-tumor necrosis factor (TNF)-α agents represent an effective treatment for chronic inflammatory diseases. However, some concerns about their potentially undesirable effects on liver function have been reported. On the other hand, evidence of their therapeutic effects on certain liver diseases is accumulating. Many data showed the safety of anti-TNF-α in patients with chronic hepatitis B and C and in liver transplanted patients even if a strict follow-up and prophylaxis are recommended in well-defined subgroups. On the other side, anti-TNF-α-induced liver injury is not a rare event. However, it is often reversible after anti-TNF-α withdrawal. Anti-TNF-α agents have been tested in advanced stages of severe alcoholic hepatitis and non-alcoholic fatty liver disease. Limited data on the efficacy of anti-TNF-α in patients with autoimmune hepatitis and primary biliary cholangitis are also available. In this review, we explored the hepatic safety concerns in patients receiving anti-TNF-α agents with and without pre-existent hepatic diseases. In addition, the available evidence on their potential benefits in the treatment of specific hepatic diseases is discussed. In the last 20 years, anti-tumor necrosis factor (TNF)-α agents have been increasingly used for the treatment of patients affected by inflammatory bowel disease (IBD), such as Crohn’s disease (CD) and ulcerative colitis (UC), as well as rheumatologic and dermatological disorders such as rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), ankylosing spondylitis (AS), psoriatic arthritis (PsA) and psoriasis. Currently, five TNF-α antagonists have been licensed by international regulatory authorities for clinical use: infliximab (IFX), adalimumab (ADA), certolizumab pegol (CER), etanercept (ETA) and golimumab (GOL). IFX  is a chimeric monoclonal immunoglobulin (Ig) G1 antibody composed of 75% human sequence and 25% mouse sequence, given intravenously. It is approved for RA, CD, UC, AS, PsA and psoriasis. ADA  is a recombinant, fully human IgG1 monoclonal antibody licensed subcutaneously for RA, CD, UC and psoriasis, whereas CER , a pegylated, humanised Fab fragment of an anti-TNF-α monoclonal antibody given subcutaneously, is licensed for RA, SA, and PsA and for CD only in the United States. Notably, all these drugs are able to neutralise both forms of TNF-α, either by binding the trans-membrane form (m-TNF-α) and/or by blocking the soluble form (s-TNF-α) . It is aThus, in this clinical review we explore the potential risks of hepatic injury induced by TNF-α antagonists, both in patients without pre-existent hepatic disorders and HBV or HCV chronic infection carriers; and the available evidence on the beneficial use of anti-TNF-α agents for the treatment of some liver diseases, such as AH or NAFLD.A literature search was conducted using Pubmed, up until November 2017. Original articles and reviews were identified using the key words: “anti-TNF-α”, “infliximab”, “adalimumab”, “certolizumab pegol”, “etanercept” and “golimumab” matched with each of the following key words: “viral chronic hepatitis”, “liver transplantation”, “drug-induced liver injury”, “autoimmune hepatitis”, “non-alcoholic fatty liver disease”, “alcoholic hepatitis”, “primary biliary cholangitis”. Additional articles were identified through a review of the reference lists of selected pertinent articles.TNF-α is a major proinflammatory cytokine that plays an important role in the integrated host defence system against infectious diseases. Given the role of TNF-α on both the innate and adaptive immunity, it is not surprising that the major toxicity concern with the anti-TNF-α agents is the risk of serious infection. Indeed, in vivo animal studies and post-marketing analysis of anti-TNF-α agents have shown that the blockage of TNF-α signalling pathway leads to an increased susceptibility to a range of pathogens , in partIt is estimated that there are nearly 100 million people worldwide with serological evidence of current or past HCV infection . A growiHowever, a word of caution is required for a number of reasons. First, these studies did not include pre- and post-treatment hepatic biopsies to exactly assess any worsening of liver damage due to anti-TNF-α therapy. Second, a long-term follow-up is not yet available and controlled trials are lacking. In addition, a small study including 6 cases with actively replicating chronic HCV infection on anti-TNF-α treatment for an associated RA showed the appearance of cryoglobulinaemia in 2/6 patients . Thus, tp = 0.02) [In contrast to HCV, experimental studies in vitro and in animal models have shown that TNF-α plays a pivotal role in clearing HBV from infected hepatocytes by inhibiting the replication and stimulation of the HBV-specific T cell response ,34,35,36 = 0.02) . In cont = 0.02) ,40,41. T = 0.02) ,43,44. I = 0.02) . As expe = 0.02) . Acute l = 0.02) . IFX was = 0.02) ,47,48,49 = 0.02) . In rece = 0.02) ,28,50,51Currently, the presence of HBV or HCV infection should not preclude the therapy with an anti-TNF-α agent . For HCVFew data are available on the ideal management of IBD when combining anti-TNF-α therapies with immunosuppression after liver transplantation (LT). After LT, about 30% of patients report an improvement of IBD, while almost the same percentage of patients get worse. In addition, occurrence of de novo IBD can develop in approximately 14–30% of patients with primary sclerosing cholangitis (PSC) . In thisp = 0.886). After correction for time since transplant, in the anti-TNF-α group it resulted 0.194 vs. 0.115 in the non-exposed (p = 0.219) [A case series evaluated six patients with IBD who required LT for PSC (4/6), AIH (1/6) or biliary atresia (1/6) . All of = 0.219) . HoweverOverall, anti-TNF-α therapy in post-LT IBD patients seems to be equally effective and safe despite the concomitant consumption of immunosuppressive medications. Nevertheless, caution should be used because of the risk of adverse effects, including cytopenia, opportunistic infections, and cancers .Abnormalities in liver functions tests, including transient and self-limiting hypertransaminasemia, cholestatic disease and hepatitis can develop during treatment with anti-TNF-α and, in some cases, they could be severe and life threatening ,7. IndeeDespite the pathogenesis of AIH induced by IFX still being unclear, it has been suggested that TNF-α blockade could impair the normal suppression of auto-reactive B cell production and apoptosis of CD8 T cells leading to an increased lymphocyte presence. This could trigger the development of autoantibodies  ,84. OtheCholestatic hepatitis induced by anti-TNF-α agents has also been described ,69,88. IAmong all these reports, there are some clear differences, including the definition of hepatic injury and the patient populations studied. Conversely, there are also some notable similarities. First, IFX appears the most common cause, with ETA and ADA involved to much lesser degrees . HoweverIn conclusion, these cases may alert physicians to the possibility of liver injury associated with the use of TNF-α blockers in an autoimmune setting, especially in the presence of pre-existent serological signs of autoimmunity such as ANA. While the incidence of liver injury due to anti-TNF-α therapy seems to be relatively low, the hepatic damage is nonetheless significant. Thus, in patients with an important elevation of transaminases or clinical signs consistent with acute hepatitis, anti-TNF-α should be stopped before the development of severe irreversible injury. In addition, based on current data, the mechanisms of liver toxicity may be different between IFX and ETA, according to their different structure. Moreover, ETA may be safe in patients who have presented liver toxicity while receiving IFX. In n = 10) or placebo (n = 10) at day 0. The authors excluded patients with DF > 55. At day 28, there was a significant improvement in the IFX group for both Maddrey’s score and IL-6 and IL-8 levels. Conversely, in the placebo group no significant difference was observed for these parameters compared with baseline. IFX was well tolerated and there were no significant differences between the two groups in terms of side effects [p = 0.04) as well as the 6-month mortality rate  [AH is a necro-inflammatory liver disease, observed in approximately 20% of heavy drinkers. Severe AH is associated with high morbidity and mortality rates ,98. In t effects . In the  effects , 36 pati effects ,118. In  effects , 12 pati effects . Serum b effects . Interes effects . In this effects . Finally effects . The inc= 0.017) . In concp < 0.05) [NAFLD includes a large spectrum of clinical and pathological liver conditions, including simple steatosis, non-alcoholic steatohepatitis (NASH) and fibrosis ,122. NAF < 0.05) . The eff < 0.05) . Authors < 0.05) . There a < 0.05) .In conclusion, although no data in patients with NASH are yet available, several experimental data seem to confirm that the blockade of TNF-α might be an important target for the treatment of NASH. Certainly, we are looking forward to new studies designed to evaluate the role of anti-TNF-α agents for the treatment of NASH.AIH is a relative rare chronic liver disease that affects mainly woman and is characterized by elevated transaminases, hypergammaglobulinaemia, circulating autoantibodies, interface hepatitis at liver histology. If untreated, it often leads to cirrhosis, liver failure and death . In the Infliximab  may be considered as rescue treatment in patients  with difficult-to-treat AIH, even if therapy may be associated with infection complications ,142.In general, patients with autoimmune liver diseases display a different cytokine expression pathway compared with healthy patients, with increased serum levels of proinflammatory cytokines, such as IL-6, IL-8, and TNF-α . FurtherWeiler-Normann et al.  describeAnyway, no strong recommendation can be drawn from these small sample size studies. On the other side, anti-TNF-α antibodies may also induce an immune-mediated liver disease resembling AIH, as previously discussed.PBC is a chronic progressive liver disease characterized by serological presence of the antimitochondrial antibodies (AMA) with a title of 1/40 or greater, chronic non-suppurative destruction with progressive loss of intrahepatic small bile ducts on liver histology, resulting in progressive fibrosis and cirrhosis. PBC is considered an immunologically mediated disease where the TNF-α plays a prominent pathogenic role . The uniFurthermore, reversible cholestatic liver disease has been noticed in a few patients receiving IFX for various autoimmune disorders .These reports could suggest a common pathogenic pathway for these diseases. In this scenario, the use of anti-TNF-α could be useful in some subsets of autoimmune colangiopathies. In particular, among these groups, those with a concomitant inflammatory arthropathy could have a successful response to anti-TNF-α drugs.Anti TNF-α agents represents a paramount treatment for several chronic inflammatory diseases. Although their clinical use and safety risk for hepatic diseases has not been completely explored, they have undoubtedly a role on liver pathophysiology. Indeed, they possess a dichotomous impact with both therapeutic and toxic effect depending on the immunological status and the concomitant diseases of the host. It should be underlined that no randomized controlled trials or well-conducted observational studies exist that assess, for example, the safety of TNF-α blockers in Hepatitis B or Hepatitis C or their overall hepatotoxic potential (even large case series from established networks do not provide enough information to quantify the risk of hepatotoxicity). Future studies should also better define the mechanisms of liver injury linked to anti-TNF-α drugs. More importantly, reliable factors are crucially needed to predict the risk of adverse hepatic effects. Conversely, the investigation on specific host variables that are connected to TNF-α involvement in inflammatory liver diseases could open new therapeutical horizon to the selection of patients who would benefit from anti-TNF-α use.


Perceptual decision-making is often modeled as the accumulation of sensory evidence over time. Recent studies using psychophysical reverse correlation have shown that even though the sensory evidence is stationary over time, subjects may exhibit a time-varying weighting strategy, weighting some stimulus epochs more heavily than others. While previous work has explained time-varying weighting as a consequence of static decision mechanisms , here we show that time-varying weighting can reflect strategic adaptation to stimulus statistics, and thus can readily take a number of forms. We characterized the temporal weighting strategies of humans and macaques performing a motion discrimination task in which the amount of information carried by the motion stimulus was manipulated over time. Both species could adapt their temporal weighting strategy to match the time-varying statistics of the sensory stimulus. When early stimulus epochs had higher mean motion strength than late, subjects adopted a pronounced early weighting strategy, where early information was weighted more heavily in guiding perceptual decisions. When the mean motion strength was greater in later stimulus epochs, in contrast, subjects shifted to a marked late weighting strategy. These results demonstrate that perceptual decisions involve a temporally flexible weighting process in both humans and monkeys, and introduce a paradigm with which to manipulate sensory weighting in decision-making tasks. During decision-making, the weight assigned by subjects to sensory information over time is not necessarily constant. Such time-varying weighting is often interpreted as a signature of a particular decision-making model . Temporal weighting may also result, however, from a strategic reweighting of the stimulus evidence itself that takes place before and/or independent of a decision-making mechanism. Here we use a psychophysical reverse correlation paradigm to both measure and manipulate temporal weighting behavior. We demonstrate that both humans and macaques adopt weighting strategies that are flexible, consistent with dynamic reweighing of the sensory stimulus.Perceptual decisions are typically thought of as resulting from some form of accumulating samples of a stimulus over time. During this process, a decision variable is updated as evidence is integrated until a choice is made. In both human and nonhuman primates, perceptual decision-making has been studied extensively in the context of motion direction discrimination tasks, where the vast majority of stimuli provide statistically uniform sensory evidence over time . DespiteThe diverse set of temporal weighting profiles observed across studies and species may be explained in a number of ways. One approach appeals to mechanistic models of decision-making. An early weighting strategy, for example, could be explained as a consequence of bounded accumulation , which pAn alternative approach to explaining the variety in weighing strategies postulates that the temporal weighting strategy is flexible and is linked to the demands or structure of the task. This notion is supported by experiments in which weighting changes systematically with variable trial length and signal timings , as wellWithout appeal to a specific decision-making mechanism, we set out to manipulate temporal weighting under the hypothesis that weights should be flexible and influenced by the dynamic features of the stimulus itself, either independent of or in addition to constraints imposed by integration mechanisms such as a bound or a leak.To test this idea, we adopted a motion stimulus designed explicitly for psychophysical reverse correlation in the presence of experimenter-controlled manipulation of temporal stimulus statistics . The stiWe found that in both time-varied conditions (early-stimulus and late-stimulus), subjects shifted their temporal weighting strategy, placing highest weight on motion pulses with the highest mean motion strength. In flat-stimulus sessions, however, subjects exhibited a large range of temporal weighting strategies despite equal mean motion strength over time. Overall, these results demonstrate that temporal weighting strategies in human and monkey observers are flexible and can be adjusted to suit temporal stimulus statistics.Data were collected from both monkeys and humans. Monkey data were collected from two adult rhesus macaques  aged 10 and 14 years, weighing 7.7 and 10 kg, respectively. All animal procedures were performed in accordance with The University of Texas at Austin animal care committee’s regulations. Both M1 and M2 had standard surgery for implantation of a head-holder. Some portion of the monkey data were presented previously . Human d2) that was corrected to have a linear gamma function. Monkeys viewed the stimulus from a distance of 118 cm . Auditory feedback was played at the end of every trial, and fluid reward was delivered through a computer-controlled solenoid. Humans viewed stimuli on a linearized 16.5-inch OLED (LG) display  at a distance of 65.3 cm .For both monkeys and humans, stimuli were presented using the Psychophysics Toolbox with Matlab (Mathworks) using a Datapixx I/O box (Vpixx) for precise temporal registration . Sample Stimulus and task design were identical between monkeys and humans unless otherwise noted. Subjects were required to discriminate the net direction of a motion stimulus and communicate their decision with an eye movement to one of two targets, placed on either side of the motion stimulus. The sequence of task events is presented in The reverse-correlation motion stimulus contained motion toward one direction or the opposite, with varying motion strength. Spatially, the stimulus consisted of a hexagonal grid of 19 Gabor elements, 5°–7° across, scaled by eccentricity B. Indivii was defined as the proportion of signal elements out of the total number of elements, the value of which was drawn from a Gaussian distribution, k is the distribution index for the five trial types  and Each motion stimulus presentation consisted of seven consecutive motion pulses lasting 150 ms each (9 frames), producing a motion sequence of 1050 ms in duration in total. For human subjects S2 and S3, each motion pulse lasted 100 ms each (6 frames), producing a 700-ms-long stimulus. On any given pulse, a number of Gabor elements would have their carrier sine waves drift in unison to produce motion , and the remaining would counterphase flicker (“noise elements”). Signal elements on any given pulse were assigned at random within the grid and all signal element drifted in the same direction. Motion strength on pulse ld be μk . The subThe distributions i = 1), and reaches its expected value . Additionally, 30 macaque sessions were excluded from analysis for having psychophysical thresholds >2 median absolute deviations about the median. Overall, 188 and 52 sessions were included for macaques and humans, respectively, with median session lengths of 632 and 295 successfully completed trials, netting a total of 129,922 and 15,275 trials overall.p and leftward choice is 1 – p, where p is given byx is the net motion strength value , All analyses were performed in Matlab (Mathworks). Subject choices in the direction-discrimination task were analyzed with a maximum likelihood fit of a three-parameter logistic function  assumingw, were computed by using all trials within a session. These include trials where The temporal weighting kernel  was computed using ridge regression via maximum likelihood. The log posterior of the psychophysical weights is given bymization . Psychopw, describes the temporal weighting adopted by the subject for a given set of trials. If the individual weights have a similar value, then that implies that the subject had weighted all pulses equally on average. If some weights are larger than others, that implies uneven weighting over time. We summarized temporal weighting by performing linear regression on the 7 weights and using the slope of the fit as a metric of temporal structure, where negative slopes indicate early psychophysical weighting and positive slopes indicate late. Comparisons of temporal weighting profiles across experimental conditions and species were assessed using the slope of the linear fit ± 95% confidence intervals. Wilcoxon sign tests were used to evaluate whether slopes differed significantly from zero. ANOVA was used to assess differences in mean slopes across experimental conditions. Bartlett’s test was used to evaluate differences in variance between distributions of slopes across experimental conditions. The vector of weights, Overall, subjects performed more than 145,000 trials of a one-interval motion direction discrimination task. After viewing a sequence of motion pulses, they indicated the net perceived direction by moving their eyes to one of two targets . In addiChanges in temporal stimulus statistics led to clear shifts in the psychophysical weighting strategy in all subjects. We consider the flat-stimulus condition as a baseline, both because of the stationary statistics of the stimulus over time, and because the vast majority of stimuli used in the study of perceptual decision-making have temporally stationary statistics. In the flat-stimulus condition, subjects exhibited an inclination toward early weighting, with the highest weight on the first three pulses and then a steady decrease as time went on D. The teDuring late-stimulus sessions, subjects shifted their strategy to place higher weight on the later pulses, which more often carried high motion information and were therefore more reliably correlated with the final trial outcome. Temporal weights in the late-stimulus condition started low, increasing to a peak at the fifth or sixth motion pulse, followed by a decreased weight on the seventh  pulse E. AlthouIn sharp contrast to the late-stimulus sessions, during early-stimulus sessions, subjects showed steep early weighting, where the first three pulses were weighted the highest followed by a large decrease F. As witThe differences in temporal weighting strategies as a function of stimulus condition were robust and consistent across species . TemporaIn addition, no differences in temporal weighting strategy were observed between species within either the early- or late-stimulus conditions. In the flat-stimulus condition, in contrast, macaques exhibited an early weighting that was substantially steeper than that exhibited by the human observers  was in conflict with the net direction of motion of the full trial . Most choices in these trials were in favor of the net direction of motion, as opposed to the direction of the extreme single pulse, in both human and macaque subjects E. We theWhen averaged across sessions and subjects, temporal weighting profiles tell a fairly straightforward story: subjects adopt a late weighting strategy for the late-stimulus, an early weighting strategy for the early-stimulus, and a flat-to-early weighting strategy for the flat-stimulus. Here we sought to quantify the weighing strategy at a higher resolution by looking at performance for individual subjects and sessions.When each subject is considered individually, results were largely consistent with the average weighting profiles reported above. In the late-stimulus condition, human and macaque subjects’ weighting was extremely similar A. All obp < 0.0001), with no single individual sessions having a slope greater than zero (p < 0.0001), with only 2 of 42 sessions having a slope less than zero. These distributions of weighting slopes reveal distinct populations across conditions , indicating that even at the resolution of single sessions, distinct strategies were adopted during the early- and late-stimulus conditions. The distribution of weighting slopes from the flat-stimulus condition had a mean of –0.0356, denoting slight early weighting , but also differed in that it had a considerably larger range of results. The standard deviation of flat-stimulus weighting slopes was more than double that of the early- or late-stimulus weighting slope distributions , indicating that subjects adopted a larger variety of temporal weighting strategies in this condition. It is worth noting that some of the variance in all three of the distributions comes from noise inherent to fitting a two-parameter linear model to the seven weights that constitute the temporal weighting strategy; nevertheless, the difference in distribution widths is substantial and therefore likely meaningful.When each session is considered individually, variability in temporal weighting strategy is evident both between and within each of three stimulus conditions. To quantify the degree of early versus late single-session weighting, we fitted a line to the seven temporal weights of the observer for each session and used the slope of this fit to summarize the temporal weighting profile: a positive slope indicates late weighting, a negative slope indicates early weighting, and a slope around zero indicates flat  weighting over time. The distribution of weighting slopes for all experimental sessions in the early-stimulus condition had an average of –0.079 , indicating that adopting an early weighting strategy is detrimental to psychophysical performance. The early-stimulus sessions exhibited a positive correlation between temporal weighting slope and psychophysical threshold , indicating that in the early-stimulus condition, an early weighting strategy is preferable. Little to no correlation was observed in the late-stimulus sessions .We next sought to examine the relationship between temporal weighting strategies and psychometric performance in the direction discrimination task. We compared the slope of the temporal weights to psychophysical threshold  for each stimulus condition B. Duringr = 0.40, p < 0.0001), demonstrating that during flat-stimulus sessions, employing weights that are highly variable from temporal uniformity  is detrimental to psychophysical performance. Late-stimulus sessions showed a moderate positive correlation , and early-stimulus sessions showed no obvious linear relationship .Perhaps more compelling was the relationship between psychophysical threshold and the energy of the temporal weights, where energy was measured as the sum of the squared residuals of each weight from the mean of the seven weights C. This mTaken together, larger variability in weighting and higher energy appear to be detrimental toward psychometric performance. These were most pronounced in the flat-stimulus condition, offering a potential explanation for the slight and unexpected decrease in psychophysical behavior during the flat-stimulus relative to early- and late-stimulus conditions C, D.We used psychophysical reverse correlation in the context of manipulations of temporal stimulus statistics to examine observers’ ability to update their temporal weighting strategy to match the time course of available evidence in a dynamic motion discrimination task. First, we found that when motion strength was systematically varied over time within a stimulus presentation, subjects changed their temporal weighting strategy to weight the periods of strong motion more heavily than those with weak motion. Second, weighting strategies were rather consistent across species and subjects, with the exception of the flat-stimulus condition. Third, session-to-session variability in strategy was greater in the flat-stimulus condition than in the late- and early-stimulus conditions. Each of these findings is discussed in more detail below.The observation of early sensory evidence exerting a larger effect on decisions than late evidence  has been identified in prior work and has been interpreted within the context of a drift diffusion decision-making model. Early weighting is often interpreted as a straightforward consequence of accumulation to a decision bound—sensory data arriving after the bound has been hit does not impact the accumulator . Just asThe shifts we identified in temporal weighting strategies show that time-varying weighting of a stimulus is a flexible strategy that adapts to the statistical structure of the stimulus. This flexibility highlights the possibility of a more direct reweighting of the sensory signal itself, regardless of downstream impacts, such as a bound or a leak in the sensory integration system. Temporal weighting strategies need not be solely the result of static decision-making mechanisms, but rather could reflect a dynamic strategy for directly weighting incoming stimulus. Another group made a similar observation , but in It remains to be seen whether the observed time-varying weighting in sensory brain areas can be changed in response to temporal manipulations of the stimulus of the sort we employed, but the well documented effects of temporal attention in multiple visual cortical areas  lend crepost hoc characterization or infrequent probe trials. Our results suggest that even individual subject averages may gloss over strategic variability within the observer that occurs over sessions. Likewise, even the relatively high-resolution session averages we present here may mask variability over single trials, variability that current trial-based psychophysical methods lack the resolution to resolve. Consequently, all temporal weighting strategies presented here  are computed as an average over multiple trials, each with a potentially unique temporal weighting strategy.Variability in temporal weighting strategy during the flat-stimulus condition was far larger than in either the early- or late-stimulus conditions. This substantial variability is of general relevance to the study of evidence accumulation, because it is typically performed using stimuli that are similar to our flat-stimulus condition, in that their expectation is stationary over time. Although the average weighting strategies for both humans and macaques in the flat-stimulus condition trend toward early weighting, session-by-session analysis of weighting slopes revealed robust variability . Few if The large session-by-session variability in weighing strategies observed here may serve to reconcile those presented elsewhere. In the flat-stimulus condition, all time points  are equally informative of the trial outcome, and thus the flat-stimulus condition is more forgiving of different temporally biased weighting strategies compared to the early and late conditions, for which only approximately half of the stimulus contained informative evidence on average. Thus, increased variability in weighting strategies during the flat-stimulus condition compared to early- and late-stimulus conditions is likely a consequence of temporally uniform stimulus statistics—a feature of most evidence accumulation studies.The consistency of temporal weighting across species displayed in the late and early stimulus conditions also suggests that, at least for humans and macaques, interspecies differences need not be a major player in variability of weighting. This is of possible broader interest, for example, in linking to rodent work .One discrepancy across species was present in the flat-stimulus condition, in which macaque subjects  displayed an early-weighting strategy (despite flat stimulus expectation) compared to the flat-weighting strategy displayed by humans. This could be for a number of reasons. Macaques performed many more trials and sessions than human subjects, raising the possibility that extensive training may result in faster decisions, based on early epochs of the stimulus. This may be further accentuated by a desire to perform more trials and obtain more liquid reward (a factor not included in experiments with human subjects). While such a strategy does not in fact change the trial duration or, in turn, the speed-accuracy trade-off, it might factor into macaques’ behavior. It is noteworthy that the species difference is present only in the flat-stimulus condition, and not the time-variant conditions. We believe this is because the flat-expectation and fixed-duration design is lenient with respect to temporal weighting, granting subjects the liberty to adopt any number of temporal weighting strategies . This isStimulus and task design must be considered to properly interpret the shape of a temporal weighting strategy. Given that single trials are always rewarded based on the true net motion presented, regardless of their underlying distribution, all motion pulses are always informative. Therefore, it is intuitive that highest overall accuracy would be realized via a strategy that assigns equal weighting across all pulses. However, this was not uniformly present in our dataset, indicating that subjects did not perform the task optimally. Importantly, the assumption of equal weighting is only one part of an optimality argument, as equal but low weighting of incoming sensory data would of course be suboptimal too. Complete optimality of the decision mechanism is a difficult standard to assess without a detailed characterization of signal and noise in both the stimulus and the sensory neural representation . Given tA similar difficulty is present in evaluating the optimality of temporal integration in fixed-duration tasks. Classically, tests of optimal temporal integration appeal to the relation between viewing duration and accuracy . HoweverOther aspects of experimental design may increase the complexity of inferences drawn from the assessment of temporal weighting as well. For example, although early weighting may be a general default state , variable duration paradigms may fortify an early weighting strategy. Variable duration paradigms can be thought of as loosely analogous to our early-stimulus condition, in that as time progresses, the expected stimulus strength falls off (owing to the end of the variable-duration stimulus). Reaction time tasks can also facilitate an early weighting strategy, as the subject is typically incentivized to respond as fast as possible, placing more weight on early samples within a stream . Lastly,Our characterizations of temporal weighting are of course inherently limited by the assumptions of logistic regression. While it is clear that subjects weigh temporal sections of the stimulus in proportion to their expected motion signal, it seems unlikely that the way the brain performs this task is completely described by logistic regression. There are likely a cascade of nonlinearities between stimulus and response that cannot be fully described by a set of linear weights passed through a sigmoid, which implies that the exact pattern and magnitudes of an individual temporal kernel are an incomplete description of the decision process. However, given the close correspondence between kernels computed using only flat-expectation, zero-mean (noise) trials and kernels computed using all trials , any nonlinearity in mapping from stimulus to sensory evidence appears to have a minimal impact on our core result: differences between temporal stimulus statistics can exert systematic and interpretable effects on temporal weighting strategies.More generally, our results provide an opportunity to reconnect perceptual decision-making models with other frameworks for information integration. For example, the dynamic temporal weighting we observed has a direct connection to classical Bayesian integration . Over reIn summary, past work has used reverse correlation and time-varied stimuli to probe temporal integration. In the present study, we used a reverse correlation task in the context of tractable manipulations of stimulus statistics, allowing for direct control over a subject’s temporal weighting strategy. Although the neural correlates of such changes remain uncertain, the ability to both manipulate and characterize temporal weighting strategies should provide a powerful tool for neurophysiological experiments to come.


Heart failure (HF) and chronic kidney disease (CKD) co-exist, and it is estimated that about 50% of HF patients suffer from CKD. Although studies have been performed on the association between CKD and HF with reduced ejection fraction (HFrEF), less is known about the link between CKD and heart failure with preserved ejection fraction (HFpEF). Approximately, 50% of all patients with HF suffer from HFpEF, and this percentage is projected to rise in the coming years. Therapies for HFrEF are long established and considered quite successful. In contrast, clinical trials for treatment of HFpEF have all shown negative or disputable results. This is likely due to the multifactorial character and the lack of pathophysiological knowledge of HFpEF. The typical co-existence of HFpEF and CKD is partially due to common underlying comorbidities, such as hypertension, dyslipidemia and diabetes. Macrovascular changes accompanying CKD, such as hypertension and arterial stiffening, have been described to contribute to HFpEF development. Furthermore, several renal factors have a direct impact on the heart and/or coronary microvasculature and may underlie the association between CKD and HFpEF. These factors include: (1) activation of the renin-angiotensin-aldosterone system, (2) anemia, (3) hypercalcemia, hyperphosphatemia and increased levels of FGF-23, and (4) uremic toxins. This review critically discusses the above factors, focusing on their potential contribution to coronary dysfunction, left ventricular stiffening, and delayed left ventricular relaxation. We further summarize the directions of novel treatment options for HFpEF based on the contribution of these renal drivers. Heart failure with preserved ejection fraction (HFpEF) is characterized by impaired relaxation of the left ventricle (LV) during diastole and accounts for over 50% of all patients with heart failure (HF) . Both thThe current paradigm for HFpEF proposes that commonly present comorbidities such as diabetes mellitus (DM), obesity, and hypertension lead to a systemic pro-inflammatory state. This pro-inflammatory state causes coronary microvascular dysfunction, evidenced by an imbalance between nitric oxide (NO) and reactive oxygen species (ROS) leading to stiffening of the LV . ExcessiIt is important to note that the impact of microvascular dysfunction on cardiac structure and function is not limited to dysfunction of the NO-cGMP-PKG pathway. Indeed, upregulation of VCAM-1 and E-selectin on the coronary microvascular endothelium induces transendothelial leucocyte migration and activation, increased transforming growth factor β (TGF-β) levels, thereby promoting pro-fibrotic pathways and differentiation of fibroblast to myofibroblasts  and incrThe so-called cardio-renal syndrome describes the co-existence of HF and chronic kidney disease (CKD). Approximately 50% of the patients with HFpEF also suffer from CKD . AlthougGiven the co-incidence of HFpEF and CKD, the present review aims to provide a mechanistic link between CKD and HFpEF, by describing potential pathways through which CKD can induce or aggravate coronary microvascular dysfunction and thereby contribute to the development and progression of left ventricular hypertrophy and diastolic dysfunction. These include mechanical effects, neurohumoral activation, systemic inflammation, anemia and changes in mineral metabolism as induced by CKD . As someCKD is defined as a progressive decline of renal function and is associated with hypertension, proteinuria, and the loss of nephron mass . CKD is It should be noted however, that in addition to decreased diastolic function, both hemodialysis and pre-dialysis CKD patients show impaired regional systolic function measured by longitudinal, circumferential, and radial strain while ejection fraction was preserved . SimilarArterial remodeling in CKD patients is characterized by arterial stiffening, increasing pulse pressure, as a consequence of premature aging, and atherosclerosis of the arteries . Prematu+ by inhibition of the cardiac Na+/H+ exchanger (NHE) and induce coronary vasodilation  in response to renal hypoxia resulting in volume overload , which mependent . Furtherependent . Myocardependent . In a stDownstream from angiotensin II in the RAAS, aldosterone regulates blood pressure and sodium/potassium homeostasis through the mineralocorticoid receptor in the kidneys, by enhancing sodium reabsorption, thereby contributing to hypertension and high plasma sodium levels. Besides the renal effects, aldosterone has been shown to directly promote myocardial fibrosis, left ventricular hypertrophy, and coronary microvascular dysfunction, acting through endothelial and myocardial mineralocorticoid receptors, independently of angiotensin II .1-blockade with Irbesartan reduced mortality and improved outcome on cardiovascular endpoints in patients with natriuretic peptides below the median, but not in patients with higher natriuretic peptide levels  and sacubitril (neprilysin inhibitor). Neprilysin inhibition exerts its beneficial effects through inhibition of the breakdown of natriuretic peptides. Entresto was superior to the standard therapy, enalapril, in patients with HFrEF in reducing mortality and number of hospitalizations for HF , which is a combination of valsartan  in the D-HART trial including 12 patients , in which myocardial oxygen supply is limited by coronary microvascular dysfunction. Indeed, INOCA is increasingly being recognized as a risk factor for development of HFpEF  have been measured in patients with CKD . This isIn the LV, AGEs are particularly prominent in the coronary microvasculature, where their presence induces a pro-inflammatory phenotype , endothede novo AGE synthesis, drugs that break pre-existing AGE crosslinks and AGE receptor blockers  is a hormone produced by osteoblasts and osteocytes, which inhibits phosphate reabsorption in the kidneys and suppresses circulating calcitriol, effectively lowering plasma phosphate levels in physiological conditions . In CKD,1 receptors by angiotensin II. High levels of FGF-23 were further shown to cause endothelial dysfunction, increase superoxide formation, and decrease NO bioavailability in mouse aortas (Mechanistically, FGF-23 induces chronic inflammation by stimulating cytokine secretion from the liver, but is also locally produced by M1 macrophages, and can thereby further modulate inflammation in the heart . FGF-23 e aortas . Finallye aortas . Hence, e aortas . Furthere aortas . FGF-23 e aortas . Furthervia several processes, as summarized in The kidneys and heart are interdependent organs that are highly connected through multiple systems on both macrovascular and microvascular level. Unfortunately, many studies on the cardiorenal connection have not been conducted in specific HFpEF populations. Pathological processes which are present in CKD, such as vascular changes, deficiencies in kidney produced factors, and impairments in renal filtration can cause and/or contribute to development of HFpEF The complexity and multitude of connections between the heart and kidney make it unlikely that there is a single causal contributor for progression from CKD to HFpEF. In addition, although HFpEF is more prevalent in women, and the effect of sex on cardiovascular disease is increasingly recognized , the speThis review was designed by JW, MB, and DM. JW and MB have written the largest body of text. OS, JJ, MV, DD, AD, and DM have made a substantial, direct, and intellectual contribution to specific topics. All authors approved the work for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A prophylactic vaccine eliciting both broad neutralizing antibodies (bNAbs) to the HIV-1 envelope glycoprotein (Env) and strong T cell responses would be optimal for preventing HIV-1 transmissions. Replication incompetent HIV-1 virus-like particles (VLPs) offer the opportunity to present authentic-structured, virion-associated Env to elicit bNAbs, and also stimulate T cell responses. Here, we optimize our DNA vaccine plasmids as VLP expression vectors for efficient Env incorporation and budding. The original vector that was used in human trials inefficiently produced VLPs, but maximized safety by inactivating RNA genome packaging, enzyme functions that are required for integration into the host genome, and deleting accessory proteins Vif, Vpr, and Nef. These original DNA vaccine vectors generated VLPs with incomplete protease-mediated cleavage of Gag and were irregularly sized. Mutations to restore function within the defective genes revealed that several of the reverse transcriptase (RT) deletions mediated this immature phenotype. Here, we made efficient budding, protease-processed, and mature-form VLPs that resembled infectious virions by introducing alternative mutations that completely removed the RT domain, but preserved most other safety mutations. These VLPs, either expressed from DNA vectors in vivo or purified after expression in vitro, are potentially useful immunogens that can be used to elicit antibody responses that target Env on fully infectious HIV-1 virions. Following the discovery of human immunodeficiency virus type 1 (HIV-1) almost thirty-five years ago and its role as the etiological agent of acquired immunodeficiency syndrome (AIDS), the resulting pandemic has become one of the most significant infectious disease outbreaks in recent human history. The introduction of combined anti-retroviral therapy (cART) more than two decades ago has delayed disease progression and reduced mortality in patients from many developed countries dTTP (PerkinElmer). The reaction was incubated at 37 °C for 2 h before 10 μL of each reaction was spotted in duplicate onto DE81 DEAE cellulose filter paper . The filter paper was washed with saline-sodium citrate buffer and then ethanol, before being air-dried and exposed to a BAS-MS-IP 2340 imaging plate and read on a Molecular Imager FX™ Pro  and then analysed with Quantity One® software (Bio-Rad).The assay was based on previously described protocols ,59. Brie5 CEM.NKR CCR5+ cells were added to wells that were loaded with viral particles containing BlaM-Vpr  diluted in Opti-MEM . The samples of immature VLPs, for which the p24 concentration could not be accurately quantified by the p24 ELISA, were loaded according to their gp120 concentration and normalized to the highest gp120-containing sample. Spinoculation of the cells and viral particles was performed at 800×g for 1 h at 18–22 °C, and then incubation for 2 h at 37 °C, 5% (v/v) CO2 to ensure fusion reached completion. The media containing excess viral particles was removed from the cells and the cells were washed with CO2 Independent Medium (Thermo Fisher Scientific) and loaded with CCF2/AM dye from the LiveBLAzer™ FRET-B/G Loading Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Briefly, the loading solution contained 2 μM CCF2/AM, 139.2 μM acetic acid, 0.8 mg/mL Pluronic-F127R in CO2 Independent Medium, and the cells were incubated in 100 μL loading solution per well for 1 h at RT. Cells were washed twice with CO2 Independent Medium and then incubated for 14–16 h at 18–22 °C in 200 μL/well CO2 Independent Medium that was supplemented with 10% (v/v) FBS and 2.5 mM probenecid. The cells were then analysed by flow cytometry using a BD LSRFortessa™  and endpoint BlaM-mediated CCF2/AM dye cleavage (% fusion) was assessed by measuring the shift in emission fluorescence detected at 525 ± 50 nm (uncleaved) to 450 ± 40 nm (cleaved) when excited by a 405 nm laser. The p24 concentration yielding 50% fusion was interpolated from three-parameter logistic curves that were fitted using GraphPad Prism v7 software.Viral particles that were used for the fusion assay were produced, as described above, with the exception that cells were co-transfected with pMM93TH at one-fifth the mass of the total DNA. The viral fusion assay was based on previously published protocols . Brieflygag and pol with a B clade env, was previously utilized as a DNA vaccine vector [gag, pol, and nef ORF to abrogate enzymatic activity, such as RT function, RNA encapsidation, and IN activity. Specifically, the NC zinc (Zn2+) fingers were deleted by removing residues C15–C18 and C36–C39 (HXB2 numbering used here and throughout this manuscript); the RT was inactivated by the deletion of dNTP thumb (K65–L74), dNTP binding (D113–Y115), and active site (Y183–D186) residues; the RNase H active site was mutated (E38Q); the entire IN domain was deleted; a frameshift mutation was introduced at D36 of Nef, which created a premature stop codon at position 37. The vpr and vif genes and both 5′ and 3′ LTR promoters were completely removed, while vpu, env, tat, and rev were left intact. Replication-competent NL(AD8) (NL4-3 virus encoding an AD8 strain env) and increasing volumes of AEB VLP preparations were compared by anti-p24 Western blotting to assess the viral composition of these VLPs,. At the lowest VLP loading, only unprocessed (Pr55Gag) Gag was detected with increased loading revealing an additional matrix-capsid partial Gag processing fragment (p41) and another approximately 45 kDa partially processed fragment  by iodixanol density gradient rate velocity centrifugation. The samples of the initial VLP concentrate (In), the uppermost iodixanol-free fraction of buffer created upon the application of sample to the gradient (S) and gradient fractions of increasing density (1–12), were analysed by anti-gp120 and anti-p24 Western blotting. The NL4-3 and 93TH253.3 particles (as indicated by p24) mainly sedimented to fractions 9–10  of transiently-transfected cells, with EGFP-expressing cells being used to confirm no endogenous particle budding from the plasma membrane D. As expgag and pol regions had potential to cause the aberrant Gag cleavage profile that was seen in the viral particles: The complete removal of the IN domain  and cellular RNA act as scaffolds to tether adjacent Gag polyproteins [gag start codon to promote gRNA dimerisation and enhance NC binding [Several of the mutations within the AEB VLP N domain A, which proteins ,65, faciproteins , and areproteins ,68,69. Fproteins ,71, specpol domain  with or without an IN domain (IN +/−) did not alter the Gag cleavage efficiency of AEB VLPs, with fairly consistent Pr55Gag expression and no p24 being detected. When these modifications were coupled with the introduction of RNA motifs for efficient RNA scaffolding and packaging located in the truncated 5′ LTR (5LTRΔU3 +), the VLPs failed to demonstrate a mature particle phenotype, with similar proportions of Pr55Gag and p41 being observed for the unmodified AEB VLPs .To assess the contribution of the RNA stem loop and RNA-interacting protein domain mutations to AEB VLP morphology, particles that were expressed from vectors with a functional NC domain A or a tr AEp55B, C were alobserved D. The prgag and pol domains were those affecting RT. It has been reported that certain mutations within RT were designed to affect p51/p15 heterodimer stabilisation often decreased the infectivity of the resulting virions due to premature PR activity producing immature virions with irregular size and morphology [Gag ratios (as measured by band densitometry) and a Gag cleavage profile that was similar to the parental 93TH253.3 virus  also demonstrated enhanced Gag cleavage relative to particles containing the IN domain, but lacking the LTR, NC, and RT domain modifications.The other major set of safety mutations within the rphology . Althoug.3 virus B. This w2+ fingers for proper particle assembly and maturation was assessed with VLP vectors expressing functional RT alone and in combination with the U5 or functional NC. Anti-p24 Western blotting showed that increased Gag processing was strongly associated with the presence of functional RT (RT +) with a similar Gag cleavage profile to the parental 93TH253.3 virus, regardless of other modifications in the VLP vector  to confirm that the Tat modification was not altering Gag processivity, and it demonstrated a similar Gag cleavage profile to the unmodified AEB VLP. Overall, the three RT domain deletions in the AEB VLPs appeared to be the specific cause of the aberrant Gag cleavage profile. Combinations of the three RT domain deletions  were subsequently investigated for their contribution to the aberrant Gag cleavage profile. Analysis by anti-p24 Western blotting showed that AEB VLPs containing any one or more of the RT deletions produced particles with a similarly deficient Gag cleavage profile to that of VLPs with all three RT mutations  were further investigated, given that they expressed a good yield of mostly mature particles without including a functional NC domain, which is advantageous from a safety perspective. The morphology of AEB.RT.Δtat VLPs was assessed by rate velocity centrifugation through an iodixanol density gradient with samples being collected, as described previously. Anti-p24 Western blotting showed that the particles were predominantly found in fraction 10 C. This sGiven that the truncation of Tat was introduced to prevent potential alterations to VLP mRNA transcription via interaction between Tat and the TAR RNA stem loop present within the truncated 5′ LTR sequence, and the truncated 5′ LTR sequence was dispensable for the efficient expression of mature-form AEB VLPs with a functional RT domain, a modified AEB.RT.Δtat VLP vector with a functional Tat ORF (AEB.RT) was investigated. These AEB.RT VLPs showed increased levels of Env expression and demonstrated a similar particle size and/or shape as AEB.RT.Δtat VLPs by rate velocity centrifugation A. Env copol truncation C-terminal to the PR domain , the AEB.NCΔRT VLPs were utilized for further analysis.Alternative approaches to expressing mature-form VLPs without including a functional RT domain were investigated to achieve a safer VLP expression plasmid. Given that the original RT safety mutations and deletions likely destabilized the Gag-Pol polyprotein and prematurely activated the PR enzyme (based on reports of RT “primer grip” mutations inducing premature PR activity ), whetheThe morphology of the mature-form AEB.NCΔRT VLPs was assessed by rate velocity centrifugation, where anti-24 Western blotting showed the VLPs sedimented between fractions 7–12, with a peak in fraction 10 C, which Virus fusion assays were performed, since viral particle fusion has been reported to be inhibited by an uncleaved Gag polyprotein shell  and to fTo assess whether the extensive safety mutations in the mature-form AEB.NCΔRT VLPs prevented the incorporation and presentation of correctly folded Env, ELISAs were performed to determine the binding of bNAbs and poorly or non-neutralizing antibodies to Env-equalized amounts of AEB.NCΔRT and AEp55B VLPs. Recombinant AD8 gp120  and Env-deficient, mature-form VLPs (AEΔB.NCΔRT)  were included for comparison. The Env-bearing VLPs and gp120 showed comparable binding curves for 2G12 A, which Typically, HIV-1 VLPs are expressed by co-transfecting an Env-deficient particle expression vector with a separate Env-expression plasmid. This facilitates the rapid assessment of various Env strains or it is used to express viral particles that resemble infectious virus, but are not themselves replication-competent ,76,77. TGag alone is sufficient to mediate the assembly and budding of HIV-1 VLPs from the cell membrane [Gag-Pol polyprotein [The expression of Pr55membrane ,80; howeyprotein ,82,83. Iyprotein . Additioyprotein . VLPs reyprotein . The cloyprotein ,87. Addiyprotein . VLPs reyprotein . TherefoGag than p24 in particles pelleted from cell supernatant, which was indicative of immature HIV-1 virion formation, despite the VLP expression vector encoding pol with a functional PR sequence. The partial Gag polyprotein cleavage products that were observed by anti-p24 Western blotting (p41 and a ~45 kDa capsid-containing fragment) indicated that the viral PR was at least partly functional. The functional PR normally exists as a dimer following autocatalytic cleavage and release from the multimerized Pr160Gag-Pol polyproteins, which forms via interactions between the N- and C-terminal regions flanking the precursor PR domain [pol sequence contained a significant truncation via removal of the IN domain. However, the restoration of the IN domain in combination with functional NC or RNA stem loop structures did not alter the immature VLP profile, which suggested that inefficient Pr160Gag-Pol multimer formation and PR activation were not responsible. Furthermore, rate velocity centrifugation showed that the AEB VLPs more rapidly sedimented than infectious 93TH253.3 virions expressed from the proviral sequences that the VLP vector was derived from. This showed that the size and/or shape of the VLPs was distinct from normal HIV-1 virions, which indicated a defect or difference in the assembly of the VLPs due to one or more of the mutations within gag and pol to remove their replicative functions. Indeed, the morphology of AEB VLPs at the cell membrane visualized by TEM showed that their size and shape was highly variable and generally different to that observed for functional HIV-1 virions, which was consistent with the VLPs having a different sedimentation rate when assessed by rate velocity centrifugation. Given the extensive safety mutations within gag and pol, it is possible that they facilitated the inappropriate folding of Pr160Gag-Pol polyproteins, such that they were excluded from budding particles, inhibited PR activity, or disordered the Gag lattice within a budded VLP. Ultimately, the presence of the RT domain deletions was found to be the specific cause of incomplete Gag cleavage for the AEB VLPs, and this was consistent with previous observations that RT mutations that destabilise the Pr160Gag-Pol polyprotein multimers can cause premature activation of the PR [Gag, p41, and p24 that had undergone partial PR-mediated cleavage within the cell cytoplasm, rather than from uniform Pr55Gag assembly that would normally occur.The initial single-plasmid AEB VLP expression vector yielded particles with incomplete proteolytic cleavage of Gag, as demonstrated by comparatively more Pr55R domain ,90. For f the PR ,91. TherGag into its functional subunits. This was consistent with particle expression vectors that are employed to produce fourth generation lentiviral vectors, which express Gag polyprotein and a Gag-PR polyprotein from one plasmid, whereas the RT, RNase H, and IN are supplied in trans as a Vpr-tagged polyprotein [The removal of the entire RT domain from the VLP expression cassettes served two purposes. Firstly, it prevented the destabilisation of Gag/Gag-Pol multimers and subsequent aberrant particle assembly. Additionally, the RT functions are not required for viral particle maturation, thus its removal was a safety advantage. The VLP expression vectors that removed the entire RT domain (ΔRT) likely represent the minimal arrangement for VLP vector expressing particles that undergo proteolytic maturation, given that the minimum requirement for HIV-1 particle expression is the Gag polyprotein and the viral PR that is required to cleave Pr55yprotein . The paryprotein . The ΔRTyprotein . This woyprotein . This isyprotein . NonetheThe parental 93TH253.3 strain AE clade virus did not generally demonstrate the complete processing of Gag polyprotein, with variable amounts of the p41 intermediate being detected in addition to p24. The presence of these Gag processing intermediates is undesirable, given that they can reduce the efficiency of viral particle maturation . This copol was untranslated. However, in the unlikely event of the premature stop codon reverting to its wild type sequence, the multiply-mutated non-functional RT and RNase H from the original pVLP vector would be expressed, maintaining safety. Nonetheless, this untranslated region leaves the option open for the further deletion of unnecessary viral sequence from these domains to lower the total viral sequence contained in the VLP vector to be less than the maximum to be considered safe. However, it is critical to consider whether the removal of native HIV-1 mRNA splice acceptor and donor sites within the pol sequence or inadvertent introduction of cryptic splice sites would interfere with the virally regulated mRNA expression that is required for these VLP expression vectors. This was clearly demonstrated in the studies here with the introduction of a Tat truncation reducing the amount of Env expression, likely via one or more of the 4a/b/c splice acceptor sites being disrupted [The final mature VLP expression vector (AEB.NCΔRT) bears 70% of the parental 93TH253.3, NL4-3, and AD8 viral sequences. This is slightly above the generally accepted safety standard of retention of no more than two-thirds of the complete virus genome that, when including removal of viral promoters, ensures that there is little possibility of replicative function. The ΔRT mutation within the VLP vector did not remove the mutated RT and RNase H domain nucleic acid sequences that were derived from the original VLP vector, rather a premature stop codon was introduced at the end of the PR domain, such that most of isrupted .An important aspect of the VLP expression vectors that were developed here was their ability to incorporate membrane-bound Env, given the large number of safety mutations. VLP-associated Env was capable of presenting both gp120- and gp41-specific bNAb epitopes, as demonstrated by VLP ELISA. The VLP-associated Env bound several anti-gp120 bNAbs more efficiently than soluble, monomeric gp120, including b12, PG16, and PGT121, likely due to the presence of trimeric Env on the VLP surface. Enhanced VLP binding as compared to soluble gp120 was also observed for the poorly neutralising antibodies F105 and 17b, indicating the likely presence of improperly folded Env on the VLP membrane, as has been observed on virions . OverallIn summary, here we describe the design of a single-plasmid expression cassette that is capable of production of mature-form VLPs that are not infectious and safe for use as vaccine immunogens without requiring inactivation. The initial AEB VLP single-plasmid vectors yielded particles, in which Gag was incompletely processed by the viral PR and displayed an aberrant morphology. We found that safety mutations within the RT domain were responsible for the immature phenotype, and that restoring a functional RT domain facilitated the expression of proteolytically mature VLPs. To increase the safety of the VLPs and maintain the mature phenotype, the entire RT domain was deleted and this vector gave rise to VLPs that mimicked the fusibility of infectious HIV-1 virions. The mature VLPs efficiently incorporate membrane-bound Env and display bNAb epitopes within gp120 and gp41. These VLPs, which resemble mature HIV-1 virions, warrant further investigation as potential vaccine immunogens that are able to be delivered either as particles or in a nucleic acid vector to elicit antibody responses against HIV-1 Env.