There is little evidence addressing the use and differential impact of respiratory support in acute heart failure (AHF) patients with preserved (HFPEF) vs reduced (HFREF) ejection fraction. Therefore, our objective was to determine the usage and clinical outcomes of critical care respiratory support in AHF across the two populations.Respiratory support would be associated with adverse outcome in both HFPEF and HFREF.We identified HFPEF, HFREF, invasive mechanical ventilation (IMV), and noninvasive ventilation (NIV) using International Classification of Disease‐Ninth Edition codes in the National Inpatient Sample between January 1, 2008 and December 31, 2014. We determined rates of IMV and NIV use. We identified predictors of need for IMV and NIV and the association between ventilation strategies and in‐hospital mortality in HFPEF vs HFREF.1.3 million AHF‐HFPEF and 1.7 million AHF‐HFREF hospitalizations were included; 5.98% of AHF HFPEF hospitalizations included NIV and 0.57% included IMV. Among HFREF hospitalizations, fewer (4.1%) included NIV and more (0.93%) included IMV. In HFPEF hospitalization, NIV use was associated with 2.24‐fold increased risk for death compared to no respiratory support in an adjusted model (HR 2.24 95% CI 2.05‐2.44) and IMV use was associated with 2.85‐fold increased risk for death (HR 2.85 95% CI 2.30‐3.53). This increased risk of in‐hospital mortality was similar among HFREF patients.Use of respiratory support is increasing among patients with both HFPEF and HFREF and associated with substantially increased mortality in both heart failure subtypes. We hypothesized that the epidemiology of usage of IMV and NIV would be different between the two diseases and the predictors of need for IMV and NIV would differ between HFPEF and HFREF patients. Finally, we hypothesized that need for respiratory support would be associated with adverse outcome in both HFPEF and HFREF.22.1Our study population included AHF hospitalizations from the National Inpatient Sample (NIS) between January 1, 2008 and December 31, 2014. The NIS is a large all‐payer inpatient care database representative of US hospitals. The Institutional Review Board of Johns Hopkins University and the Healthcare Cost and Utilization Project approved the project.All diagnoses and procedures in the NIS are identified by International Classification of Disease‐Ninth Edition‐ Clinical Modification (ICD‐9‐CM) codes. The first diagnosis listed is the primary reason for hospital admission. We included all hospitalizations with a principal diagnosis of AHF with HFPEF or HFREF based on ICD‐9 codes 428.31 or 428.33 for HFPEF and 428.21 or 428.23 for HFREF which have been validated for identification of heart failure admissions.2.2Our exposure of interest was receipt of IMV or NIV compared to no support in patients with HFPEF and HFREF. IMV use was identified using ICD‐9‐CM code 96.7x for mechanical ventilation or 96.0 for endotracheal intubation which are specific for mechanical ventilation.We first considered the outcome of use of IMV or NIV vs neither modality in both the HFPEF and HFREF populations. Thus, our primary outcome for an initial set of analyses was IMV or NIV use stratified by HFPEF vs HFREF. Then, to determine the association of respiratory support with outcome in each of HFPEF and HFREF, we divided each of the HFPEF and HFREF populations into three groups: AHF using IMV, AHF using NIV, and AHF using no ventilation. To assess the association of IMV and NIV use with outcome in the HFPEF and HFREF populations, we used a primary outcome of in‐hospital mortality within 30 days.2.32 test for categorical variables and one‐way analysis of variance for continuous variables. Annual rates of IMV and NIV use within HFPEF and HFREF were calculated per 1000 AHF patients during the study period. Multivariable regression models adjusting for age and sex were constructed according to mode of ventilation in the HFPEF and HFREF populations to identify changes in mortality over time, adjusting for clustering of patients within hospitals. To determine the predictors of need for IMV and NIV in each heart failure population, we performed survey‐weighted logistic regression models with IMV and NIV as dependent variables and adjusted for factors of a priori interest based on our conceptual model. To determine the association between ventilation strategies and in‐hospital mortality in HFPEF and HFREF, we performed univariable and adjusted Cox proportional hazards models censoring at hospital discharge or 30 days of hospital stay, whichever came first. Survival curves were generated with the Kaplan‐Meier method and compared using the log‐rank test. Analysis was performed using Stata/MP version 13.0 . A two‐tailed P value less than .05 was considered statistically significant.Clinical characteristics and outcomes were compared between groups with the Pearson χ33.11 331 236 AHF‐HFPEF and 1 707 762 AHF‐HFREF hospitalizations were included. 5.98% of AHF‐HFPEF hospitalizations included NIV and 0.57% included IMV. Among HFREF hospitalizations, 4.1% included NIV and 0.93% included IMV. Rates of IMV and NIV use increased over the study period in both populations  and moderately increased odds of NIV use (OR 2.39 95% CI 1.85‐3.08). Among HFPEF patients, in‐hospital arrest was associated with very high odds of IMV  while in‐hospital arrest was not associated with NIV use.For HFREF patients, increasing age was associated with lower odds of treatment with IMV but higher odds of treatment with NIV, female sex was associated with higher odds of support with both modalities. For HFREF patients, Hispanic and Asian‐Pacific Islander race were associated with higher odds of respiratory support as was chronic lung disease. Obesity was associated with higher odds of NIV but not IMV. Similar to HFPEF, cardiogenic shock was associated with substantially higher odds of IMV use and moderately increased odds of NIV use while in‐hospital arrest was associated with very high odds of IMV and not associated with NIV use.Overall, chronic lung and kidney disease, diabetes, and obesity were associated with NIV use in both HFPEF and HFREF populations while cardiogenic shock and in hospital arrest as well as chronic lung disease were associated with IMV in both populations.3.4In HFPEF patients, NIV use was associated with 2.03‐fold increased risk for in hospital death compared to no respiratory support  and this increased risk persisted (HR 2.24 95% CI 2.05‐2.44) in a model adjusted for age, sex, race, comorbidity index, cardiogenic shock and in hospital arrest, chronic lung and kidney disease, diabetes and hospital size. In HFPEF patients, IMV use was associated with 4.06‐fold increased risk for in hospital death compared to no respiratory support  and this increased risk persisted (HR 2.85 95% CI 2.30‐3.53) in a model adjusted for age, sex, race, comorbidity index, cardiogenic shock and in hospital arrest, chronic lung and kidney disease, diabetes and hospital size. Kaplan‐Meier survival curves for NIV and IMV use compared to neither modality in both HFREF and HFPEF patients are shown in Figure This increased risk of in‐hospital mortality was similar among HFREF patients. In HFREF patients, NIV use was associated with 2.34‐fold increased risk for in hospital death compared to no respiratory support  and this increased risk persisted (HR 2.30 95% CI 2.11‐2.50) in adjusted models. IMV use was associated with 5.57‐fold increased risk for in hospital death compared to no respiratory support  and this increased risk persisted (HR 2.96 95% CI 2.57‐3.40) in adjusted models.4In our analysis of respiratory support in AHF stratified by HFPEF vs HFREF, we report several major findings. First, respiratory support with IMV and NIV is increasing in use in AHF hospitalizations with both HFPEF and HFREF, and HFPEF patients have higher rates of NIV use and lower rates of invasive ventilation use compared to HFREF. Patients with AHF who require support are at high risk for in‐hospital mortality irrespective of underlying EF and mortality is not declining over time. Finally, chronic lung disease and other patient comorbidities are associated with need for respiratory support in both populations as are cardiac comorbidities of cardiac arrest and cardiogenic shock. Our findings provide insights into important critical care complications in the HFPEF population which is important yet understudied to date.4.1Our data support that respiratory support in AHF admissions is common irrespective of LVEF, and HFPEF patients are more likely to require NIV while HFREF patients slightly more likely to require IMV. Given the fact that over one million patients are hospitalized with heart failure each year nationallyThe greater requirement for NIV in HFPEF may reflect the burden of obesity and pulmonary vascular disease,4.2Requirement for NIV and IMV in AHF connotes poor prognosis, with high risk of in‐hospital death. While respiratory support per se does not cause adverse outcome, a conceptual model would be that heart failure patients who require respiratory support are at high risk for poor outcome due to the severity of underlying condition, due to comorbidities, and due to any risk of harm inherent in critical care support. Herein, we report that the adverse prognosis of heart failure complicated by respiratory failure is similar in both HFPEF and HFREF. While the overall death rate is slightly lower for HFPEF compared to HFREF,4.3Pulmonary disease was mildly associated with IMV use in both populations, however by far the strongest predictor of need for IMV was cardiac complications of cardiac arrest and cardiogenic shock, irrespective of LVEF. We also identify shared risk factors for NIV use across HFPEF and HFREF, including chronic lung and kidney disease, diabetes, and obesity. These shared risk factors for respiratory failure may point to a common mechanism of respiratory failure among AHF patients, irrespective of LVEF. Novel treatments to improve outcome in patients with HFPEF and respiratory failure are needed, and targeting comorbidities such as lung disease may be a promising treatment strategy.4.4The main limitations of our study are related to the NIS as an administrative database. Thus, our study is unable to assess patient‐level factors such as specific LVEF or other echo parameters, labs assessed and drugs delivered during the hospital stay and disease‐specific cause of death. The NIS also does not provide an opportunity to evaluate postdischarge events or the specific mode of in‐hospital death. Classification of heart failure relies on accurate coding; however, the ICD‐9 diagnosis and procedure codes we use are consistent with prior literature and are well validated. Finally, as an observational risk factor analysis, our study can suggest associations but not demonstrate causality and as such, future studies in these patient populations are needed. Specifically, while our findings describe the association of respiratory support with outcome in HFPEF and HFREF, that is not to imply that patients who require respiratory support should not receive it. Rather, our findings should serve as an impetus to study the optimal mode of respiratory support and also provide general prognostic information for clinicians.5We demonstrate that the use of respiratory support with IMV and NIV is increasing in AHF hospitalizations with both HFPEF and HFREF. Requirement for respiratory support is associated with substantially higher risk for mortality irrespective of underlying HFPEF or HFREF, and mortality has not declined over time in either population. Studies assessing the mechanism of respiratory failure in HFPEF and HFREF and novel treatment strategies are needed to address these enlarging and high‐risk patient population.Dr Thomas S. Metkus reports personal fees from BestDoctors/TelaDoc Inc., personal fees from Oakstone/EBIX, personal fees from McGraw‐Hill publishing, outside the submitted work; Dr David A. Morrow reports grants and personal fees from Abbot Laboratories, grants from Amgen, grants and personal fees from AstraZeneca, grants from Daiichi Sankyo, grants from Eisai, grants and personal fees from GlaxoSmithKline, grants and personal fees from Merck, grants from Norvartis, grants and personal fees from Roche Diagnostics, grants from Medicines Company, grants from Quark Pharmaceuticals, personal fees from Verseon, personal fees from Peloton, personal fees from Bayer, personal fees from Aralez, personal fees from InCarda Therapeutics, outside the submitted work. Other authors have nothing to disclose.Table S1 ICD‐9‐CM Codes usedTable S2 (a) Association Between Select Factors and Type of Ventilation For patients with HFPEF, NIS 2002‐2013. (b) Association Between Select Factors and Type of Ventilation For patients with HFrEF, NIS 2002‐2013Click here for additional data file.


Nicotinamide riboside (NR) is widely used as an NAD+ precursor and known to increase NAD+ level in several tissues. The present study aimed to examine the effect of NR on tumor necrosis factor (TNF)-induced optic nerve degeneration and to investigate whether it alters SIRT1 expression and autophagic status in optic nerve. We also examined the localization of nicotinamide riboside kinase 1 (NRK1), which is a downstream enzyme for NR biosynthesis pathway in retina and optic nerve. Intravitreal injection of TNF or TNF plus NR was performed on rats. The p62 and LC3-II protein levels were examined to evaluate autophagic flux in optic nerve. Immunohistochemical analysis was performed to localize NRK1 expression. Morphometric analysis showed substantial axonal protection by NR against TNF-induced axon loss. TNF-induced increment of p62 protein level was significantly inhibited by NR administration. NR administration alone significantly increased the LC3-II levels and reduced p62 levels compared with the basal levels, and upregulated SIRT1 levels in optic nerve. Immunohistochemical analysis showed that NRK1 exists in retinal ganglion cells (RGCs) and nerve fibers in retina and optic nerve. NR administration apparently upregulated NRK1 levels in the TNF-treated eyes as well as the control eyes. Pre-injection of an SIRT1 inhibitor resulted in a significant increase of p62 levels in the NR plus TNF treatment group, implicating that SIRT1 regulates autophagy status. In conclusion, NRK1 exists in RGCs and optic nerve axons. NR exerted protection against axon loss induced by TNF with possible involvement of upregulated NRK1 and SIRT1-autophagy pathway.Nicotinamide adenine dinucleotide (NADThe online version of this article (10.1007/s12035-020-02063-5) contains supplementary material, which is available to authorized users. NAD+ is synthesized by salvage of vitamin precursors, nicotinic acid (NA), nicotinamide, and nicotinamide riboside (NR). Nicotinamide phosphoribosyltransferase (Nampt) converts nicotinamide into nicotinamide mononucleotide (NMN), whereas nicotinamide riboside kinase 1 (NRK1) converts NR into NMN. Then, NMN is converted to NAD+ by nicotinamide nucleotide adenylyltransferase1–3 (Nmnat1–3) [Nicotinamide adenine dinucleotide (NADmnat1–3) –6. Nmnatmnat1–3) –9, and Nmnat1–3) . Our premnat1–3) . On the mnat1–3) . This ismnat1–3) . Interesmnat1–3) . Since omnat1–3) , it is r+ precursor supplementation and has been reported to increase the blood NAD+ level in humans [+ synthesis from NR [+ and its precursors on autophagy machinery [+ levels, enhances Sir2 functions, and extends lifespan [+, and its precursors [NR is widely used as an NADn humans . NRK1 is from NR . Howeverachinery –17. Autoachinery –21. For achinery . In yeaslifespan . Sirtuinlifespan , NAD+, aecursors . Thus, tExperiments were carried out on 8-week-old male Wistar rats. All studies were conducted according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by Ethics Committee of the Institute of Experimental Animals of St. Marianna University School of Medicine. The animals were kept in the controlled rooms .Intravitreal injection of TNF  was performed as described previously . PhosphaOptic nerve specimens (4-mm lengths) were gathered and homogenized in protein extraction buffer 1 week after injection. Homogenized samples were then centrifuged at 15,000×g for 15 min at 4 °C. Protein concentrations were determined with the supernatants. Each sample (3 μg) was applied and subjected to the mini gel (Bio-Rad Laboratories) and transferred to enhanced chemiluminescent membrane . The membranes were blocked with 5% skim milk with tris buffered saline (TBS) containing Tween-20 and reacted with anti-p62 antibody , anti-LC3 antibody (MBL Life Science), anti-SIRT1 antibody (Santa Cruz Biotechnology), anti-NRK1 antibody  or anti-β-actin antibody (Sigma-Aldrich). After three times washing, the membranes were reacted with anti-rabbit or anti-mouse peroxidase-labeled secondary antibody . Immunoblotting was visualized with a chemiluminescence detection system .Three eyes 1 week after intravitreal injection of NR or three normal eyes were collected and fixed by immersion in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Sections were made through the optic disc and blocked with 1% bovine serum . The primary antibodies were against NRK1 , neurofilament-L , or Thy-1 . The secondary antibodies were FITC-labeled or rhodamine-labeled antibodies . The sections were mounted on slides in DAPI-containing medium with cover glass.2, and total area is 29,250 μm2 per eye) were used for quantification with the Aphelion image processing software . The number of axons was averaged in each eye and each group, and data were presented as the number per square millimeter. After quantification, representative color photos were obtained.Optic nerve specimens (4-mm lengths from 1 mm behind the globe) were collected and soaked in Karnovsky’s solution for 24 h at 4 °C 2 weeks after injection. Several dehydrations were performed, and samples were embedded in acrylic resin at 70 °C two overnight. Then, samples were sectioned and stained with 1% paraphenylene-diamine (Sigma-Aldrich) in absolute methanol . This cap < 0.05.Data are expressed as mean ± SEM. Differences among groups were analyzed by one-way ANOVA with post-hoc Tukey’s HSD test or Mann–Whitney method. A probability value was considered statistically significant when p = 0.1850 vs. TNF; Fig. p = 0.0002 vs. TNF, and p < 0.0001 vs. TNF, respectively; Fig. As shown previously , histoloThere was a significant increase in the LC3-II level in the treatment with 200 pmol NR plus TNF as compared with those in the treatment with TNF at 1 week Fig. . MoreoveIn agreement with our previous findings , p62 proAs we recently found , no signTo examine the effect of NR and its metabolic pathway further, we investigated the localization of NRK1 in the retina and optic nerve. In the normal retina, the NRK1 immunoreactive pattern was similar to that of Thy-1 immunoreactivity .High resolution image (TIF 4757 kb).


Virus-like particles (VLPs) have been shown to be strong activators of dendritic cells (DCs). DCs are the most potent antigen presenting cells (APCs) and their activation prompts the priming of immunity mediators based on B and T cells. The first step for the activation of DCs is the binding of VLPs to pattern recognition receptors (PRRs) on the surface of DCs, followed by VLP internalization. Like wild-type viruses, VLPs use specific PRRs from the DC; however, these recognition interactions between VLPs and PRRs from DCs have not been thoroughly reviewed. In this review, we focused on the interaction between proteins that form VLPs and PRRs from DCs. Several proteins that form VLP contain glycosylations that allow the direct interaction with PRRs sensing carbohydrates, prompting DC maturation and leading to the development of strong adaptive immune responses. We also discussed how the knowledge of the molecular interaction between VLPs and PRRs from DCs can lead to the smart design of VLPs, whether based on the fusion of foreign epitopes or their chemical conjugation, as well as other modifications that have been shown to induce a stronger adaptive immune response and protection against infectious pathogens of importance in human and veterinary medicine. Finally, we address the use of VLPs as tools against cancer and allergic diseases. Virus-like particles (VLPs) have attracted the attention of many researchers, most significantly virologists and immunologists. VLPs are supramolecular structures that closely resemble viruses , but are replication-deficient because they contain no viral genetic material (DNA or RNA) , 2. VLPs+ or CD4+ T cell responses, respectively. CD4+ T cells help B cells to produce antibodies (Th2 cells) and enhance CD8+ T cells (Th1 cells) cytotoxicity  which are expressed on the surface , 7 or entoxicity –15. Sincbodies Th cells an T cells , 16–24. The aim of the present review is to summarize the findings regarding the interactions between VLPs and pattern recognition receptors (PRRs) from DCs, in an attempt to highlight avenues directed to the more efficient VLP design.VLPs are supramolecular assemblies with the same or similar structure as native virions of about 10–200 nm in diameter , 24–26. VLPs can be produced by heterologous expression systems such as yeast or baculovirus , 36–38 aVLPs can be used as an antigen delivery system by themselves but they can also carry individual epitopes from other pathogens –48. FirsIt is also important to know that some VLPs can elicit a stronger immune response than others and this fact is a very important point when choosing the VLP. The hepatitis B virus (HBV) core antigen (HBcAg) dimerizes and forms spikes found in the surface of HBV, and it has been used as a strategy for displaying exogenous antigens to increase their immunogenicity . For insThe first VLP-based vaccines were designed because of some viruses cannot replicate efficiently in cell culture and VLPs showed to be better than traditional vaccines in terms of safety . CurrentThe commercial vaccines Engerix® (GlaxoSmithKline) and Recombivax HB® (Merck & Co) against HBV were approved in the 1980s and they are based on VLPs . The secThe HPV capsid is formed by two proteins, L1 and L2. L1 is the major capsid protein and its single expression or with L2 lead to the generation of 40-nm VLPs , 70. AltCervarix® (GlaxoSmithKline) and Gardasil® (Merck & Co) are commercial vaccines against HPV. Cervarix® protects against HPV types 16 and 18 whereas Gardasil® protects against HPV types 6, 11, 16, and 18 , 71–73. VLPs are also being used in veterinary medicine. The first commercially available veterinary vaccine based on VLPs was against porcine circovirus type 2 (PCV2) . PCV2 geThere are other prototype vaccines based on VLP tested in veterinary medicine. VLPs of porcine parvovirus (PPV) have been used as antigen delivery system and they are based on the expression of VP2 capsid protein which self-assembles into 25-nm VLPs –48. ImmuA pathogenic virus that infects mammalian species including both humans and animals is rotavirus which causes diarrhea . RotavirTo generate enveloped VLPs, the structural proteins of the virus must be produced in cells to simulate a natural infection and promote self-assembly of the proteins. As an example, to generate influenza A VLPs, hemagglutinin (HA), neuraminidase (NA), and matrix proteins (M) from influenza are expressed in the baculovirus expression system. Influenza viruses are classified according to HA and NA proteins located in their surface , 37. InfThe use of algorithms is another strategy that can assist the design of improved VLPs. For instance, COBRA  was used for the development of VLP-based vaccines against subtypes H1, H3, and H5 of influenza A virus. These vaccines induced antibodies that block HA antigen, and inhibit the infection and pathogenesis of wild type influenza virus in mice , 86.in vitro generation of DCs, monocytes are cultured with cytokines: granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) (DCs are a heterogeneous population of cells . Differe4 (IL-4) –91 or GM4 (IL-4)  but LCs 4 (IL-4) , 93. In 4 (IL-4) .+ and CD8α+ DCs. mDCs express chemokine receptor 7 (CCr7), zinc finger and BTB domain containing 46 (Zbtb46) and FMS-like tyrosine kinase 3 ligand (Fl3) throughout their development (The macrophage—dendritic cell progenitor (MDP) is a common myeloid progenitor that differentiates in: monocytoid lineage and common DC progenitors (CDPs), depending on its environment . CDPs can be differentiated in plasmacytoid DCs (pDCs) and pre-dendritic cells (pre-mDCs). Pre-mDCs are precursors of CD11belopment . mDCs seelopment  whereas elopment , 23, 95.+ Lymphoid DCs are the most abundant DCs in the lymphoid organs  that interact with proteins containing sugars such as mannose or galactose in a calcium dependent manner , 43. TheDC-SIGN is a CLR expressed on the surface of DC, interacts with mannose residues of glycoproteins through its CRD located in its C-terminal and also acts as an adhesion molecule. DC-SIGN participates in migration of the DC and in the activation of T cells . DC-SIGNSome viruses can bind to different types of DCs and/or use different PRRs as binding sites. HPV VLPs can bind to human DCs and LCs , 50 but Another PRR of immature LCs is the FcγR. FcγR is also expressed on the surface of monocytes, DCs, macrophages, natural killer cells, among others , 128 andImmature DCs have high endocytic capacity to uptake antigens  capturinDCs can uptake antigens by different endocytic pathways: through macropinocytosis where antigens and fluids are taken up forming pinocytic vesicles ; DCs canPhagocytosis and macropinocytosis are the two main mechanisms by which VLPs are taken up by DCs , 139, trUpon VLP internalization, activation of PRRs leads to pro-inflammatory responses . This reA very important point is to differentiate between DCs and macrophages. Unlike macrophages, the main function of DCs is not to clear the infection but to prepare the immune system through inflammatory stimuli to recruit inflammatory cells and migrate to lymph nodes to activate T cells . DCs areGiardia lamblia variant surface proteins increase the expression of CD40 and CD86 signaling through TLR-4 (Binding of PAMPs (such as VLPs) to PRRs expressed on the DCs, including TLRs , 131 andgh TLR-4 .For DC activation, it is also important the particulate nature of VLPs. Particles are easily taken up by DCs and this event increases the expression of costimulatory molecules in DCs . For insDuring maturation, DCs also overexpress CCR7 that drives DC migration through lymphatic vessels reaching the T cells in the lymph nodes for their activation , 143. Ho+ T cells directing them against intracellular pathogens and cancer cells  to secondary lymphoid organs to present epitopes to T cells . There aer cells , 25, 142er cells .+ T cells (The other presentation pathway is through MHCII. This presentation pathway depends on the processing of the invariant chain (li) in the endo-lysosomal compartment where peptides of 3 kD are generated  . CLIPs a T cells , 153.+ T cells is dependent on the presentation of peptides bound to MHCI molecules on DCs  and loaded on phagosomal MHCI molecules –155. Lat+ and CD4+ T cell responses. This is a favorable result, because immune responses based on CD8+ T cell are important to eliminate HIV infections  lipopeptide, DCs increase the expression of MHCII molecules and elicit the production of neutralizing antibodies and CTL responses against HCV. These immune responses were higher than that elicited with HCV VLPs co-administrated with aluminum hydroxide  only recognize short peptides bound to MHC molecules. CD4 T cells , 24, 175 T cells , whereas T cells , 46.Overall, Th cells are essential for generating immune responses based on antibodies and CTL , 177. Be+ T cell knockout (KO) mice with chimeric SHIV VLPs coupled to HA from influenza virus induced higher titers of IgA and IgG (including IgG2a) than CD4+ T cell KO mice immunized with SHIV VLPs alone. These antibodies had a neutralizing effect. In addition, splenocytes from CD4 KO mice immunized with chimeric SHIV VLPs containing HA resulted in more lysis of cells expressing proteins from HIV than splenocytes of CD4 KO mice immunized with SHIV VLPs alone  while pDCs produce both IL-12 and IFN-α in response to several virus infections. IFN-γ enhances the production of IL-12 during inflammatory responses. Interestingly, IL-4 and IL-13 (Th2 cytokines) also induce the production of IL-12 , as occur during infection of intracellular pathogens  induced a strong immune response based on CTL without adjuvant use. These VLPs were captured by CD8α− and CD8α+ DCs where macropinocytosis and lipid rafts participate. Processing of these VLPs requires vacuolar acidification, proteasome activity, TAP translocation, and synthesis of MHCI molecules . It was observed that DCs of mice injected with these VLPs carried out cross-presentation. For this process, macrophages cross-presented p33-VLP efficiently in the absence of TAP-1, while the cross-presentation was reduced in TAP-1 deficient DCs .+ T cells reduces the severity of symptoms and prompt elimination of infected cells , which is also the target of neutralizing antibodies , 195. ThAn adequate vaccine against dengue virus must generate an effective immune response against all 4 serotypes –199. TheZika virus is another important flavivirus, its infection is generally asymptomatic or produces mild disease, but during pregnancy, it is associated with defects at birth and in infants it produces visual and hearing defects –207. ZikJapanese encephalitis is also important, and as it occur with other flavivirus, the envelope glycoprotein is important for the attachment and entry of the virus into target cells . ExpressIn the case of West Nile virus (WNV), its infection is facilitated by endothelial cells until it reaches the nervous system . WNV infChikungunya and Venezuelan equine encephalitis (VEE) viruses are also transmitted by mosquito bites , 221. ChVEE virus targets DCs and LCs resident of skin; upon infection, these APCs migrate to lymph nodes and later infect the central nervous system leading to lethal encephalitis in horses. In humans, VEE virus produces flu-like symptoms, although encephalitis is also observed in older people and children , 164. VEVLPs against emerging diseases such as Crimean-Congo Hemorrhagic Fever (CCHF) and hantavirus have also been produced. CCHF causes hemorrhagic fever in humans, with high mortality. CCHF VLPs were developed expressing the glycoproteins Gc and Gn, the viral polymerase and the nucleoprotein encapsulating a minigenome. Immunization with these VLPs has been shown to induce 100% protection in challenge with transgenic mice . The immune response generated was humoral and cellular, but dominant Th1 and balanced Th2 responses correlated with protection . These Vin vitro by using DCs indicated that these VLPs induces the expression of CD80, CD86, MHCI, MHCII as well as cytokines IL-6 and IL-10, primordially  is caused by the coronavirus SARS-CoV. SARS-CoV binds to angiotensin-converting enzyme 2 (ACE-2) through their receptor-binding domain (RBD) found in the S1 region of the spike (S) protein . SARS-Co+ T cells and DCs stimulated with SARS-CoV VLPs induces IFN-γ and IL-4 synthesis by CD4+ T cells  have been designed. Mice immunized with these VLPs containing HA were protected from the challenge. Additionally, western blot analysis showed that these VLPs withstand to proteolytic digestion, adverse pH and temperatures . This side effect is a rare even in subjects with IgE anti-yeast antibodies , 249. InAlthough VLPs can be internalized by other mechanisms such as macropinocytosis  and phagocytosis , in this work we focus on analyzing the main proteins used to produce VLPs targeting DC through their PRRs, favoring the receptor-dependent endocytosis mechanism.Here, we analyze several VLPs utilized either as a preventive agent or as a therapeutic agent according to their abilities to prime innate immune responses. The first point to consider for developing smart VLP-based vaccines is based on the selection of a VLP candidate according to the type of immune response that the individual requires. Thus, if it is necessary to prevent any infection by a pathogen that can be blocked by antibodies, the most appropriate VLP should be selected for this case. If the pathogen is intracellular, VLPs targeting DCs that can perform cross-presentation with high efficiency should be selected. For example, RHDV VLPs have been shown to be very effective in carrying cancer epitopes. These VLPs increase the production of MHC molecules as well as costimulatory molecules . Therefo+ DC subsets: CD8α+ and CD8α−  was recombinantly produced as a fusion protein, whereas in the other one, the gp33 peptide was chemically conjugated to the RHDV VLP. RHDV VLPs with gp33 incorporated as a fusion protein generated stronger cytotoxicity than RHDV VLPs chemically conjugated with gp33. Interestingly, these VLPs were internalized by two DC11cnd CD8α− . Thus, iVLPs can also be modified to increase their uptake and improve the processing of incorporated epitopes. As an example, RHDV VLPs containing a melanoma epitope (KVPRNQDWL derived from glycoprotein 100) were developed. The melanoma epitope containing proteasome cleavable linkers was fused to N-terminus and C-terminus from VP60 protein. These VLPs were also conjugated with mono and dimannosides to favor their internalization. It was observed that these VLPs entered in APCs through more than one pathway, increasing the internalization of the antigen through phagocytosis and macropinocytosis. Furthermore, the use of linkers allowed proper processing of the epitope, as indicated by experiments in mice vaccinated with these VLPs that were challenged with tumor cells after vaccination, where specific lysis, decreased tumor size, as well as increased survival percentage were observed . Therefo+ T cells, production of Th1 cytokines and class switch from IgE to IgG. Similarly, VLP-based vaccines designed against cancer should contribute to restoring the immune response. VLPs co-administrated with a suitable adjuvant activate DCs, promote their maturation and proper presentation of cancer antigens to CD4+ and CD8+ T cells.The correct selection of an adjuvant is also important to promote DC maturation for a suitable presentation of the antigen, and therefore, for the generation of a strong adaptive immune response. Although many VLPs do not require adjuvant co-administration, its use can favor the type of response desired. In the case of allergic diseases, in which the response is favored toward a Th2 profile, an adjuvant used for the formulation of VLP-based vaccines must favor a Th1 response as well as the immunoglobulin class switch from IgE to IgG. VLP-based vaccines against allergic diseases are primarily based on bacteriophage Q-beta proteins that assemble in the presence of nucleic acids to form VLPs . This prNowadays, vaccine production against emerging tropical viral diseases is a concern. There are several vaccines produced in a traditional way, including inactivated or attenuated viruses, but many of them have not been commercialized due to safety issues , 251, whAll this knowledge together may be useful to design smart VLPs as a tool for personalized medicine, in which a VLP can be selected in a particular way and modified according to the individual needs of each patient.The smart design of VLPs could include the insertion of modules in VLP proteins. The modular VLPs design may allow that a single VLP backbone can be decorated with additional proteins as needed for each specific case to incorporate several modules such as: carriers, T and/or B cell epitopes, glycosylated epitopes, epitopes containing proteasome cleavable linkers for improving their processing, and one or several PRR ligands to enhance their uptake by DCs.in vitro transduction of cells (Although the main aim of this work was focus on VLP-based vaccines targeting DC PRRs to prompt a strong immunogenicity, modular VLPs could also be designed with DNA binding modules for efficiently carry nucleic acids for transduction of other specific target cells. VLPs containing genes have been designed for of cells , but we The right design of VLPs should not be based only in the overexpression of structural proteins of viruses. Instead, the smart design of VLPs must be based on the molecular mechanism of infection of the pathogen, the target PRRs to which VLPs will attach for their internalization, and the type of immune response desired. VLPs can also be designed for their use in the treatment of allergic diseases, because the immunization with VLPs can switch the immune response from Th2 to Th1. Since VLPs can restore the immune response, be cross-presented and induce a CTL response, they can also be used for future cancer therapies. The ability to engineer VLPs with exquisite detail makes them a very attractive candidate for the design of platforms aimed at the production of vaccines against infectious pathogens as well as against allergic and cancer diseases used in personalized and/or translational medicine.JZ-C conceived the main idea of this work, conducted the search of bibliography, designed and wrote the most topics of this review. JZ-C, JR-J, and LV wrote the final draft and designed the figures of this review article. LV corrected the manuscript and provided important contributions during the development of this work. All authors approved the final manuscript.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.


We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer–small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF–TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein–protein interactions. TNF can be inhibited by small molecules that stabilize the TNF trimer in an asymmetric conformation. Here, the authors develop a monoclonal antibody that selectively binds this inactive form of TNF, enabling both target engagement assessment and structural characterization of TNF binding to TNF receptor 1. TNF is a pleiotropic cytokine that exerts broad functional activity via binding and activation of two distinct receptors: TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2)2. TNFR1 is ubiquitously expressed and is activated by both soluble and membrane-associated TNF. Expression of TNFR2 is restricted to specific cell types, such as immune cells, neurons, and endothelial cells and is primarily activated by membrane-associated TNF3. It has been proposed that TNFR1 primarily promotes the TNF-induced inflammatory response, whereas TNFR2 controls local homeostatic effects4. This study therefore focused on the TNF–TNFR1 interaction.Tumour necrosis factor (TNF) is an important therapeutic target for treating a range of autoimmune and inflammatory disorders. The biology of TNF and its role in disease have been extensively studied and reviewed elsewhere5. The small molecule compounds bind in a pocket at the centre of the TNF trimer and stabilise a conformation where a single TNF monomer subunit is perturbed, thereby inducing a molecular asymmetry in the trimer which in turn affects TNFR1 receptor interaction stoichiometry and subsequent signalling. Data from analytical size exclusion chromatography suggests that the compound-bound asymmetric TNF trimer only associates with two TNFR1 molecules instead of three. This is consistent with the crystal structure of the TNF-small molecule complex which identified significant distortion at one of the three receptor binding interfaces. Rather than inducing a conformational change, molecular dynamic simulations support a mechanism whereby small molecules stabilise a naturally occurring signalling-deficient conformer of TNF.The development of a number of orally active small molecule inhibitors of human TNF that exhibit activity both in vitro and in vivo has recently been described6. Although determination of a compound-bound human TNF-human TNFR1 was not possible, the crystal structure of compound-bound mouse TNF in complex with human TNFR1 revealed a distorted TNF trimer with two copies of receptor bound. Interestingly, the bound receptors existed as dimers, an observation which, along with data from a solution-based network assembly assay, led to the formation of a model for TNF-TNFR1 network signalling and its inhibition by small molecules.In a subsequent study with the same small molecule inhibitor series, crystallography and solution-based techniques enabled characterisation of the mechanism of action6. Furthermore, we show that the antibody, in addition to providing insights into the structure and dynamics of TNF and the mode of action of the small molecules, has potential utility as a tool for measuring target occupancy. This may enable increased understanding of target dynamics and PK-PD effects in preclinical models and when moving into learn-phase clinical studies to confirm proof of mechanism in humans. Collectively, this work highlights the utility of monoclonal antibodies as reagents to support discovery and clinical development of small molecule drug inhibitors of protein–protein interactions.To facilitate further characterisation of these small molecule compounds in a variety of contexts, we here generate a monoclonal antibody, known as CA1974, that selectively binds with high affinity to the TNF trimer–small molecule complex. We solve a crystal structure of the conformation-selective antibody in complex with TNF trimer, small molecule inhibitor, and TNFR1 extracellular domain. The structure confirms other studies suggesting that the small molecule inhibitors stabilise a perturbed conformation of the TNF trimer, resulting in a reduction in receptor stoichiometry, which likely leads to the subsequent inhibition of signalling7 to efficiently screen the antibody repertoire from five rats which had been immunised with human TNF pre-complexed with the small molecule inhibitor UCB-9260. A monoclonal antibody, CA1974, was identified and shown to have excellent selectivity for the TNF–small molecule complex compared to the apo form of TNF. Consistent with previous observations that small molecules do not ablate engagement with all three receptor subunits6, binding specificity was initially demonstrated using a human TNFR1-capture ELISA in order to present the distorted A/C interface of the trimer. As shown in Fig. UCB-9260 or UCB-8733.We employed a high-throughput single B-cell platformTo determine if the small molecule inhibitor–TNF complex was able to bind to TNFR1 on the cell membrane, we performed flow-cytometry experiments using human embryonic kidney (HEK)-293 Jump In cells which overexpress TNFR1 after induction with doxycycline . For those complexes which exhibited 100-fold enhanced KD versus apo TNF, the measured off-rates (kd) were close to the accurate limit of detection of the Biacore device (1.00E-05 s−1), so the difference in affinity between the TNF–small molecule complex and apo TNF may have been even greater than the 2-logs recorded. Over 10-fold lower affinity was observed for antibody binding to mouse TNF–small molecule complex compared to human and cynomolgus complexes. However, an approximate 100-fold greater KD for mouse TNF–UCB-8733 complex compared to the mouse apo TNF was observed.Furthermore, to accurately measure the level of specificity of CA1974, we performed affinity measurements by Biacore using human, cynomolgus monkey, and mouse TNF with and without the small molecule inhibitors 6. We rationalised that this probably represented a region of the trimeric molecule which could be bound by the monoclonal antibody. We tested this theory by performing SE-HPLC to assess complex formation prior to crystallography (see below) and to determine stoichiometry of binding.We wanted to understand the nature of the interaction between the antibody and the perturbed TNF trimer. Structures of TNF in association with small-molecule indicate that the normally symmetric trimer becomes distorted, resulting in a breakdown in trimerous symmetry. A prominent feature of this perturbation is the generation of an expanded cleft at the altered interface between two of the monomer subunits (subunits A and C)UCB-9260 small molecule complex and then analysed by SE-HPLC. As can be seen in Fig. Fab fragment of CA1974 was incubated at various molar-ratios with TNF-UCB-8733 small molecule inhibitor, and human TNFR1 extracellular domain . Only two residues of CDR1 contact TNF, one binding monomer C and the other monomer A, on the inner faces of the cleft. Light chain CDR3 associates solely with monomer A, interacting with residues towards the top of the cleft (thick end of TNF) Fig. .We have demonstrated that CA1974 is capable of binding apo TNF, so it was of interest to model the symmetric apo TNF structure (PDB code 1TNF) bound with CA1974. When symmetric apo TNF was aligned with TNF in our complex structure  there were clear clashes between apo TNF and the Fab. Unsurprisingly, given the degree of distortion in the compound-bound TNF, there were a number of very significant structural clashes between several surface loops on monomer C of apo TNF and the CDR and framework regions of CA1974 Fig. . When al9. In particular, an overlay of the TNF and receptors from this structure with those from the compound-bound mouse TNF/human TNFR1 structure (7KP8)6 reveal a striking similarity: RMSD = 0.75 Å UCB-8733 in the absence of CA1974 and receptors (PDB code 7KPA)  Fig. . OverallFab Fig. . This suAs described, CA1974 also recognised cynomolgus monkey and, to a lesser degree, mouse TNF–small molecule complexes. To understand the nature of the interaction with TNF from these different species, we determined the epitope on the human TNF trimer and identified those residues that were different in the cynomolgus and mouse molecules Fig. . As expeUCB-8733 complex diluted in neat human plasma. As can be seen in Fig. One of the main objectives of this work was to generate a reagent that would allow for measurement of target occupancy in plasma samples taken from animals and eventually humans. An ELISA was established utilising CA1974 as a capture reagent to measure a range of concentrations of recombinant human TNF-5 and DEER analysis of spin-labelled trimer10.Despite the structural analyses suggesting that CA1974 is unlikely to interact with the symmetric form of apo TNF due to major side-chain clashes that would likely prevent association, Biacore measurements  binding to TNF trimer–small molecule complex. The data clearly suggested that the TNF trimer–small molecule complex accommodates a single Fab fragment. This observation was consistent with the hypothesis that a single epitope on the TNF trimer, which arises or is stabilised through small molecule binding, is recognised by the antibody reagent.UCB-8733 small molecule inhibitor, and human TNFR1 extracellular domain. Previous attempts to obtain a structure of the human TNF–TNFR1 complex had failed6. The elucidation of a crystal structure was only made possible through the inclusion of the small molecule inhibitor and the conformation-selective antibody CA1974.In order to understand the mechanism with which CA1974 antibody recognised the distorted TNF trimer and to gain further insights into the interaction of the perturbed trimer with receptor, we generated an X-ray crystal structure of a complex consisting of CA1974 Fab fragment, human TNF trimer, Consistent with previous work suggesting that small molecules reduce TNF:TNFR1 stoichiometry from 1:3 to 1:2, which leads to a disruption in productive receptor assembly and signalling, the work described here, using a fully human TNF-receptor system, supports that conclusion. As predicted, the structure revealed that CA1974 recognises a unique epitope at the perturbed A/C receptor-binding interface, formed as compound captures TNF in a distorted conformation where monomer A has tilted and twisted down and out.5 and DEER experiments with spin-labelled TNF which predict 6% of trimers at any one time adopt a naturally sampled asymmetric open state10. The presence of CA1974 in the SEC HPLC experiment probably draws the equilibrium of conformational sampling towards the open state, increasing the percentage of asymmetric trimer. The low fraction of open conformer in the apo TNF sample and the reduced affinity compared to the small molecule-bound form are also likely to account for the lack of signal with apo TNF seen in the ELISA screens  and 3′ TGATAAGCTT (HindIII is underlined), such that two stop codons follow the C-terminal residue. The final gene inserts were then synthesized by DNA 2.0 and delivered in a shuttle vector. Following synthesis, the gene inserts were digested with BamHI and HindIII and subcloned to vector pEMB54, which is an ampicillin-resistant, arabinose-inducible vector with pMB1 origin of replication and 6XHis-Smt3 under the PBAD promoter, followed by a multiple cloning site that includes BamHI followed by HindIII. After BamHI/HindIII cloning into pEMB54, gene inserts are fused in-frame with the 6XHis-Smt3 sequence. Following digestion of both pEMB54 and the respective gene inserts with BamHI/HindIII, DNA fragments were gel-purified, and ligated into the vector and then the ligation was transformed to chemically competent TOP10 E. coli cells . One transformant was mini-prepped per construct and submitted for DNA sequencing of the Open Reading Frame.Human TNF (UniProt P01375 residues 77–233), cynomolgus monkey TNF , and mouse TNF  genes were codon-engineered in silico using GeneComposer™ for E. coli cells . A starter culture containing 100 µg/ml  ampicillin (Teknova) was inoculated with a single colony and grown for 16 h at 37 °C. This was then transferred to 8 litres of terrific broth (Teknova) containing 100 µg/ml  ampicillin and grown to OD600 = 0.6. Protein expression was induced by adding arabinose to a final concentration of 0.1% (VWR) and grown for 16 h at 25 °C. The cells were harvested by centrifugation (Beckman) at 6200 × g for 15 min and the pellets were collected and stored at −80 °C.Briefly, the target specific vector was transformed into TOP10 g for 30 min at 4 °C (Beckman) and filtered with a 0.2 µm bottle-top filter . The supernatant was applied to two 5 ml Ni2+ charged HiTrap Chelating HP  columns and the protein eluted with a 500 mM imidazole gradient over 20 column volumes. The fractions of interest were pooled and the His-Smt tag was removed via cleavage with Ubiquitin-like-specific protease 1 (Ulp-1) while dialysing against 2 L of 25 mM Tris pH 8.0 and 200 mM NaCl overnight at 4 °C using 3.5 kDa MWCO snakeskin dialysis tubing. The affinity tag was removed by applying the digested pool over two 5 ml Ni2+ charged HiTrap Chelating HP columns. The flow-through contained the cleaved protein of interest. The protein was concentrated for size exclusion chromatography via centrifugal concentration  to 13.5 mg/ml for injection over a HiPrep 16/60 Sephacryl S-100 HR  in 10 mM HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), pH 7.5 and 150 mM NaCl. Fractions of interest were pooled and concentrated via centrifugal concentration  to 20 mg/ml, aliquoted, and stored at −80 °C.Cells were resuspended 1 g:4 ml in 25 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) pH 8.0 (Teknova), 200 mM NaCl (Teknova), 0.02% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (JT Baker), 50 mM L-arginine (Sigma), 500U of benzonase (Novagen), 100 mg lysozyme (Sigma) and one complete EDTA-free protease inhibitor tablet (Roche). The cells were lysed via sonication (Misonix) and clarified via centrifugation at 142,000 × UCB-9260. Knowledge that the small molecule–TNF interaction had a sufficiently slow off-rate (koff 0.019 s−1) and evidence that the complex could exist in vivo following mouse efficacy models5 supported the immunisation approach taken here. Complete Freund’s adjuvant was used for the first shot, while subsequent immunisations used incomplete Freund’s adjuvant both at a 1:1 v/v ratio with TNF–small molecule complex. Approval for use of animals for immunisation was provided through the UCB Pharma, UK Animal Welfare and Ethical Review Body (AWERB), and the license was granted by the UK Home Office. At the end of the study the mice were anaesthetised with isoflurane, terminal bleeds taken, and then sacrificed using a Schedule 1 method in accordance with the Animals Scientific Procedures Act (ASPA).Five female Sprague Dawley rats (150–180 g) (approx. 8-weeks of age) were immunised subcutaneously with three shots of 50 μg human TNF pre-complexed for 2 h at room temperature (RT) with 50-fold molar excess of 7. Briefly, splenocyte suspensions from the immunised rats containing immune B cells were cultured in 96-well plates at 1000–5000 cells per well in order to induce clonal expansion and antibody secretion. IgG in culture supernatants were then screened in a homogeneous fluorescence-based bead assay for binding to apo human TNF and human TNF in complex with UCB-9260 (at a 50-fold molar excess). In the homogeneous assay, human TNF  was captured onto beads using human TNFR1-Fc  coated onto Superavidin beads (Bangs Laboratories) coated with 2 µg/ml of biotinylated goat F(ab)2 anti-human Fc reagent . Selective binding to human TNF in complex with small molecule was confirmed by ELISA using TNF in complex with UCB-9260 and UCB-8733 small molecules captured onto plates coated with TNFR1-Fc (see below). Antibodies that demonstrated preferential binding to the TNF-compound complex were taken forward for cloning. The Fluorescent Foci method28 (US Patent 7993864/ Europe EP1570267B1) utilising beads coated with pre-complexed TNF-UCB-9260 small molecule inhibitor (50-fold excess) was used to identify and isolate antigen-specific B cells from positive wells. Specific antibody variable-region genes were recovered from single cells by reverse transcription (RT)-PCR using heavy and light chain variable- region-specific primers. The following PCR primers were used: CTGGTTTCCAGGCACCAGGTGTGACATCCAGATGACCCAGTCTCC (VK primary forward); CTTTCATATTCAACCTTGGTCAAC (VK primary reverse); TTGTTGCTCTGGTTTCCAGGCACCAGGTG (VK secondary forward); TGGATACAGTTGGTGCAGCATCCGTACGTTTCAGTTCCAGCTTGG (VK secondary reverse); CAGTAACTACCGGTGTCCATTCTGAGGTGAAGCTGTTGGAATCTGG (VH primary forward); GATAGACHGATGGGGSTGTTG (VH primary reverse); TTCTCTTCTTCCTGTCAGTAACTACCGGTGTCCATTC (VH secondary forward); GATGGGGGTGTTGTTTTAGCACTCGAGACAGTGACCAGAGTGCC (VH secondary reverse). Secondary PCR primers contained restriction sites allowing cloning of the variable region into a mouse IgG1 (VH), mouse Fab (VH), or mouse kappa (VK) mammalian expression vector under the control of a human cytomegalovirus (HCMV) promoter. Heavy and light chain constructs were co-transfected and expressed using the Freestyle 293 expression system  following manufacturer’s guidelines and recombinant chimeric IgG1 antibody or Fab (composed of rat variable domains and mouse constant domains) expressed in 30 ml cultures. After expression for 7 days, supernatants were harvested and antibody rescreened for selectivity using the specificity assays described above and also tested by Biacore to determine affinity for apo and small molecule-bound TNF (see below). CA1974 was selected as the lead molecule based on its selective binding and affinity profile. To support further analysis, CA1974 IgG and Fab were purified on an AKTA system  using affinity chromatography  followed by SE-HPLC to produce a product >98% pure monomer species.Antibody discovery was essentially performed using a B cell screening method as previously describedIn order to identify conformation-specific antibodies in B-cell culture supernatants, HEK-293  culture supernatants or as purified preparations, a binding ELISA was developed. Nunc maxisorp 384-well ELISA plates  were coated with 10 μl per well of 1 μg/ml human TNFR1-Fc  overnight at 4 °C. Plates were washed and then blocked for 1 h with 1% BSA in PBS. After washing plates again, 10 μl of 100 ng/ml apo TNF or TNF pre-incubated with compound at a 50-fold molar excess in 1% DMSO  in PBS for 2 h at room temperature, were added to each well of the plate and incubated for a further 1 h at room temperature. After washing again, mouse IgG or mouse Fab at a range of concentrations diluted in 1% BSA in PBS were added to the plate and incubated for 1 h at room temperature. Antibody binding was detected with either an HRP-conjugated goat anti-mouse IgG Fcγ-specific antibody  or a goat anti-mouse IgG F(ab)2-specific antibody  at a 1:5000 dilution in 1% BSA in PBS. Binding was revealed with 3,3′,5,5′-Tetramethylbenzidine (TMB) Substrate and absorbance at 630 nm measured on a synergy 2 microplate reader (BioTek).n = 2 experiments, with duplicate data points within each) were analysed on the logarithmic scale to satisfy the analysis assumptions. The linear model included fixed effects of group, concentration, and associated interactions and allowed for experimental differences. Pre-planned comparisons between the groups at each concentration were performed and results of the unadjusted two-sided comparisons are presented as ratios of geometric means and 95% confidence intervalsThe OD responses (UCB-9260 or UCB-8733) in HBS-EP + (1% DMSO) for 5 h.Surface plasmon resonance (SPR) was performed at 25 °C using a Biacore T200 . Goat anti-mouse IgG Fcγ-specific antibody  was immobilised on a CM5 Sensor Chip  via amine coupling chemistry to a capture level of approx. 6000 response units. HBS-EP buffer  surfactant P20 ) +1% DMSO was used as the running buffer. A 10 μl injection of CA1974 IgG at 1 μg/ml was used for capture by the immobilised anti-mouse Fcγ reagent to create the TNF-binding surface. Human, cynomolgus monkey or mouse TNF at 50 nM was pre-incubated with 2 µM compound  following standard procedures. Kinetic parameters were determined from the fitting algorithm.UCB-68765 or UCB-9260 to either directly immobilised TNF or CA1974-captured TNF, surface plasmon resonance was performed at 25 °C using a BIAcore T200 . Human TNF was tethered onto flow-channel (Fc)2 (~2000 RU), CA1974 IgG  tethered onto Fc3,4 and Fc1 blank immobilisation on a CM5 Sensor Chip  via amine coupling chemistry. The surface was allowed to stabilise in HBS-P buffer  surfactant P20-GE Healthcare) + 5% DMSO was used as the running buffer. Human TNF was flowed over Fc4 to a capture level of ~2,000 RU. Compound UCB-6876 was flowed over all four work cells in series  at 30 µl/min. Compound UCB-9260 was flowed over all four flow cells in series  at 100 µl/min. Double-referenced, background-subtracted binding curves were produced using the T200 Evaluation software (version 1.0) following standard procedures.For single-cycle kinetics profiling of UCB-9260 or 0.4% DMSO  for 1 h at 37 °C. The pre-incubation mix was added to a 3-fold v/v excess of the cells, giving a TNF concentration of either 25 or 250 ng/ml. The sample was incubated for 1 h on ice. Cells were washed, fixed (1.5% PFA), and stained with 10 µg/ml CA1974 antibody for 1 h on ice. After washing again, binding was revealed using a secondary anti-mouse-Alexa fluor-488 antibody . Samples were run on a BD Biosciences Canto II Flow Cytometer with three lasers and a standard 4:2:2 configuration. The Alexa fluor-488 was excited by a 488 nm laser and fluorescence collected through the 530/30 BP filter. Gating of single viable cells (excluding doublets and other cell debris) was made using a forward scatter versus side scatter plot -293 Jump In cells , which overexpress TNFR1 after induction with doxycycline at 1 µg/ml for 2.5 h   for 18 h at 4 °C. To this complex was added CA1974 mouse Fab (70 µM) in PBS pH 7.4 at a range of different molar excesses and maintained at room temperature for 2 h. As a control we also tested CA1974 binding to apo TNF which had been pre-incubated with DMSO only . Samples were analysed by size exclusion-HPLC by injection of 25 µL onto a Superdex S200 10/300  column connected in series to an Agilent 1100 HPLC and eluted isocratically with PBS pH 7.4 at 1.0 ml/min for 30 min. Control samples containing Fab only and TNF-UCB-9260 complex only were also prepared for calibration of the size exclusion-HPLC analysis. The molar excess at which unbound Fab appeared was equated to the saturation of binding.Human TNF trimer (20 µM) in PBS pH 7.4 was pre-incubated with a 5-fold molar excess of Trichoplusia ni insect cells  using ESF-921 insect cell media . An extra glycine was present at the N-terminus of the protein due to a cloning artifact. This is not part of the native sequence of the TNFR1. The fusion protein plasmid was cloned into the pEMB50 expression vector, which encodes a cleavable N-terminal secretion signal and His-tagged fusion protein. Virus was generated using the baculovirus expression system. Infected insect cells secreted the fusion protein into the media. The fusion protein was purified by Ni-NTA chelate chromatography and eluted from the Ni column using an imidazole gradient. The eluted protein was cleaved with protease to release the N-terminal His-fusion tag. The cleaved TNFR1 was subsequently purified by a subtractive Ni chelate chromatography step and further purified by size exclusion chromatography. The final TNFR1 product was typically concentrated to 10.0 mg/ml and flash-frozen in liquid nitrogen.The soluble form of human TNF  was generated as described above. The product contained an N-terminal serine residue as a cloning artefact but this was not part of the native sequence of the TNF molecule. The extracellular domain of human TNFR1  with N54D and C182S mutations was expressed as a secreted protein in baculovirus-infected UCB-8733  and incubated at 4 °C overnight. The following day, the complex was formed by adding 4080 µl of SEC buffer to 700 µl TNFR1, 5,000 µl of TNF/UCB-8733 mix, and 220 µl CA1974 Fab fragment at 500 mM. Total volume of the reaction was 10 ml with a final molar ratio of 3 TNF monomers : 2.5 TNFR1 receptors: 1.2 Fab. The ternary complex  was incubated for 1 h at 4 °C. Analytical SEC was used to assess complex formation  and 100 µL of UCB-8733 was achieved using methods previously described5. In brief, TNF at 4-7 mg/ml was incubated with 0.5 mM UCB-8733 overnight at 4 °C. Crystals were grown at 16 °C by sitting drop vapour diffusion with a mixture of 0.5 µl protein:compound and 0.5 µl of 24.4% w/v PEG 3350, 0.1 M HEPES, pH 7.0. Crystals were cryo-protected in 10% ethylene glycol and frozen in liquid nitrogen for data collection. The data set was collected at APS, LS-CAT, beamline 21-ID-G . The structure of the human TNF (CID2043), human TNFR1 (CID5602), and CA1974 Fab complex with small-molecule UCB-8733 was solved by molecular replacement using Phenix.Phaser29 with input models based upon previously determined unpublished structures . Diffraction data for TNF and small-molecule UCB-8733 were reduced and scaled as above and the structure was solved by rigid body refinement in Refmac32 using a previously determined structure as the input model. Model building was performed using iterative rounds of Coot and Refmac until R and Rfree converged at 0.184/0.270, respectively . Model quality was validated using Coot and MolProbity prior to deposition in the Protein Data Bank (PDB codes 7KPB/7KPA)35. Final data processing and refinement statistics are listed in Supplementary Table UCB-8733) in complex with human TNFR1 and CA1974 Fab) and 6 (human TNF bound with UCB-8733).Crystallization of TNF with UCB-8733 in 0.1% (w/v) BSA/PBS, resulting in an approximately 50-fold molar excess of small molecule inhibitor over TNF. Following overnight incubation the complex was diluted to a range of concentrations in human plasma that had been depleted of endogenous TNF by immunoprecipitation using an in-house anti-TNF monoclonal antibody. Small molecule-bound TNF was measured using a sandwich ELISA. A Nunc 96 well maxisorp ELISA plate  was coated with 0.1 µg/ml CA1974 in PBS overnight at 4 °C. After washing three times in wash buffer (0.05% [v/v] Tween-20/PBS) the plate was blocked in 2% (w/v) BSA/PBS for at least 1 h at RT. Each sample (100 µl) was loaded to the blocked ELISA plate and incubated for 30 min at room temperature (RT) with shaking at 200 rpm . The plate was washed three times in wash buffer before adding 100 µl per well of biotinylated anti-TNF detection antibody , at a concentration of 0.1 µg/ml in 1% (w/v) BSA/wash buffer, for 1 h at RT with shaking at 200 rpm. Following three washes, 100 µl per well of 100 ng/ml Streptavidin-horseradish peroxidase  in 1% BSA/wash buffer was added and incubated for 45 min at RT with shaking at 200 rpm. The plate was washed three times in wash buffer before including a biotin-tyramide (B-T) amplification step to provide additional amplification, using the ELAST system from Perkin Elmer . B-T solution was diluted 1 in 100 in ELAST buffer, 100 µl added per well and the plate incubated for exactly 15 min at RT with shaking at 200 rpm before washing four times in wash buffer. An incubation with ELAST SA-HRP was performed, followed by four washes and the addition of 100 µl TMB per well. When appropriate colour change had been reached, the reaction was stopped by the addition of 100 µl per well of 2.5 M sulphuric acid. Absorbance at 450 nm was measured using a Thermo Multiskan EX plate reader with Ascent Software version 2.6.We initially tested the ability of CA1974 to detect a range of different recombinant human TNF concentrations pre-incubated with saturating levels of small molecule. Pre-incubation of TNF with excess NCE was achieved by mixing 10 µg/ml TNF with either 1% (v/v) DMSO or 10 µM To determine the concentration at which TNF plus excess small molecule inhibitor could be detected above apo TNF background, three standard deviations of the TNF plus DMSO OD results were added to the mean of these values. All TNF plus small molecule spike results greater than this value were considered to be significantly different to apo TNF, with a probability of at least 0.999.Further information on research design is available in the Supplementary InformationReporting SummaryPeer Review File


Epidermal keratinocytes are considered as the most important neighboring cells that modify melanogenesis. Our previous study used microarray to show that guanine deaminase (GDA) gene expression is highly increased in melasma lesions. Hence, we investigated the role of GDA in skin pigmentation. We examined GDA expression in post-inflammatory hyperpigmentation (PIH) lesions, diagnosed as Riehl’s melanosis. We further investigated the possible role of keratinocyte-derived GDA in melanogenesis by quantitative PCR, immunofluorescence staining, small interfering RNA-based GDA knockdown, and adenovirus-mediated GDA overexpression. We found higher GDA positivity in the hyperpigmentary lesional epidermis than in the perilesional epidermis. Both UVB irradiation and stem cell factor (SCF) plus endothelin-1 (ET-1) were used, which are well-known melanogenic stimuli upregulating GDA expression in both keratinocyte culture alone and keratinocyte and melanocyte coculture. GDA knockdown downregulated melanin content, while GDA overexpression promoted melanogenesis in the coculture. When melanocytes were treated with UVB-exposed keratinocyte-conditioned media, the melanin content was increased. Also, GDA knockdown lowered SCF and ET-1 expression levels in keratinocytes. GDA in epidermal keratinocytes may promote melanogenesis by upregulating SCF and ET-1, suggesting its role in skin hyperpigmentary disorders. Skin hyperpigmentation is caused by the interplay between melanocytes and neighboring cells of keratinocytes (KCs), fibroblasts, endothelial cells, and inflammatory cells ,3,4,5,6.The search for other unknown melanogenic factors produced by epidermal KCs is ongoing. Melasma is a UV irradiation and/or inflammation-aggravated hyperpigmentary disorder . SimilarThis study was approved by the Institutional Ethics Review Board of the Asan Medical Center (2013-0673), and all patients provided informed written consent before participating in the study. When next generation sequencing (NGS) was performed with punch biopsy specimens of lesional and perilesional facial skin tissue obtained from three patients with RM, GDA gene expression level increased 1,283.4, 3.7, and 26.4-fold in the facial lesions of 59-, 72-, and 59-year-old female patients with RM, respectively, than that in the non-lesional control areas .We also performed quantitative PCR (qRT-PCR) in samples from four RM lesions and perilesional controls. The clinical information on the patients who participated in NGS and qRT-PCR analyses are shown in Next, we attempted to compare GDA expression in primary human KCs, fibroblasts, and melanocytes. To investigate whether GDA is constitutively expressed in cultured skin cells, we performed qRT-PCR. GDA mRNA expression in normal human KCs (NHKs) was nearly 100-fold higher than that in NHMs and human dermal fibroblasts HDFs, A. Thus, When GDA was visualized by immunofluorescence staining in KC and melanocyte coculture, it was predominantly localized in the cytosol of KCs. Transfecting the coculture with a small interfering RNA for GDA (siGDA) suppressed the GDA signal in KCs B. In addTo determine whether GDA expression can be associated with inflammatory stimuli, human KCs were treated with various inflammatory cytokines, and the GDA mRNA expression level was evaluated by qRT-PCR. The expression level of RELA, a member of the nuclear factor-kappa B (NF-κB) family known to be involved in the inflammatory response ,34,35, wKCs are the well-known neighboring cells to melanocytes, which secrete melanogenic mediators to melanocytes in the epidermis ,2,3. To UVB irradiation is known to increase melanogenesis by upregulating SCF and ET-1, melanogenic growth factors in KCs . When weTo examine the association between GDA and melanogenesis, we silenced GDA using siGDA in the coculture, which significantly reduced the melanin content and tyrosinase mRNA expression on day 5 A,B.Since we found that GDA mRNA level is increased under the UVB melanogenesis-stimulatory conditions in KCs, we sought to elucidate whether suppression of GDA expression also decreases the melanin content in the UVB-stimulated coculture. Melanin content was even lower in the UVB-stimulated coculture of KCs and melanocytes after GDA suppression in KCs by siGDA than in the coculture without UVB stimulation, suggesting the regulating role of GDA in UVB-mediated melanogenesis C.Next, we attempted to induce GDA overexpression using an adenovirus vector. While NHMs and NHKs could not be infected with GDA–adenovirus, HaCaT cells were successfully infected. Increased GDA expression in HaCaT cells promoted melanogenesis in a 1:1 coculture of NHMs and GDA-overexpressed HaCaT cells D.To investigate how KC GDA regulates melanocyte function, we treated melanocytes with UVB-treated KC-conditioned media, which increased the melanin content A. ET-1 aSkin pigmentation is regulated by many factors and is complicated by the interconnections between melanocytes, KCs, and fibroblasts. Interactions between KCs and melanocytes under UVB exposure are reported ,4,5,6,11In our previous study using DNA microarray from melasma lesions, GDA was shown to be one of the most upregulated genes, which was validated by qRT-PCR analysis . KCs werBased on the above results, we hypothesize that epidermal KC GDA expression promotes melanogenesis. Our present study showed that GDA in epidermal KCs may promote pigmentation and play a role in UV-induced melanogenesis by interacting with KC-derived melanogenic growth factors ET-1 and SCF, suggesting its role in UV-aggravated skin hyperpigmented disorders. These results suggest that inhibiting keratinocyte GDA expression can be a novel approach for treating skin hyperpigmentary disorders.We included patients who were clinically diagnosed with RM in the Dermatology Department of the Asan Medical Center . The patients were excluded if they had other possible causes of hyperpigmentation such as Addison’s disease, hyperthyroidism, and hemochromatosis. We also excluded patients with hyperpigmentation in sites other than the face and neck. Clinical features such as age, sex, location of the lesion, Fitzpatrick skin type, morphology of the lesion , and color of the lesion  were identified.NHMs, NHKs, and HDFs were obtained from Invitrogen . Anti-GDA and anti-Melan-A antibodies were obtained from GeneTex  and Cell Marque , respectively. SCF, ET-1, IL-1α, and IFN-γ were purchased from R&D systems , and TNF-α was purchased from BioSource . siGDA, siNC and all primers  were purchased from Bioneer .We performed punch biopsies of lesional skin and normal adjacent skin on 3 RM patients . Total RNA was isolated from tissue using the Trizol based method. One microgram of total RNA was processed for preparing the mRNA sequencing library using the TruSeq stranded mRNA sample preparation kit  according to the manufacturer’s instructions. The first step involves purifying the poly-A containing mRNA molecules using poly-T oligo attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity is achieved by replacing dTTP with dUTP in the second strand marking mix (SMM), followed by second strand cDNA synthesis using DNA polymerase I and RNase H. These cDNA fragments then have the addition of a single ’A’ base and subsequent ligation of the adapter. The products are then purified and enriched with PCR to create the final cDNA library. Finally, quality and band size of libraries were assessed using an Agilent 2100 bioanalyzer . Libraries were quantified by qPCR using the CFX96 Real Time System . After normalization, sequencing of the prepared library was conducted on the Nextseq system (Illumina) with 75 bp paired-end reads. The result was aligned using the reference human genome (hg19). Three independent analyses were performed per biopsy specimen.® 480II machine coupled with SYBR Green . For qRT-PCR, the initial denaturation was performed at 95 °C for 5 min, followed by amplification at 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s for 45 cycles. Relative gene expression levels were calculated after normalization to the RPLP0 gene using the ΔΔCt method. Three independent analyses were performed per biopsy specimen. Three independent cellular experiments were performed in triplicate.Total RNA was isolated from skin biopsies  or cells using the RNeasy Mini kit . Then, 1 μg RNA was reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit (Invitrogen). qRT-PCR was performed using the LightCyclerPrimers for RPLP0 were used for loading control amplifications. Specific primer sets used for each gene are shown in For immunofluorescence staining, the samples obtained from RM lesional skin and adjacent perilesional skin were fixed in 10% neutral buffered formalin and embedded in paraffin. The specimens were cut into 4 μm-thick sections, and serial sections were deparaffinized and rehydrated. For antigen retrieval, the sections were heated in antigen unmasking solution  using a pressure cooker  at 120.5 °C for 30 s and 90 °C for 10 s. Then, they were immunofluorescently stained with the primary antibodies for GDA (1:200) and Melan-A (1:50) at 4 °C for 8 h. We used a FITC-conjugated anti-mouse (1:500) secondary antibody to detect Melan-A and anti-rabbit Alexa Fluor 546 (1:500) to detect GDA at 4 °C for 30 min. Images were acquired using a Zeiss LSM 780 laser scanning confocal microscope .g for 10 min at 4 °C and stored at −70 °C. ET-1 and SCF levels in the serum were measured using the Human Magnetic Luminex Screening Assay kit  according to the manufacturer’s instructions. ACTH and α-MSH serum levels were measured using the MILLIPLEX MAP Human Pituitary Magnetic Bead Panel  and the MILLIPLEX MAP Human Neuropeptide Magnetic Bead Panel , respectively, according to the manufacturer’s instructions.Blood samples were obtained from 15 patients with RM : 23.6 ± 2.7) and 6 healthy volunteers . The serum was separated by centrifugation at 4000× 2. For coculture of NHMs and NHKs, 6 × 104 NHM cells were seeded in each well of a 6-well plate. The next day, 3 × 105 NHK cells were added to each well for the coculture. NHMs and NHKs were cocultured at a 5:1 seeding ratio in KC growth medium.NHMs were maintained in Medium 254 (Invitrogen) containing human melanocyte growth supplement (Invitrogen). NHKs were cultured in KC growth medium  supplemented with human KC growth supplement (Invitrogen). HDFs and HaCaTs were cultured in Dulbecco′s modified Eagle′s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). All cells were maintained at 37 °C in a humidified atmosphere with 5% COCells were dissolved in 550 μL 1 N NaOH at 100 °C for 30 min and centrifuged at 13,000 rpm for 5 min. The optical density of the supernatant was measured at 405 nm using a microplate reader .® RNAi MAX reagent (Invitrogen) according to the manufacturer’s instructions. The siGDA sequence is AGUUGUCAGGAGAACACUA. The detailed knockdown protocol is shown in siGDA and siNC were transfected into cells using the LipofectamineFor ectopic expression of GDA, human GDA cDNA was cloned into lentiviral vectors ) and lentiviruses containing either empty vector (Empty) or human GDA (GDA) were produced and introduced into HaCaT cells. After puromycin selection and confirmation of GDA overexpression by qRT-PCR, HaCaT cells overexpressing GDA or infected with empty virus were cocultured with NHMs for 5 d and melanin content was measured as described above.The cells were exposed twice to a narrow band-UVB lamp . The cells were washed and resuspended in phosphate-buffered saline (PBS) prior to UVB radiation exposure. Non-exposed control samples were maintained in the dark under the same conditions. Following UVB radiation exposure, the cells were grown in fresh medium.5) were seeded into a 60 mm dish, cultured for 24 h, washed with PBS, and treated UVB or siGDA. After treatment, the cells were incubated in KC growth medium for 48 h; the conditioned media was harvested, centrifuged, and stored at −20 °C.NHK cells  according to the manufacturer’s instructions.p-Values < 0.05 were considered significant.The experimental data were expressed as means and standard deviations for three independent experiments performed in triplicate. Differences between results were assessed using the Kruskal–Wallis test in


Human papillomaviruses (HPVs) are the most common sexually transmitted infections worldwide. Ninety percent of infected individuals clear the infection within two years; however, in the remaining 10% of infected individuals, the infection(s) persists and ultimately leads to cancers  and genital warts. Fortunately, three prophylactic vaccines have been approved to protect against HPV infections. The most recent HPV vaccine, Gardasil-9 , protects against seven HPV types associated with ~90% of cervical cancer and against two HPV types associated with ~90% genital warts with little cross-protection against non-vaccine HPV types. The current vaccines are based on virus-like particles (VLPs) derived from the major capsid protein, L1. The L1 protein is not conserved among HPV types. The minor capsid protein, L2, on the other hand, is highly conserved among HPV types and has been an alternative target antigen, for over two decades, to develop a broadly protective HPV vaccine. The L2 protein, unlike the L1, cannot form VLPs and as such, it is less immunogenic. This review summarizes current studies aimed at developing HPV L2 vaccines by multivalently displaying L2 peptides on VLPs derived from bacteriophages and eukaryotic viruses. Recent data show that a monovalent HPV L1 VLP as well as bivalent MS2 VLPs displaying HPV L2 peptides (representing amino acids 17–36 and/or consensus amino acids 69–86) elicit robust broadly protective antibodies against diverse HPV types (6/11/16/18/26/31/33/34/35/39/43/44/45/51/52/53/56/58/59/66/68/73) associated with cancers and genital warts. Thus, VLP-based L2 vaccines look promising and may be favorable, in the near future, over current L1-based HPV vaccines and should be explored further. Multivalent display enhances cross-linking of B-cell receptors on naïve B cells, leading to a stronger B-cell activation, proliferation, and differentiation, secretion of high-affinity antibodies, and the generation of long-lived memory B cells ,7,8,9.(ii)VLPs have virally encoded T-helper cell epitopes, which enhance T-cell activation. Presentation of these epitopes by antigen-presenting cells (APCs) in association with major histocompatibility complex class II to T-helper cells leads to the activation of T-helper cells. In addition to this, co-stimulatory molecules from APCs help to activate the T-helper cells. Activated T-helper cells then secret cytokines, leading to the activation of B-cells, T-cells, and macrophages .(iii)Most VLPs are between 25 and 100 nm. This size range is very important for the following reasons. Firstly, nanoparticles between 10 and 200 nm, unlike those >200 nm, have been shown to efficiently enter the lymphatic system by direct diffusion through cell junctions. This allows VLPs to be efficiently exposed to immune cells. It is worth mentioning that VLPs can also be taken up and transported by APCs to the lymphatic system. Secondly, the small size of VLPs enables them to be transported to the lymphoid organs within a short period of time and to be efficiently taken up by APCs for presentation to T-helper cells .(iv)Some VLPs  contain single-stranded (ss)RNA, which serves as endogenous adjuvant. Bacteriophage coat proteins  have the potential to encapsudate ssRNA that codes for its capsid/coat protein during VLP assembly ,11. ThisVirus-like particles (VLPs) are empty viral shells derived from the expression, in a suitable host cell, of viral structural proteins such as capsid or coat proteins. Over-expression of these proteins allows them to spontaneously self-assemble into VLPs . VLPs caThe aforementioned features, in addition to the fact that VLPs are noninfectious and are safe, have made VLPs attractive biological agents for vaccine design and development. A number of VLP-based prophylactic vaccines have been approved by the Food and Drug Administration to protect against human papillomaviruses (HPVs) and hepatitis B virus (HBV) infections. Two VLP-based HBV vaccines are credited for reducing incidences of HBV-related hepatocellular carcinomas and mortalities, worldwide, with protection lasting for up to 30 years in some individuals ,15. On tApproximately 42 HPV types can be transmitted sexually via anogenital to anogenital sex or anogenital to oral sex . Out of Although current L1-based HPV vaccines are highly immunogenic, they protect mostly against the HPV types included in the vaccines ,34,35,36Immunization with the N-terminus of L2 protein (out of the context of the L1 protein) elicits antibodies that protect and cross-protect against heterologous HPV types. For example, unlike antibodies elicited against L1 VLPs, antibodies elicited against an L2 peptide representing amino acids 1–88 from HPV type 18 or even from L2 of bovine papillomavirus (BPV) cross-protect, albeit at low titers, against diverse mucosal  and cutaneous HPV types (HPV5) ,39,40,41Not only can VLPs be used to develop vaccines against the virus from which the structural proteins are derived from, they can also be used as platforms to display heterologous antigens from other viruses , bacteriDifferent HPV L2 peptides shown in  have bee(i)L2 peptides derived from different individual HPV types, especially HPV16 and HPV18. They correspond to aa 17–31, 20–29, 14–40, 34–52, 49–71, 56–75, 108–120, and so forth. ,58,61.(ii)L2 peptides derived from a concatemer of aa 17–31 and aa 20–31 from HPV16/HPV31 and a concatemer of aa 17–38 from five HPV types  ,60.(iii)Peptides derived from a consensus sequence of aa 65–85 or aa 69–86 following the alignment of cancer-causing and wart-causing HPV types  57,61].,61.57,61As mentioned above, peptides representing different L2 epitopes have been displayed on bacteriophage VLPs. These peptides, which range in size from 12 amino acids (aa) to 110 aa, can be grouped into three categories based on the source of peptides:Of all the above HPV L2 peptides that have been displayed on bacteriophage VLPs, only bacteriophage VLPs displaying peptides representing aa 17–31 from HPV5, 6, 16, 18 ,59, bactL2 peptides have also been displayed on VLPs derived from eukaryotic viruses such as HPV, bovine papillomavirus type 1 (BPV1), adenovirus, adeno-associated virus, hepatitis B virus, and potato virus A . All L2 Of all these VLP insertions, HPV16 L1 VLPs displaying on the DE loop, L2 peptides representing aa 17–36 from HPV16 elicited antibodies that efficiently neutralized/protected against diverse HPV pseudovirus types (6/16/18/26/31/33/34/35/39/43/44/45/51/52/53/56/58/59/66/68/73) . This isIn summary, the type of peptide and the location of peptide insertion on eukaryotic VLPs make a difference in the neutralization potential. L2 peptide (aa 17–36) inserted on the DE loop elicits efficient broadly protective antibodies against diverse HPV types.Current HPV vaccines based on L1 VLPs are highly immunogenic and offer robust protection against the HPV types included in the vaccines. However, they offer little cross-protection against non-vaccine HPV types ,34,35,36Among all the L2 peptides that have been displayed on VLPs, peptides representing amino acids 17–31 or 17–36 from HPV16 (and consensus sequence amino acid 69–86 to an extent) elicit antibodies with the broadest and the most efficient level of protection against diverse HPV types, regardless of whether a bacteriophage or eukaryotic VLP-platform was used to display the peptides. Within bacteriophage VLP platforms, MS2 VLPs displaying HPV16 L2 peptide (amino acids 17–31) elicit better cross-protection, while within eukaryotic VLP-platforms, HPV16 L1 VLPs displaying the HPV16 L2 peptide (amino acids 17–36) elicit better cross-protection. It is worth mentioning that the location of L2 insertion on the coat protein affects the immunogenicity of the peptide as well as the level of cross-protection. For bacteriophage VLPs, the insertion of amino acids 17–31 on the N-terminus seems to elicit better cross-protective antibodies against diverse HPV types compared to the same peptide inserted on the AB-loop  and our  65,72].,72.65,72As already highlighted, peptides representing amino acids 17–31 or 17–36 from HPV16 (compared to the same peptide from other HPV types) elicit robust broadly protective antibodies against diverse HPV types (6/11/16/18/26/31/33/34/35/39/43/44/45/51/52/53/56/58/59/66/68/73) ,67,68,69Overall, VLP-based L2 vaccines against diverse HPV infections look promising and may be favorable, in the near future, over current L1-based HPV vaccines. HPV16 L1-16L2 (aa 17–31) VLPs and mixed MS2-L2 VLPs ((MS2–16L2/31L2 and MS2–consL2 69–86)) can be formulated as a monovalent vaccine ,69 or bi–86) can Although aforementioned preclinical protective studies look promising, there are still some questions that remain to be answered with both candidate vaccines. For example, we still do not know if immune responses elicited by HPV16 L1-16L2 (aa 17–31) VLPs or mixed MS2-L2 VLPs will last 3–8 years, as has been shown with a single dose of Gardasil-4 . Recent Additionally, studies are required to assess the thermostability of candidate vaccines. Gardasil-9 has recently been shown to be thermostable at temperatures of up to 40 °C for three months ,82. InseStudies are also required to assess the effect of pre-existing L1 antibodies  to the immunogenicity of HPV16 L1-16L2(aa 17–31) VLPs. Studies have shown that pre-existing antibodies to some platforms can reduce the immunogenicity of the platforms and consequently, the immunogenicity of peptide displayed on the platforms ,87,88,89


The association between serum TNF-α and disease severity in adult patients with IgAV nephritis (IgAV-N) has been inadequately evaluated.  Tumor necrosis factor-α was measured by chemiluminescence immunoassay in 53 renal biopsy-proved IgAV-N patients, 53 healthy controls, and 53 IgA nephropathy (IgAN) patients. The correlations of clinicopathologic parameters of IgAV-N patients with serum TNF-α were analyzed.  Serum TNF-α was significantly higher in the IgAV-N group than in the healthy group , but comparable with sex, age, and estimated glomerular filtration rate (eGFR) grade-matched IgAN patients. Serum creatinine (P = 0.006) and serum cystatin C (P = 0.001) were positively correlated with serum TNF-α level, while albumin (P = 0.014) and eGFR (P = 0.021) were negatively correlated with serum TNF-α level. Multivariate linear regression analysis revealed that eGFR (P = 0.007) was an independent clinical predictor of serum TNF-α. Patients with higher pathological classification grade also had higher serum TNF-α.  In this cross-sectional study, the median age of IgAV-N patients was 29 (25-37) years, and 67.9% were female. Serum TNF-α is associated with renal function and the pathological classification of adult patients with IgAV-N. TNF-α is a potential biomarker for the assessment of IgAV-N severity. Serum TNF- TNF-α can regulate the mucosal immune response from multiple aspects   . Renal p2] . Treatmeα was adjusted by logarithmic transformation before multiple linear regression. The clinical indicators showing correlation in univariate analysis will be included in multiple linear regression with stepwise analysis. Statistical analysis was performed using SPSS statistical software version 23.0 . P < 0.05 was considered statistically significant.Data were expressed as the median (interquartile range) or absolute numbers (frequency). The Mann–Whitney test, Kruskal-Wallis test, or Chi-square test was used to analyze differences between groups. Spearman's rank test was used to determine the correlations of continuous variables. TNF-α was significantly higher in the IgAV-N group  (The median age of the 53 IgAV-N patients was 29 (25-37) years, and 67.9% were female. The median age of the 53 healthy controls was 30 (27-34) years, and 60.3% were female. Serum TNF-= 0.001] . Most Igα groups according to the upper normal limit of TNF-α detection (8.1 pg/mL) (α group had higher sCr (P = 0.049) and Cys-C (P = 0.036) levels. Although the average eGFR was not different between the groups, more patients in the high TNF-α group were CKD G3 and fewer were CKD G1 than in the low TNF-α group (P = 0.032). The high TNF-α group also had higher IgA levels (P = 0.004), while proteinuria and hematuria were comparable. More patients received glucocorticoid treatment in the high TNF-α group (P = 0.037). Correlation analysis showed that sCr (P = 0.006) and Cys-C (P = 0.001) were positively correlated with serum TNF-α level, while albumin (P = 0.014) and eGFR (P = 0.021) were negatively correlated with serum TNF-α level (Patients were divided into the high and low TNF-1 pg/mL) . The hig-α level . Multivaα test, and the biopsy-test interval was 28 (7-85) months. According to the ISKD classification, most patients were Class II-IV, with 15.1%, 73.6%, and 7.5%, respectively. Class IV patients had significantly higher average TNF-α levels than Class II and III patients (P = 0.027 for Class II and P = 0.009 for Class III).All patients underwent biopsy before TNF-α level was comparable in the two groups (We compared IgAV-N with IgAN patients matched for age, sex, and eGFR grade. The serum TNF-o groups . Other cα is an important inflammatory factor and has been studied in pediatric IgAV-N patients [α was increased in IgAV patients, and IgAV-N patients had the highest level of TNF-α compared with patients without kidney involvement. The level of TNF-α was also positively correlated with the degree of ISKD pathological grade [α level in IgAV children compared with the healthy group, and the level was higher in patients with renal function impairment than in those without renal function impairment. In addition, the TNF-α level in the acute nephritis group was higher than that that in those with chronic nephritis and nephritic syndrome [α was significantly higher in proteinuric IgAV children in the acute phase compared with those without renal involvement and those with hematuric IgAV. TNF-α levels declined in the convalescent phase compared with the acute phase in those with proteinuric IgAV using matched serum samples from the same patients [α was a biomarker of disease severity. However, some studies have shown that serum TNF-α was comparable in patients with IgAV-N and those without renal involvement [TNF-patients , 16, 17.al grade . Yuan etsyndrome . Ha repopatients . These rα levels in adult patients with IgAV-N. Berthelot et al. reported similar TNF-α serum concentrations in IgAV patients and controls. However, in their IgAV-N group, patients were older with more hypertension and diabetes, and not all patients with renal impairment underwent renal biopsy [α and the clinicopathologic manifestations in biopsy-proven IgAV-N was carried out. It was found that serum TNF-α was significantly higher in the IgAV-N group than in healthy controls and was correlated with renal function and ISKD pathological classification. Albumin was also negatively correlated with serum TNF-α, although the relationship between TNF-α and urine protein excretion was not significant. This may be because lower albumin indicated inflammatory status. As serum TNF-α was correlated with renal function and histopathology, we suspect that TNF-α might be a biomarker of renal prognosis in IgAV-N, which we will investigate in future research.There are few studies on serum TNF-l biopsy . In the α, secreted mainly by monocytes/macrophages, is a proinflammatory cytokine that plays a critical role in mucosal functions including impacting paracellular permeability, wound healing and guiding immune cell attraction, and function [α was also proved to cause direct renal injury. Raised serum TNF-α concentration may damage vascular endothelium [α in IgAV. Ding et al. reported that the A/A genotype frequency at the +308G/A position of TNF-α in IgAV patients was higher than that in surgical patients without renal diseases or rheumatic immune diseases, which indicated that A/A homozygosity of TNF-α may result in a genetic predisposition to IgAV [α levels and other factors such as interleukin 6 and malondialdehyde to a greater extent. The hemoperfusion group reduced the glucocorticoid dosage and the rate of renal involvement in children with severe IgAV, which may benefit from the removal of inflammatory factors [IgAV is frequently reported to follow respiratory tract infections which can activate the immune system and induce IgA production. As the deposition of IgA complexes is a characteristic of IgAV-N, the IgA mucosal immune system may be a key element in the pathogenesis of IgAV-N . Downstrfunction , 5. Abnoothelium , 19 and othelium . Genetic to IgAV . Zhu et α in IgAV-N and IgAN patients as these two diseases share similar renal clinicopathologic manifestations and pathogenesis. Serum TNF-α levels were comparable in our study. Sugiyama et al. reported significantly higher TNF-α in IgAV-N than in IgAN patients [α was significantly correlated with eGFR, urine protein excretion, mesangial hypercellularity, and tubular atrophy according to the Oxford classification [α, was effective in ameliorating proteinuria in selected patients with IgAN [α plays a role in IgAN and is a useful biomarker of disease severity. Thus, it is reasonable that serum TNF-α was correlated with renal function in IgAV-N and comparable in these two diseases. We also suspect that hydroxychloroquine may be effective in IgAV-N, which requires a further study.In the present study, we compared serum TNF-patients . Howeverfication . Our pilith IgAN . These rα can impact or predict renal prognosis requires further research. Second, this was a single center study, and the enrolled patients had relatively mild to moderate disease with well-preserved renal function. Therefore, caution should be used when extending these conclusions to more severe patients. Third, as this was a retrospective study, the biopsy data was obtained before TNF-α test with a variable range, which may have had an impact on the relationship between the pathological manifestations and TNF-α level. A prospective study enrolling more patients and a follow-up period of at least 3 years to confirm the value of serum TNF-α in IgAV-N patients are planned.Our research had some limitations. First, it was a cross-sectional design and only provided a correlation analysis. How TNF-α is correlated with renal function and pathological classification in IgAV-N patients. TNF-α is a potential biomarker for the assessment of IgAV-N severity.Serum TNF-