Devicesare fabricated starting from high-quality single-crystal monolayersgrown by chemical vapor deposition. The heterojunction was investigatedby scanning Raman and photoluminescence spectroscopies. Moreover,transconductance curves of MoS2 are compared with the current–voltagecharacteristics of graphene contact stripes, revealing a significantsuppression of transport on the n-side of the transconductancecurve. On the basis of ab initio modeling, the effectis understood in terms of trapping by sulfur vacancies, which counterintuitivelydepends on the field effect, even though the graphene contact layeris positioned between the backgate and the MoS2 channel.We demonstrate agraphene–MoS Within this family, peculiar junctions can be obtained whengraphene is used as a contact material for a TMD monolayer. Whilethe interface between a TMD and a conventional, bulk metallic electrodetends to display Schottky behavior due to intrinsic and extrinsicFermi pinning phenomena,4 vdW graphene–TMD junctionsyield well-behaved linear transport characteristics.5 This contacting approach has been successful in improvingTMD-based devices,6 and together with theside-contacting approach,9 it is commonly used for the realizationof most devices based on 2D materials. Graphene–TMDs heterostructureswere employed for many other applications, e.g., in flexible photodetectors.10 Nevertheless, the exact physics behind graphene–TMDvdW junctions is still debated13 and difficult to probe in a directway. In particular, devices typically include only two contacts, whichmakes the effects on the transport characteristics due to the interfacenot easy to distinguish from those due to the resistivity of the 2Dmaterials, since typically only the global conductance of the devicecan be measured. Charge transfer phenomena,14 strain,15 and charge trapping in defects18 might also play an important role. Furthermore, in the case of field-effectdevices, the low density of states in the vicinity of the Dirac pointleads to weak screening properties despite the metallic nature ofgraphene.19 This implies that a nontrivialresponse to field effect can be observed and exploited in device concepts.21 Gating on graphene–MoS2 heterostructures has beenwidely investigated from a numerical22 andexperimental25 point of viewand in different stacking configurations. Indeed, the reciprocal electrostaticscreening of the junction materials can affect the contact resistance,and it was shown theoretically that MoS2 can screen thefield effect on graphene, or not, depending on the order of the specificstacking sequence.22 Nevertheless, to thebest of our knowledge, direct experimental evidence of how the formationof vdW interfaces leads to changes in the transport properties ofthe individual materials involved in field-effect transistor (FET)devices is still missing.Formed whentwo or more atomicallythin crystals are bonded by van der Waals (vdW) interaction,27 and TMDs29 can be reproducibly grown. Whenthis technique is associated with a patterning of the seed points,predictable flake arrays of chosen sizes can be achieved,30 enabling the fabrication of multiple paralleldevices combining different 2D materials. Here, we take advantageof this opportunity to demonstrate a graphene–dichalcogenidearchitecture where a monocrystalline MoS2 channel is contactedby a large number of monocrystalline graphene stripes, each of themcrossing the whole MoS2 channel as schematized in 2 backgated FET  techniquesgives us the opportunity to investigate vdW interfaces from a differentangle. High-quality and large-scale monocrystalline flakes of graphene2 systems,highlighting the suppression of the electron-side transconductancein MoS2-covered graphene, in coincidence with the conductingthreshold of free MoS2 in the main device channel. Thisbehavior is apparently at odds with recent predictions for defect-freeMoS2;22 nonetheless, it canbe understood in terms of a gate-driven trapping by sulfur vacancieswhich further highlights the nontrivial weak screening propertiesof graphene.Such an arrangement allows us to study the conducting propertiesfor the different components of graphene–MoS2 flakeand analyze the influence of the graphene contact stripes. We analyzea MoS2 flake with a small bilayer island both in regionswith and without graphene. The Raman spectra of bilayer MoS2–graphene heterostructure and bare MoS2 bilayerare reported in the Supporting Information. The characteristic A1g and E2g modes visiblein 33 andtheir separation of Δω ≃ 19 cm–1 is in good agreement with the expected monolayer nature of the MoS2 flake. The shift of the Ag1 modein 2 and graphene, as reported in ref  .31 Interestingly, Raman shifts fromregions where MoS2 overlaps graphene (E2g =383.4 ± 0.5 cm–1 and A1g = 404.3± 0.3 cm–1) indicate a variation in both tensilestrain distribution of ≈0.10–0.35% and a sizable electronreduction of (3.0 ± 1.8) × 1012 cm–2. Thanks to the sensitivity of the A1g peak on doping,36 the spatial doping modulation of MoS2 due to graphene can be directly appreciated in the map of the A1g position in 2 are compared. Both curves show a single sharp Lorentzian-shaped2D peak, which is a typical signature of monolayer graphene,37 and no D peak, which indicates a negligibledensity of defects.38 The absence of defectswas confirmed for all the fabrication steps (see the Supporting Information). We also note that when graphene iscovered in MoS2 (orange curve), a strong baseline appearsbelow the Raman peaks due to the MoS2 PL signal. As inthe case of MoS2, the strain and doping profiles can bederived from the Raman data,32 based onthe correlation plot of the 2D and G modes reported in Supporting Information for additional correlation plots). The positions of the G and 2Dpeaks in regions free from MoS2 are 1582.7 ± 0.9 and2676.2 ± 2.2 cm–1, respectively, correspondingto a p-type doping. In contrast, Raman data collected in MoS2-covered regions  fall on thestrain line, thus indicating a neutralization of graphene. The variationof mean peaks position corresponds to an electron increase of (2.3± 1.5) × 1012 cm–2 and to avariation of strain nature from tensile to compressive. The spatialmodulation of the doping can be seen from the 2D peak position mapin 2 flake.A modified strain is also observed, turning from slightly tensileto compressive, ≈ 0.10–0.30%. We note that the dopingand strain trends observed where MoS2 and graphene overlapare opposite and are thus consistent. We further highlight that whilethe discussed analysis of the Raman data indicates an electron transferfrom MoS2 to graphene35 theabsolute equilibrium carrier densities are not obvious to quantify.This is due to the presence of photoexcited carriers during the Ramanmeasurements, and to the unknown exact calibration of the zero-strainand zero-doping points  with respect to stand-aloneMoS2 (blue curve): indeed, a lowering of the A intensityby ∼30% and a line shape broadening from ∼66 meV to∼96 meV is retrieved. The spatial modulation of the effectcan be directly appreciated from the maps of the intensity and widthof the A exciton in Supporting Information.Both the patterning procedures and the formationof vdW interfacescan significantly perturb the properties of the 2D materials. Forthis reason, photoluminescence (PL) and Raman spectroscopy were employedto characterize the 2D crystals at relevant device processing steps.In d in ref . Neverth2 channel and lead, at room temperature and in vacuumconditions (P < 10–5 mbar),to highly linear two-wire I–V curves over the ±2 V range.In ISD versus VSD characteristics of a representative MoS2 FET,measured as a function of the gate voltage (VG) in the 0–80 V range. The transfer characteristicin 45 as well as in Kelvin probe microscopy experiments.46 The effect is generally ascribed to the slow dynamics oftrap states leading to a time-dependent screening of the field effectof the gate. Trap states may have several origins, including defectsat the SiO2 substrate interface,45 adsorbates,44 or MoS2 pointdefects.43 In our devices, possible sourcesof traps include interfaces between MoS2, graphene, andSiO2 (see the AFM data in the Supporting Information), as well as S vacancies in MoS2, whichare known to occur in quite large densities  in CVD flakes.48 The field-effect mobility of the MoS2 carrier can beestimated from the transfer characteristic according toCG is thecapacitance and the MoS2 trapezoid channel sketched inthe inset of L = 5.5 ± 0.3 μm and width W = 19.5 ±0.5 μm. Considering both curves in the hysteresis loop, we extracttwo mobility values, and similar analysis on different FETs yieldedfield-effect mobilities in the range of 5.3–6.6 cm2/(V s). Given that gate hysteresis generally indicates that partof the gate-induced carriers end in charge traps, field-effect measurementsare known to overestimate the carrier density induced in the channeland to underestimate mobility.49 Both the mobility valuesabove should thus be considered as a lower bound to the true room-temperatureelectron mobility in the specific MoS2 flake. The methodalso neglects the effect of contact resistances, which may lead toa mobility underestimation but are not expected to have a significanteffect in the explored transport regime, based on preliminary four-wiremeasurement data.The graphene stripes form ohmic contacts to theMoS2 transport characteristics with the electron configurationin the graphene stripes, which play here the dual role of the contactin the MoS2 FET and of the channel in MoS2-coveredgraphene FETs. The I–V curvesof all our graphene stripes are found to be highly linear (a representativemeasurement is reported in the Supporting Information), and in VG, from which we obtaina mobility. These measurements are carried out at a fixed VSD (0.2 V), as a function of VG. In the plot sequence of 2 coverage: Conductivityis calculated from the total resistance using the geometrical formfactor of the stripe, and contact resistances are estimated by comparingthe p-side of the gate sweeps. MoS2 coverage is quantifiedfrom the ratio between the area of the MoS2–grapheneand the graphene regions; see the optical pictures in 2-free graphene in 2 and MoS2–graphene heterostructures and in graphenefunctionalized with different materials, such as TiO2 ororganic molecules.52 The important role ofMoS2 on electron transport suppression is clear as no deepsuppression is observed in bare graphene stripes on SiO2 devices.53 However, the observed behavioris somewhat puzzling since, ideally, MoS2 should not affectcarrier density in graphene when positioned on top of back-gated graphenedue to the reciprocal screening in the vdW heterostructure.22Our multi-FET devices were specifically designed for comparingthe MoS48 This is a reasonable assumption, since wementioned above that another possible source of trap states couldbe the SiO2 substrate. Nevertheless, the effect of theSiO2 substrate is secondary. In fact, the not-covered graphenestripe reported in 2 substrate. Moreover, the major contribution tothe electron transport suppression from the sulfur vacancies can bededuced from the behavior reported in 2 coverage increases.Furthermore, it is worth noting that the electrical transport of exfoliatedgraphene covered by exfoliated MoS2 usually does not presentthis suppression of the electron transport.54 In order to corroborate our hypothesis, and highlight the effectof sulfur vacancies, we perform density functional theory (DFT) calculations.We ascribe the origin of this apparent discrepancy as due to vacanciesin TMDs.55 we made an ab initio analysis of the electronic states of graphene–MoS256 in the presence of sulfur vacancies:These have an energy that falls in the gap57 of MoS2 and are located at a distance of few Angstromsfrom graphene, so their effect is hard to evaluate without a first-principlesapproach (see the “Supporting Information forfurther details). Numerical calculations were performed using a densityof S-vacancies of ρv ≈ 1.8 × 1013 cm–2. In 2 heterostructurefor VG = 0: As visible in the plot, theFermi energy of the system lays in the proximity of the MoS2 midgap states generated by the S-vacancies. This suggests that suchstates may influence the mobile carrier density induced in the graphenelayer by the gate when VG ≠ 0.This is confirmed by calculations performed at different values of VG. As shown in 2 is placed on topof graphene, it affects the carrier density induced by the gate inthe graphene layer. In particular, the n-side of the field-effectresponse is reduced by ∼50%; this, combined with the likelyincreased scattering59 caused by the large DOS close to the Fermi energy, clearly reproducesthe behavior reported in neff isthe effective density of the charge carrier in the graphene sheet(∼50% of the induced doping charge), kF is the Fermi momentum due to the effective density of thecharge carrier neff, ρv is the density of S-vacancies, and R = 3 Åis the average radius of the vacancy.48 In v similar to the one used in the ab initio calculations. We plot the data only for positive gate voltages becausethe midgap states affect the transport properties only in that sector.In 22 Calculations also show that no charge transfer is obtained in thecase of defect-free MoS2 since in that limit the TMD cannotsupport any electron state in the relevant energy range (see the Supporting Information). Nonetheless, it is interestingto highlight that in the presence of vacancies a TMD overlayer canindeed have an impact on the backgate response of these vdW heterostructuresand of devices based on them, extending the range of nontrivial consequencesof the weak screening properties of graphene.Using DFT,2 architectureintegrating multiple graphene-contacted MoS2 FETs and MoS2-covered graphene FETs and used it to correlate the field-effectcharacteristics of a MoS2 monolayer with the conductingproperties of graphene used to contact it. Such a study cannot beperformed in a conventional FET structure since the individual resistivecontributes cannot be discriminated in any obvious and direct way.Our results show that MoS2 can affect the field-effectconduction of a back-gated graphene monolayer, even when placed ontop of the vdW stack, and the suppression of conduction in the graphenestripes is observed over a gate voltage range which is consistentwith the conduction threshold of the MoS2 channel. Thisbehavior is explained in terms of a filling of sulfur vacancies inthe MoS2, as supported by ab initio calculations.The suppression of the electron transport can be exploited for thedevelopment of engineered optoelectronic devices based on van derWaals heterostructures,10 taking advantageof the low contact resistance of graphene in our multi-FET heterostructure.We have demonstrated a graphene–MoS30 and then transferredon a p++ Si substrate covered by 300 nm thermal SiO2 usinga delamination procedure and a semidry method based on a PMMA vector.26 Before the MoS2 transfer, the sampleswere cleaned from PMMA using an overnight immersion in acetone, followedby 2 min rinse in isopropanol, 3 min in AR 600–71 remover,and finally in deionized water. The next fabrication step was thepatterning of graphene into a set of 5 μm wide and 5 μmspaced stripes. To this aim, we spun PMMA AR-P679.04 and baked thesamples at 120 °C for 5 min. The stripe patterns were definedvia electron-beam litography (EBL) using a SEM Zeiss Ultraplus witha Raith lithographic module, an energy of 20 keV and a dose of 300μC/cm2. The samples were then developed in AR 600–56for 2 min and a half. Then, graphene was etched by means of reactiveion etching (RIE) using Ar and O2 (5:80 sccm). Finally,the samples were again cleaned from PMMA with an overnight immersionin acetone and isopropanol rinsing. Single-crystal MoS2 monolayer flakes with an average size of 50 μm were grownvia CVD following refs  and chloroform for 1.5 h. Tocomplete the devices, we defined a set of Cr/Au (10/50 nm) metallicelectrodes, via EBL, evaporation, and lift-off. Using this method,10 devices were fabricated in two batches and three multi-FETs weremeasured.The multi-FET fabrication starts froma square array of ∼150 μm wide single-crystal monolayergraphene flakes, with a spacing of 200 μm. Arrays are grownon Cu foil via CVDing refs . Single-2 were monitored by Raman and photoluminescence spectroscopy,using a Renishaw InVia spectrometer equipped with a 532 nm laser anda 100× objective lens (N.A. 0.85). Laser power was ∼1mWand the typical acquisition time was 4 s.62 Transport measurements were performed in a vacuum chamber usingsource-measure units K4200 and K2614B and a Femto DDPCA-300 currentpreamplifier.The properties of graphene andMoS67 which uses a plane wave basis set. The pseudopotentials were takenfrom the standard solid-state pseudopotential (SSSP) accuracy library72 with increased cutoffs of 50 and 400 Ry for the wave functions andthe density. The exchange-correlation potential was treated in theGGA, as parametrized by the Perdew–Burke–Ernzerhof (PBE)formula,73 with vdW-D2 correction as proposedby Grimme.74 For the BZ integrations, weemployed a Marzari–Vanderbilt smearing75 of 10–3 Ry with a Monkhorst–Pack (MP)76k-point grid with18 × 18 × 1 (24 × 24 × 1) points for self-consistentcalculations of the charge density (density of states). The heterostructureof monolayer MoS2 on top of monolayer graphene is shownin 2 supercell is placed on a 10 × 10supercell of graphene. The considered heterostructure model contains391 (392) atoms in the unit cell for the simulation with (without)S-vacancy, corresponding to a density of S-vacancies of ρv ≈ 1.8 × 1013 cm–2. We keep the lattice constant of graphene unchanged at a0 = 2.46 Å77 and compressed thelattice constant of MoS2 by roughly ∼2.4%: from3.15 Å78 to 3.075 Å. We considereda supercell with about 18 Å of vacuum along the c-direction between periodic images. We optimize the geometrical structuresby relaxing only the atomic positions until the components of allthe forces on the ions are less than 10–3 Ry/Bohr,while we keep fixed the lattice parameters.We carried out DFT calculationsby using QUANTUMESPRESSO (QE),2 is placed in front of a metal gate. The layers are then chargedwith the same amount of opposite charge, leading to a finite electricfield in the region between the heterostructure and the gate. In orderto avoid spurious and artificial electric field between the differentslabs of the repeated unit cell, an electric field generated by adipole plate of opposite charge has been included next to the gate.Furthermore, to avoid the direct interaction between the charge-densityof the system and the gate, a potential barrier has been included.67 In order to mimic the experimental values, in VG considering that in the real experiment there is a 300nm thick layer of SiO2 between the metal gate and the graphene–MoS2 interface. We use the VESTA79 codeto visualize the geometrical structure and the isosurfaces and toproduce the plots in 2), we performed a topologicalanalysis of the electron density by means of the Bader procedure83 as implemented in CRITIC2.85In


Raman Spectroscopy was performed on the devices to determine the quality of the graphene. The GFETs were annealed to improve their performance before the channels were functionalized by immobilising the graphene surface with linker molecules and anti-Clusterin antibodies. Concentration of linker molecules was also independently verified by absorption spectroscopy using the highly collimated micro-beam light of Diamond B23 beamline. The detection was achieved through the binding reaction between the antibody and varying concentrations of Clusterin antigen from 1 to 100 pg/mL, as well as specificity tests using human chorionic gonadotropin (hCG), a glycoprotein risk biomarker of certain cancers. The GFETs were characterized using direct current (DC) 4-probe electrical resistance (4-PER) measurements, which demonstrated a limit of detection of the biosensors to be ∼ 300 fg/mL (4 fM). Comparison with back-gated Dirac voltage shifts with varying concentration of Clusterin show 4-PER measurements to be more accurate, at present, and point to a requirement for further optimisation of the fabrication processes for our next generation of GFET sensors. Thus, we have successfully fabricated a promising set of GFET biosensors for the detection of Clusterin protein biomarker. The developed GFET biosensors are entirely generic and also have the potential to be applied to a variety of other disease detection applications such as Parkinson’s, cancer, and cardiovascular.We report on the fabrication and characterisation of graphene field-effect transistor (GFET) biosensors for the detection of Clusterin, a prominent protein biomarker of Alzheimer’s disease (AD). The GFET sensors were fabricated on Si/SiO Graphene has the potential to advance many technological areas because of its outstanding material properties such as its high carrier mobility  and LG Electronics Inc. . The chemicals used for the GFET fabrication were photoresist, lift-off resist (LoR), Microposit developer and Microposit remover, purchased from A-Gas Electronic Materials .Monolayer graphene was produced by chemical vapour deposition (CVD) method on a 300 nm Si/SiORecombinant human Clusterin protein and anti-Clusterin antibody were purchased from Abcam . Linker molecule 1-pyrenebutanoic acid succinimidyl ester (Pyr-NHS), bovine serum albumin (BSA) blocking solution and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich . All measurements were performed at room temperature, pH of 7.4 and using 10 μL samples deposited on the GFET sensors.The quality of graphene was evaluated using an XPLORA Raman spectroscopy system . All measurements on the XPLORA system were performed at a wavelength of 532 nm, with ∼ 4 mW of incident power and a grating of 1200T. The XPLORA Raman system was interfaced with an OLYMPUS BX41 microscope .D–VD and ID–VG) were acquired as a function of gate voltage (VG) from −100 V to +100 V with ID–VD curves from −50 to +50 mV with a 100 μA compliance. The ID–VD output and ID–VG transfer curves were measured at each functionalisation stage.The electrical characterisation of the GFETs was performed under ambient conditions using a Keysight B1500A semiconductor device parameter analyser interfaced to a MPS150 probe station . The 4-probe current–voltage measurements  over the 180–400 nm wavelength range, using a wavelength increment of 1 nm, with cell path length of 1 mm and at room temperature ∼ 22°C .2 substrate through the processes of photolithographic patterning and metal lift-off techniques with evaporated chromium and sputtered gold contacts. There are two major stages in the fabrication process, the first stage involves forming the graphene channel whereas the second stage involves forming the source, drain, and voltage electrodes. A representation of the fabrication process is shown in The GFET sensors were fabricated on a p++ Si/SiO2 substrate involves dicing the samples into sizes of 1 cm × 1 cm chips before spin-coating the samples with LoR at 3000 revolutions-per-minute (rpm) for 30 s. The samples are then pre-baked in a fan oven at 175°C for 15 min. Pre-baking the samples solidify the LoR as it eases in lifting off the photoresist (PR) while protecting the graphene channels formed. The next step is spin-coating the samples with a layer of positive PR at 3000 rpm for 30 s and then post-baking on a hotplate at 100°C for 60 s. The post-bake step is to solidify the PR and remove any solvents on the samples. The samples are then positioned in a mask-aligner under a patterned mask for creating the graphene channels, and exposed to ultra-violet (UV) radiation for 25 s. The samples are then rinsed in a chemical developer mixed with di-ionized water (20 mL developer mixed with 30 mL di-ionized water), until the graphene channels are visible under a microscope. There is always residue of PR/PMMA (poly-methyl methacrylate) on the sample at this stage, which degrades the quality of the graphene, which can be reduced by post-baking the samples on a hotplate at 180°C for 8 min under deep ultraviolet (DUV) light of 254 nm. The DUV dissociates the bonds between PR/PMMA and graphene and reduces contact resistance of the sample.The formation of the graphene channels on the Si/SiO–7 Torr at 50 W RF power for 2.5 min before the samples are treated with a chemical remover. Subsequently, the samples were placed in a chemical remover in an ultrasonic bath for ∼1 h at 60°C, the ultrasonic bath was turned off and the samples were left in the chemical remover for 15 h. The samples were then rinsed in di-ionised water and left to dry in a vacuum chamber for an hour.Next, the samples are transferred into a sputtering machine for Ar plasma etching. The unprotected graphene samples (not protected by PR) are then etched by plasma formed from the ionization of Ar gas particles in a vacuum of 6 × 10–6 Torr to form a 5 nm layer of Cr on the graphene samples. Thermal evaporation is a gentle way of depositing Cr on the samples without destroying the graphene channels already formed, and the Cr layer functions as an adhesive layer between graphene and the Au metallic contacts  of the antibody  used belongs to N-Hydroxysuccinimide (NHS) group known as Pyr-NHS ester. It has an aromatic pyrenyl group that strongly interacts with the surface of graphene via non-covalent π–π interactions and the succinimidyl ester group covalently reacts with the amino group  measurements of the ID–VD curves. In contrast, 2 substrate. D–VD results.Following the functionalisation of the GFETs, p < 0.001). At subsequent concentrations of Clusterin, 10 and 100 pg/mL, a clear reduction in the Dirac voltage shifts are observed. However, the standard deviations of the measurements are relatively large, as also observed by We also tested the three GFET sensors for their specificity by introducing a three-orders-of-magnitude higher concentration (compared to 100 pg/mL of Clusterin) hCG antigen at a concentration of 100 ng/mL; data are presented in D–VG measurements, although the standard deviation of these results were relatively higher than the results from 4-PER measurements. The GFET sensors were also found to have a repeatable performance over an extensive range of functionalisation stages from bare to 100 pg/mL of Clusterin using 4-PER measurements. In addition, the sensors were found to be highly specific, showing only a −6 ± 3% resistance change compared to 100 pg/mL of Clusterin when a three-orders-of-magnitude higher concentration of hCG was applied (100 ng/mL) to the GFET sensors. Future work includes deploying the sensors to detect a panel of biomarkers for the early detection of AD  using a novel multiplexing platform we are currently developing, which will be reported in a future study. In addition, as the GFET sensors are generic transducers, we anticipate their future applications in a variety of other disease biomarker detection also, such as cancer and cardiovascular.We have reported the fabrication, functionalization, and characterization of graphene FET sensors using Raman spectroscopy, four-probe electrical measurements and absorbance spectra using the highly collimated microbeam of Diamond B23 beamline for the detection of a prominent AD protein biomarker, Clusterin. The fabrication and functionalisation protocols have enabled detection of Clusterin from 1 to 100 pg/mL, with a limit-of-detection of ∼300 fg/mL (4 fM) using 4-PER measurement technique. In contrast, a characteristic curve for the Dirac voltage shift with Clusterin concentration has also been demonstrated using back-gated IThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.SAA: conceptualization, formal analysis, data curation, project administration, and funding acquisition. SAA, TB, CH, SD, and BO’D: methodology. BO’D and TB: software. SAA, TB, GS, and JC: validation. SAA and TB: writing—original draft preparation. TB, CH, SD, BO’D, TW, PD, GS, JC, and SAA: writing—review and editing. SAA, TW, and PD: supervision. All authors have read and agreed to the published version of the 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.


The role that the desire for self‐worth plays in international relations has become a prominent topic in contemporary political theory. Contemporary accounts are based on the notion of national self‐worth as a function of status; therefore, the desire for national self‐worth is seen as a source of anxiety and conflict over status. By contrast, according to Christian Wolff, there exists a duty to take care that both one's own and other political communities deserve to be esteemed. In his view, this duty is grounded in the duty of self‐perfection because the qualities for which communities deserve to be esteemed are those that promote the self‐perfection of individuals. From this perspective, he argues that duties of esteem toward political communities should not be seen as an outcome of power relations but rather as an outcome of the fulfillment of functions of the law of peoples. Wolff's line of argument draws attention to the possibility that anger over a violation of the desire for honor could be mistaken for an expression of specific culture‐specific sensitivities, while what really may be expressed is a diverging interpretation of the implications of dependence in international relations. What is perhaps most surprising about this literature is that concepts such as honor, which had seemed to be obsolete, have turned out to be illuminating tools for analyzing how certain cultural attitudes influence attitudes toward one's own people and toward other peoples Donelan, . DisregaFrom a historical point of view, the resurgence of these issues in contemporary political thought is striking because early modern cultures were honor cultures and questions of honor, reputation, respect, esteem, and glory were prominent topics in early modern political theory. However, read from the perspective of their early modern counterparts, the contemporary approaches to the role of honor in international politics seem to overlook two issues that were central to early modern discussions and still are central to present‐day political practice. The first issue concerns the connection between honor and status; the second issue concerns the connection between independence and sovereignty. As to the first issue, contemporary political theory seems to take it to be a conceptual truth that honor reflects nothing but a position in a social hierarchy. By contrast, early modern natural law theorists understood honor as an expression of esteem for naturally good qualities, both of individuals and of nations. It seems to be a worthwhile enterprise to ask whether such conceptions of esteem between nations could offer an alternative to conflict‐laden conceptions of national self‐worth. As to the second issue, status‐oriented approaches to honor seem to imply that dependent political communities cannot make the same claims to self‐worth as the communities on which they depend. In political practice, this intuition is expressed by withholding full diplomatic rights to the representatives of dependent communities. By contrast, early modern natural law theorists considered the possibility that even dependent communities can fulfill the natural goals of civil government and therefore have all the diplomatic rights that derive from sovereignty. There may be situations where such intercultural accounts of international relations ascribe difficulties in international communication to cultural differences whereas what is really at stake are the diverging interpretations of the implications of relations of political dependence.In what follows, I will substantiate this suggestion through an analysis of the treatment of the question of esteem for political communities in the thought of Christian Wolff (1679–1754). Focusing on Wolff may draw attention to the possibility of thinking about honor and esteem in international relations in an alternative, non‐competitive way. Honor, it may turn out, could not only be understood, as it is sometimes done . According to Bodin, sovereignty requires independence from external interference and therefore cannot be divided between different political actors Bodin, , I. 8. W2Famously, Wolff is part of the early modern person‐of‐the‐state tradition that treats peoples, like other associations, as legal persons Wolff, , § 2. AcKeeping the presence of both aspects of Wolff's notion of a people in mind lends plausibility to his strategy of construing duties between political communities as analogous to duties between individuals. Generally, he claims that political communities owe to each other exactly what individuals owe to each other Wolff, , § 156—aTo aggravate this problem, Wolff develops his account of the duties of esteem toward other political communities in the context of a cosmopolitan theory of relations between peoples. As in his conception of the nature of peoples, the theory of legal fictions plays a significant role in Wolff's conception of the civitas maxima—the political community that encompasses all political communities. Francis Cheneval has made a strong case for the usefulness of the theory of legal fictions for Wolff's cosmopolitanism  of their people. As Wolff notes, what holds true for the majority of a people usually is predicated upon its single members; in this sense “it cannot be avoided that what is predicated upon all belongs, as it were, to each individual” Wolff, , § 43; aForms of competition between scholars that take the form of mutual detraction thus are detrimental for the reputation of a whole community and thereby also for the reputation of those who compete for esteem. On the contrary, letting other scholars of the same community enjoy the reputation that they deserve increases the reputation in which this community is held, and thereby the reputation of each of its members. This gives an interesting hint at how non‐competitive practices of esteem could enhance the esteem in which all members of a community are held. This line of argument shows why we may be wrong to assume that we enhance the esteem in which we are held by trying to diminish the esteem in which other members of our political community are held. On the contrary, for Wolff there is not only a duty to care about one's own reputation (fama) but also a duty to care about the reputation of one's own people Wolff, , § 46. AAn analogous argument can be built for duties to uphold the glory of one's own people, where Wolff understands glory (gloria) as the “consistent praise of the good and experienced” Wolff, , § 47. CGlory is attributed to a people primarily and per se in so far as it is regarded as a single person, to whom one's own actions that depend upon intellectual and moral virtues are attributed; but it is also attributed in so far as the praise that belongs to individuals due to their actions or deeds that are regarded as belonging to individuals.  . This conception of duties of esteem can easily be extended to duties toward one's own political community and toward other communities, and this is exactly what happens in Wolff's discussion of these duties in the law of peoples.Wolff thereby formulates a natural law‐based conception of duties of esteem in the law of peoples that give hints at how the problem of international competition for status could be solved. His conception of honor does not make esteem among peoples a function of power relations. It is comparative in the sense that it allows for assessing degrees in which political communities fulfill duties of the law of peoples. At the same time, the value of how well political communities fulfill these functions does not derive from being better than the average performance in a competition. Rather, the value of fulfilling such functions derives from the role that doing so has in meeting basic human needs; and (apart from matters of precedence) power is only relevant for the capability of meeting these needs. This way of thinking thus reduces the status anxiety experienced by dependent communities and lowers the potential for conflicts over status.From this perspective, Wolff develops a compelling conception of the duties of esteem due to dependent political communities—those communities who have lost some of their sovereignty rights to more powerful communities. Wolff defends a conception of sovereignty that allows for geographically overlapping sovereignty rights. What from Bodin's perspective must look like a dissolution of sovereignty may bring serious advantages in political practice because it offers the theoretical foundations that would allow recognition of the functions of the law of peoples that dependent political communities still may be able to fulfill. Those functions of civil government that political communities still can exert independently from more powerful communities still count as an expression of sovereignty rights, with all the duties of esteem that these rights bring with them.This seems to be a view that still is thought‐provoking when read from the perspective of present‐day intercultural approaches to international relations. To be sure, it may be a good idea not to give the representatives of a former superpower who cultivate an honor culture the impression that one takes them to be political actors of merely regional importance. And it may be a good idea not to let political actors from a face‐oriented culture lose their face. But there is also the possibility that resentful reactions may be taken to be an expression of culture‐specific sensibilities when in fact something different is at stake. If signs of esteem that are due to independent peoples are denied to political communities that do not possess independence but nevertheless are able to fulfill functions under the law of peoples, then this constitutes a violation of a natural right to esteem. If so, then there is a genuine possibility that the anger caused by a lack of esteem could be mistaken for an expression of culture‐specific sensitivities to matters of honor, whereas what really occurs is anger over the lack of recognition for the fulfillment of functions of the law of peoples. In such cases, greater caution not to offend honor‐ or face‐related sensitivities may not only be inefficient to avoid tensions; it may also exacerbate the feeling of being offended in one's natural rights.


This paper describes an attempt to analyze, with the aid of bioinformatics resources (programs and databases), the probable cause of the cross-interaction of antibodies against HPV16 L1 with antigenic protein HPV6 L1, which has been revealed in the investigation of the candidate vaccine obtained on the base of a plant expression system (tomato plants). In our opinion, the most likely reason for the cross-interaction of antibodies with antigens of different pathogenic HPV types is the similarity of their antigenic determinants. In this work, the amino acid sequences of HPV16 L1 and HPV6 L1 used for the development of a binary vaccine against cervical cancer and anogenital papillomatosis have been analyzed. For the analysis of antigenic determinants, the programs BepiPred-2.0: Sequential B-Cell Epitope Predictor, DiscoTope 2.0 Server and SYFPEITHI have been used. As a result of the analysis of probable B-cell linear determinants (epitopes), it has been found that in both types of HPV the proteins have approximately the same location and size of linear antigenic determinants; the difference is observed only in the form of small shifts in the size of several amino acid residues. However, there are some differences in the amino acid composition of epitopes; therefore, the possibility for cross-interaction of the antibodies withthe antigens due to the similarity of linear antigenic determinants for B-cells is very small. The analysis of potential threedimensionalepitopes for B-cells has shown that due to little difference between them the HPV16 L1 and HPV6 L1 proteinshave no prerequisites for cross-interaction of the antibodies with the antigens belonging to the two different pathogenicHPV types. The analysis of probable linear epitopes for T-cells has revealed a common antigenic determinant in the two proteinsequences. According to the rank made with the SYFPEITHI program, the amino acid sequence AQL(I)FNKPYWL is thesecond most likely antigenic determinant for T-cells. Meanwhile, the amino acid sequences of this determinant in HPV16 L1and HPV6 L1 are virtually identical. There is a difference in only one position, but it is not critical due to the similarity ofthe physicochemical properties of amino acids, for which there is a replacement in the amino acid sequence of antigenicdeterminants. Consequently, some moderate cross-interaction of the antibodies to HPV16 L1 with the antigens of HPV6 L1may be expected. Tens of millions of people are infected every year with varioustypes of human papillomavirus (HPV), and this accountsonly for regions of the world where appropriate medical observationsand statistics are conducted . Therefore, the development of preventivevaccines against HPV is one of the current challenges to curbthe increase in the number of diseases caused by this type ofinfectious agents.The development of candidate vaccines based on plantexpression systems is a relatively new field of biofarming.Plant expression systems have certain advantages over othersystems. First of all, these advantages are related to safety dueto the absence of prions, mammalian pathogens, transposonsand dangerous viruses in a latent state, as well as the relativecheapness of obtaining vaccines, which generally contributesto wider commercialization and scaling. In our previousinvestigation, we attempted to develop candidate tetravalentoral vaccine based on transgenic plants against four types ofHPV  capable of causing cervical cancer. Inthis work, we planned to develop a vaccine that would providemaximum protection against cervical cancer by usingthe main antigenic protein L1 of the viral envelope of fourhighly oncogenic types of human papillomaviruses , which are responsible for mostcases of cervical cancer.It has been revealed that the antibodies to the antigenicprotein HPV16 L1 successfully interact with the HPV18 L1,HPV31 L1 and HPV45 L1 antigens .Based on the data obtained, it was assumed that the crossinteractionof the antibodies with the antigens belonging todifferent pathogenic types of HPV may be due to the similarityof antigenic determinants. This assumption was verified witha bioinformatic approach, where common linear determinantsfor T cells and B cells were found in all four types of L1 viralproteins. In addition, similar three-dimensional antigenic determinantswere found for B cells in HPV16 L1 and HPV18 L1. When working on the binary vaccinecontaining HPV16 L1 and HPV6 L1 antigenic proteins,Westernblot hybridization revealed a cross-interaction ofserum antibodies against HPV16 L1 with antigenic proteinHPV6 L1 .Human papillomavirus type 6 does not cause cancer, but can lead to the development of anogenital and respiratory papillomatoses.Despite the fact that these diseases rarely lead todeath, they are widespread and highly contagious .Such a wide range of cross-interaction between antigens andantibodies, which goes beyond the viruses that cause cervicalcancer and belong to another family, seemed extremelyinteresting to us. In this regard, in this work, the antigenicdeterminants of HPV16 L1 and HPV6 L1 have been subjectedto a comparative bioinformatic analysis. The data obtainedduring this work can be used to optimize the development ofcandidate vaccines using fewer HPV types due to the crossinteractionbetween antibodies and antigens of unrelatedtypes, which, in turn, will reduce the labor intensity and costof production of vaccines against dangerous types of humanpapillomaviruses.Alignment of the amino acid sequences of HPV16 L1 andHPV6 L1. As the first stage of the analysis of the antigenicdeterminants of HPV16 L1 and HPV6 L1, paired alignmentof HPV isolates of each type was conducted. For this purpose,their full-size amino acid sequences encoded by nucleotidesequences previously used in genetic constructs in the developmentof the binary vaccine against cervical cancer andanogenital papillomatosis were found and processed in theNCBI database (GenBank) . This wasnecessary for the subsequent determination of the difference inthe antigenic determinants of the two types of HPV. Whole setof full-size amino acid sequences of HPV16 L1 and HPV6 L1was also extracted from the GenBank database. The alignmentof amino acid sequences was carried out using the editor ofmultiple alignment of nucleotide and amino acid sequencesBioEdit. The phylogenetic tree was constructed using the program“Simple Phylogeny” (EMBL-EBI) by Nearest NeighborAlgorithms (the neighbor-joining method) and unweightedpairwise mean (UPGMA).. This bioinformatic resource allowed usto identify potential linear antigenic determinants for B cells.Identification of potential antigenic determinants. Forthe second stage of the analysis of antigenic determinants, theprogram “BepiPred-2.0: Sequential B-Cell Epitope Predictor”was used To determine the three-dimensional antigenic determinantsfor B cells, the program “DiscoTope 2.0 Server” was used(http://www.cbs.dtu.dk/services/DiscoTope/) . When working with this program, the strictestconditions were set: sensitivity 47 %, specificity 75 %. Threedimensionalmodels of proteins that were analyzed using theprogram DiscoTope 2.0 Server were found in the Protein DataBank (PDB) database. The programs BepiPred-2.0: SequentialB-Cell Epitope Predictor and DiscoTope 2.0 Server werepublicly available on the server of the Danish Technical University(DTU). The search for potential antigenic determinantsfor T cells was performed using the SYFPEITHI program,which is in the public domain http://www.syfpeithi.com. Thisbionformatic resource ranks all possible variants of antigenicdeterminants according to the probability of their interactionwith T cells .Amino acid alignmentThe amino acid sequences of the L1 capsid proteins of HPV16and HPV6 viruses were downloaded from the NCBI internationaldatabase and aligned in the BioEdit program .Analysis of linear antigenic determinants for B cellsThe amino acid sequences of two viral proteins HPV6 L1and HPV16 L1 were analyzed for the presence of potentiallinear antigenic determinants for B cells using the programBepiPred-2.0: Sequential B-Cell Epitope Predictor. The studyshowed that the HPV6 L1 protein has the following antigenicdeterminants: 14–25, 79–83, 85–91, 120–141, 162–177,208–216, 230–238, 260–283, 308–314, 345–358, 391–439,447–457, 468–497 .Previously, the following antigenic determinants wereidentified in HPV16 L1: 8–28, 83–95, 123–143, 166–177, 213–220, 234–243, 264–286, 350–369, 396–421, 426–444,452–462, 473–502 .As one can see, the analysis of linear determinants gaveevidence that in the HPV types studied, the proteins haveapproximately the same location and size of linear antigenicdeterminants, the difference is observed only in the form ofsmall shifts in several amino acid residues. A substantial differenceconsists only in the presence of linear determinants inthe HPV6 L1 protein: 79–83, 308–314, which are not observedin HPV16 L1. In addition, the antigenic determinant 426–444is present in the HPV type 16 protein, which has not beenrevealed in the virus type 6 protein.In order to draw the conclusion that there are similar linearantigenic determinants in the two HPV types under consideration,it was necessary to compare the amino acid compositionof the proposed epitopes. The difference in the amino acidcomposition can lead to a decrease in the degree of affinitywith antibodies. The absence of substitutions in amino acidsequences or substitutions with amino acids similar in propertiescan preserve the level of antibody affinity. Therefore, it isimportant to determine the presence and evaluate the qualityof amino acid substitutions in the proposed epitopes of theHPV6 L1 and HPV16 L1 proteins.The paired alignment of protein sequences demonstratedsubstantial difference in the amino acid composition in mostof the antigenic determinants. However, definite similarity wasfound between some amino acid sequences located within theboundaries of antigenic determinants close to both proteins.For eхample, in the determinant 166–177 for HPV16 L1,there was a difference in two amino acids: lysine was replaced with serine, glutamine with proline . Serine is a polaruncharged oxycarboxylic amino acid, and lysine is a polarpositively charged one. Proline is a heterocyclic nonpolaramino acid, and glutamine is a polar uncharged one. Takinginto account the difference in the physico-chemical propertiesof amino acids, for which there are differences in the supposedantigenic determinants, as well as the difference in the lengthof linear epitopes, we may assume a weak cross-interactionwith antibodies.In the determinant 213–220 for HPV16 L1, some differenceof two amino acids was found to be relative to the HPV6 L1sequence: threonine was replaced with alanine, and asparticacid was replaced with glutamic acid . Since threoninesubstantially differs from alanine in physical and chemicalproperties, such a replacement can affect the antigenic propertiesof the L1 protein. In case of the second substitution, wedeal with polar negatively charged amino acids with almostidentical physical and chemical properties. Considering thisfact, as well as the fact that the amino acid substitutions inthe sequences are located at some distance from each other,we can assume that there are similar antigenic properties ofthis region for the two viral proteins Search for antigenic determinants for T cellsIn order to obtain additional evidence of influence of the similarityof the antigenic determinants of viral proteins HPV6 L1and HPV16 L1 on the cross-interaction of the antibodieswith the antigens, which belong to the two pathogenic typesof human papillomavirus, a search for potential antigenicdeterminants for T cells was conducted with the aid of thebioinformatic resource SYFPEITHI. This program rankspossible variants of antigenic determinants according to theprobabilityof their interaction with T cells.Analysis of the protein sequences of the two scrutinizedviral proteins allowed us to reveal a common probable antigenicdeterminant for T cells, which was located in HPV6 L1at position 300–309, and in HPV16 L1 at position 304–313of the amino acid sequence. According to the rating compiledby program SYFPEITHI, the amino acid sequence AQL(I)FNKPYWL was the second most likely antigenic determinantfor T cells .When comparing the amino acid sequence of this determinant,a difference of only one amino acid was revealedin the two proteins, in HPV16 L1 leucine was replaced withisoleucine. Such a replacement should not lead to a change inthe antigenic properties of the determinant, since leucine andisoleucine belong to the same class of amino acids and havesimilar physico-chemical properties. As a result, it shouldbe noted that this antigenic determinant in the HPV6 L1 andHPV16 L1 proteins may have similar antigenic properties.Furthermore, multiple alignment of all the full-length aminoacid sequences of HPV16 L1 and HPV6 L1 presented in NCBIhas shown that this segment of the sequence is probably highlyconserved and is almost identical in these two proteins.Investigation of potentialthree-dimensional epitopes for B cellsOnly one three-dimensional model of the HPV6 L1 proteinwas found in the PDB database . Unfortunately, this model was not informative for theprogram DiscoTope 2.0 Server, so we conducted a comparativeanalysis of three-dimensional antigenic determinantsusing literary data.According to some scientific publications, the followingdomains are isolated from the HPV6 L1 protein, forming threedimensionalepitopes that can interact with B cells: F49, R53,A54; K52, R53, A54, N55; Y123, N128; G130, S131, G132;K169, T172, N173, P175, V176, Q177, A178; E262, V263,E265, P266; V344, T345, T346; S353. Critical paratopes forrecognition are domains F49, R53, A54 and K169, T172,N173, P175, V176, Q177, A178 .It was shown in our previous publication that the HPV16 L1protein has a spatial epitope in domain K53–L61, which partiallycoincides with the critical domain F49, R53, A54 of theHPV6 L1 protein. In addition, the location of the epitope S353of the HPV6 L1 protein coincides with the three-dimensionalantigenic determinant T350–Y355 of the HPV16 L1 protein. In order to determine the level of similarityof the immunological properties of these two proteins,we analyzed the paired alignment of the amino acid sequencesin the domains of their assumed three-dimensional antigenicdeterminants. Inconsistencies in amino acid residues werefound in the supposed epitopes of antigenic proteins. In theposition of 53 amino acid sequence in HPV16 L1, arginineis replaced with lysine, and in the position of 353 – serinewith glutamic acid. These amino acid substitutions may beconsideredinsignificant due to the similarity of the physicochemicalcharacteristics of the corresponding amino acids.According to the results obtained by us, it can be stated thatthere is a definite similarity between the antigenic determinantsof HPV16 L1 and HPV6 L1 proteins. At the same time, withregard to B cells, the potential for cross-interaction of antibodieswith antigens due to the similarity of linear antigenicdeterminants and three-dimensional epitopes is not substantial.However, for two types (16 and 6) of L1 viral proteins, thereis a substantial similarity of linear antigenic determinantsfor T cells. According to the results obtained with the aid ofprogram SYFPEITHI, these determinants are in the second position, but, despite this, these have a fairly high probabilityscore. At the same time, the amino acid sequences of theseepitopes are almost identical. There is some difference onlyin one position, but it is not critical due to the similarity ofthe physico-chemical properties of amino acids, accordingto which there is a replacement of antigenic determinants inthe amino acid sequence. Based on the above results, whenimmunizing HPV16 L1, we can expect a fairly reasonablecross-interaction of antibodies with HPV6 L1 antigens.The results obtained give a definite explanation of the causeof the effect of cross-interaction of antibodies with antigens belongingto different pathogenic types of HPV identified earlier.However, in order to obtain a more complete understandingof the mechanism of cross-interaction, it is desirable to studythe phenomenon of polymorphic distribution of epitopesand the process of induction of de novo antibody synthesis.The efficiency and expediency of the approaches and methodsused in this work is confirmed by publications of other scientists.For example, it is known from the literary sources thata team of authors  have used the samebioinformatic resources as our team to conduct an investigation of the cross-immune response tothe surface proteins L1 and L2 of human papillomaviruses ofhighly oncogenic types 16 and 18. In this work, much attentionwas paid to the comparison of amino acid properties, such ashydrophobicity, the surface area accessible to the solvent, thecharge and the secondary structure of the identified similarantigenic determinants of two different types of HPV. As aresult of this investigation, a candidate multiepitope vaccinewas created, the tests of which on laboratory mice showedrather good results. Application of this recombinant vaccinehelped to induce a sufficiently strong immune response andprotected mice from tumor cells with an efficiency of about66.67 % .In conclusion, it should be noted that application of theresearch methods discussed above can substantially acceleratethe development of efficient broad-spectrum vaccines againsthighly dangerous types of HPV. To date, there is a vaccine‘Gardasil-9’ that provides protection against 9 types of oncogenicHPV, but it already contains the maximum permissibleamount of antigenic proteins (270 μg of protein in one dose), while it does not provide protection in about 10 % of cases . Therefore, an extended study of cross-interactionof antibodies with antigens belonging to different pathogenictypes of HPV conducted with the aid of bioinformatic analysistechniques can help in development of multi-epitope wideactionvaccines without increasing the number of antigenicproteins in the vaccine preparations.The authors declare no conflict of interest.Brown D.R., Kjaer S.K., Sigurdsson K., Iversen O.E., Hernandes-AvilaM., Wheeler C.M., Perez G., Koutsky L.A., Tay E.H., Garcia P.,Ault K.A., Garland S.M., Leodolter S., Olsson S.E., Tang G.W., FerrisD.G., Paavonen J., Steben M., Bosch F.X., Dillner J., Joura E.A.,Kurman R.J., Majewski S., Muñoz N., Myers E.R., Villa L.L.,Taddeo F.J., Roberts C., Tadesse A., Bryan J., Lupinacci L.C.,Giacoletti K.E., Sings H.L., James M., Hesley T.M., Barr E. Theimpact of quadrivalent human papillomavirus  L1 virus-like particle vaccine on infection and diseasedue to oncogenic nonvaccine HPV types in generally HPV-naivewomen aged 16–26 years. J. Infect. Dis. 2009;199(7):926-935. DOI10.1086/597307.Jespersen M.C., Peters B., Nielsen M., Marcatili P. BepiPred-2.0: improvingsequence-based B-cell epitope prediction using conformationalepitopes. Nucleic Acids Res. 2017;45(W1):W24-W29. DOI10.1093/nar/gkx346.Kemp T.J., Hildesheim A., Safaeian M., Dauner J.G., Pan Y., Porras C.,Schiller J.T., Lowy D.R., Herrero R., Pinto L.A. HPV16/18 L1 VLPvaccine induces cross-neutralizing antibodies that may mediate crossprotection.Vaccine. 2011;29(11):2011-2014. DOI 10.1016/j.vaccine.2011.01.001.Kringelum J.V., Lundegaard C., Lund O., Nielsen M. Reliable B cellepitope predictions: impacts of method development and improvedbenchmarking. PLoS Comput. Biol. 2012;8(12):e1002829. DOI10.1371/journal.pcbi.1002829.Li Z., Song S., He M., Wang D., Shi J., Liu X., Li Y., Chi X., Wei S.,Yang Y., Wang Z., Li J., Qian H., Yu H., Zheng Q., Yan X., Zhao Q.,Zhang J., Gu Y., Li S., Xia N. Rational design of a triple-type humanpapillomavirus vaccine by compromising viral-type specificity. Nat.Commun. 2018;9(1):5360. DOI 10.1038/s41467-018-07199-6.McClements W.L., Wang X.M., Ling J.C., Skulsky D.M., ChristensenN.D., Jansen K.U., Ludmerer S.W. A novel human papillomavirustype 6 neutralizing domain comprising two discrete regions ofthe major capsid protein L1. Virology. 2001;289(2):262-268. DOI10.1006/viro.2001.1146.McLaughlin-Drubin M.E., Münger K. Oncogenic activities of humanpapillomaviruses. Virus Res. 2009;143(2):195-208. DOI 10.1016/j.virusres.2009.06.008.Nakagawa M., Greenfield W., Moerman-Herzog A., Coleman H.M.Cross-reactivity, epitope spreading, and de novo immune stimulationare possible mechanisms of cross-protection of nonvaccine humanpapillomavirus (HPV) types in recipients of HPV therapeutic vaccines.Clin. Vaccine Immunol. 2015;22(7):679-687. DOI 10.1128/CVI.00149-15.Namvar A., Bolhassani A., Javadi G., Noormohammadi Z. In silico/in vivo analysis of high-risk papillomavirus L1 and L2 conservedsequences for development of cross-subtype prophylactic vaccine.Sci. Rep. 2019;9(1):15225. DOI 10.1038/s41598-019-51679-8.Rammensee H.G., Bachmann J., Emmerich N.P.N., Bacho O.A., StevanovicS. SYFPEITHI: database for MHC ligands and peptidemotifs. Immunogenetics. 1999;50:213-219. DOI 10.1007/s002510050595.Rekoslavskaya N.I., Salyaev R.K., Stolbikov A.S. The synthesis ofmain capsid protein of anogenital type HPV6 L1 in plant expressionsystem on the basis of tomato fruits. Doklady Biochemistry and Biophysics.2021;498(1):193-198. DOI 10.1134/S160767292103008X.Salyaev R.K., Rekoslavskaya N.I., Stolbikov A.S. Cross-reactivity ofantigens and antibodies belonging to different pathogenic types ofhuman papillomaviruses. Doklady Biochemistry and Biophysics.2017;477(1):372-376. DOI 10.1134/S1607672917060084.Scherpenisse M., Schepp R.M., Mollers M., Meijer C.J.L.M., BerbersG.A.M. Characteristics of HPV-specific antibody responsesinduced by infection and vaccination: cross-reactivity, neutralizingactivity, avidity and IgG subclasses. PLoS One. 2013;8(9):e74797.DOI 10.1371/journal.pone.0074797.Stolbikov A.S., Salyaev R.K., Rekoslavskaya N.I. Bioinformatics analysisof putative causes for сross-reactive antibodies interacting withantigens derived from various pathogenic human papillomaviruses.Infektsiya i Immunitet = Infection and Immunity. 2020;10(4):695-706. DOI 10.15789/2220-7619-TBA-1263. (in Russian)


Graphene can be used as a drug carrier of doxorubicin (DOX) to reduce the side effects of doxorubicin. However, there is limited research on the surface chemical modifications and biological effects of graphene oxide (GO). Therefore, it is necessary to explore the DOX affinity of different oxygen-containing functional groups in the graphene system. We constructed graphene system models and studied the structure and distribution of epoxy and hydroxyl groups on the carbon surface. Based on molecular dynamics simulations and density functional theory (DFT), we investigated the interaction between DOX and either pristine graphene or GO with different ratios of oxygen-containing groups. The hydroxyl groups exhibited a stronger affinity for DOX than the epoxy groups. Therefore, the DOX loading capacity of graphene systems can be adjusted by increasing the ratio of hydroxyl to epoxy groups on the carbon surface. Graphene and its derivatives have great research value and application prospects in various fields because of their unique structures ,2. In paDOX, a common clinical anticancer drug, inhibits the DNA replication of cancer cells . DOX hasGO contains both sp3- and sp2-hybridized carbon atoms, with the sp3-hybridized carbon atoms mainly involved in oxygen-containing functional groups. Therefore, different interaction mechanisms are involved in the adsorption process between DOX and GO. Tonel et al. reported that the most stable conformation is achieved when DOX is parallel to pristine graphene . TherefoUnderstanding the interaction between the graphene system and DOX, especially the role of the functional groups in the adsorption process, is key. A previous study confirmed that functional groups play important roles in the adsorption of DOX to carbon nanotube drug carriers ,19. The Research on molecular interactions and the ratio of epoxy to hydroxyl groups on the surface of GO is limited. Therefore, we constructed graphene system models and studied the structure and distribution of epoxy and hydroxyl groups on the carbon surface. DFT was used to investigate the mechanism of the interaction between DOX and pristine graphene, GO with different ratios of oxygen-containing groups. DFT and charge density difference analyses were also conducted. By exploring the affinities between DOX and different oxygen-containing functional groups and adjusting the ratio of epoxy to hydroxyl groups, we aimed to provide a theoretical basis for increasing the DOX loading capacity of graphene systems.The chemical structure of DOX is shown in The pristine graphene surface is composed of 308 carbon atoms, with a surface area of 25.926 Å × 28.495 Å, and the length of the C-C bonds is 1.42 Å . The sur8O2(OH)2 units containing epoxy and hydroxyl groups [8O2(OH)2 is considered a stable unit of fully oxidized GO structures [8O2(OH)2 has not been reported. The two hydroxyl groups are above the carbon plane; the epoxy groups can either both be below the carbon plane or opposite each other (one on each side of the plane) [Due to the non-stoichiometric chemical composition of GO, it is difficult to establish a GO model. The GO surface contains many hydroxyl and epoxy groups, while the edges contain a small number of carbonyl and carboxyl groups; therefore, this study only considered Cl groups . C8O2(OHructures . Howevere plane) ,24.8O2(OH)2 unit structures were established. In one, both epoxy groups are below the carbon plane, and both hydroxyl groups are above the carbon plane (8O2(OH)2 units, the GO model used in this study contained three C8O2(OH)2 units, oriented to form an equilateral triangle.Here, two different Con plane a,b. In ton plane c. In ordTo establish a more accurate adsorption model, we use molecular dynamics simulations to simulate the adsorption trajectory and search for rough global optimal adsorption structures, then use this structure as a starting point for DFT optimization. All DFT calculations were performed using CP2K. The Nosé thermostat was used to maintain the temperature at 300 K. A constant volume was maintained, and 2000 steps were calculated for sampling.−4 Bohr/Hartree; (b) the force between root mean square atoms is less than 3 × 10−4 Bohr/Hartree; (c) the maximum displacement is less than 3 × 10−3 Bohr; (d) the root mean square atomic displacement is less than 1.5 × 10−3 Bohr.The structure with the least energy is selected from the molecular dynamics simulation for optimization. DFT calculations were based on a mixed Gaussian and plane wave (GPW) approach, Perdew-Burke-Ernzerhof (PBE) exchange-related functionals, and the corresponding pseudo potentials ,26,27. AEGS+DOX, EGS, and EDOX represent the energies of the complex, graphene, and DOX, respectively. A negative value of Eads indicates that the adsorption system is stable. The strength of DOX adsorption to the graphene surface can be directly reflected by the adsorption energy, which is obtained according to the following equation:ρ(r) represents electron density, and |∇ρ(r)| represents the norm of the electron density gradient vector.RDG analysis is an extremely useful weak interaction analysis method . It can To visually observe the change in electron density when DOX is adsorbed onto the graphene system surface, a charge density difference analysis was conducted, using the following equation:We selected a number of relatively stable adsorption structures from the structures sampled using the molecular dynamics simulation and then optimized them to obtain the most stable structure for analysis. In this study, the adsorption energies of the DOX and graphene systems were slightly higher than those observed in a previous study. By using a molecular dynamics simulation as the sampling method, we found that the orientation of DOX changed so that the anthraquinone and aminoglycoside rings faced the graphene. Thus, DOX had more sites of interaction with the carbon surface, which is the main reason for the higher adsorption energies observed. Conversely, in the study by Vovusha et al., only one of the two rings faced the graphene . The adsThe maximum adsorption energy was observed between DOX and pristine graphene, which indicates that the adsorption of DOX onto pristine graphene was the most favorable . The π-cIn the GO-OH-DOX complex , the twoThe interactions between DOX and the graphene system can have a significant effect on the hydrogen bonds within the DOX molecules. Lewis acidic hydrogen atoms on the hydroxyl-rich GO surface. Conversely, the epoxy-rich GO surface provided fewer acidic hydrogen atoms and, subsequently, formed weaker hydrogen bonds, resulting in less affinity for DOX.The adsorption energy between GO and DOX was smaller than that between pristine graphene and DOX. This was mainly because of the strong π–π stacking interaction between pristine graphene and DOX. Our results were consistent with previous studies of the interaction between aromatic compounds and pristine graphene ,33. HoweThe CH-π and OH-π interactions between the anthraquinone ring in DOX and pristine graphene can be clearly observed in the color-filled RDG isosurface plots . The intTo observe the charge transfer between graphene and DOX, we performed a charge density difference analysis . There wUsing DFT, we analyzed the interaction between DOX and either pristine graphene or GO loaded with different ratios of oxygen-containing functional groups. We observed that different oxygen-containing functional groups had different affinities for DOX. The order of the different graphene systems was as follows, in terms of adsorption energy: G-DOX > GO-OH-DOX > GO-OH-O-DOX > GO-O-DOX. Therefore, increasing the ratio of hydroxyl to epoxy groups can increase the DOX loading capacity of GO. This provides a theoretical basis for optimizing the surfaces of graphene substrate materials used to load DOX.


The human endogenous retrovirus-K (HERV-K) and TAR DNA-binding protein 43 (TDP-43) have been associated with the pathophysiology of amyotrophic lateral sclerosis (ALS). Given these findings, we investigated the humoral response against HERV-K envelope surface (env-su) glycoprotein antigens and TDP-43 in the plasma of ALS patients and healthy controls (HCs). The measured levels of Abs against the different epitopes’ fragments were significantly elevated in ALS patients, both in long-survivor (LS) and newly diagnosed (ND) patients, compared to HCs. We observed a positive correlation between HERV-K and TDP-43 antibodies (Abs) levels, which seemed to strengthen with disease progression, that was not found in HCs. The TDP-43 and HERV-K epitopes identified in this study are highly immunogenic and recognized by the humoral response of ALS patients. Increased circulating levels of Abs directed against specific HERV-K- and TDP-43-derived epitopes could serve as possible biomarkers. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown etiology described for the first time in 1874 by Jean-Martin Charcot. It is characterized by the progressive degeneration of both the upper and lower motor neurons, which display cytoplasmatic inclusions . PopulatEmerging evidence has indicated that human endogenous retrovirus K (HERV-K) may play a role in the disease’s etiopathogenesis. Human endogenous retroviruses (HERVs), considered until recently as junk DNA, constitute nearly 8% of the human genome. Proviral genome integrations into the DNA are remnants of infections that occurred over several million years ago. Nevertheless, numerous nonsense mutations have contributed to their defection .pol transcripts in post-mortem brain samples of patients affected by ALS compared to other pathological conditions such as Parkinson’s disease, chronic systemic illnesses, and healthy subjects [env gene was expressed, developed progressive motor dysfunction [HERV-K, in particular the HML-2 subgroup, is present in hundreds of copies in the human genome and is one of the most recently acquired and the most transcriptionally active among HERVs. The involvement of HERV-K in the pathophysiology of ALS has been documented by numerous groups. Douville et al., (2011) observed an altered expression of HERV-K subjects . Transgefunction .(20–38) were significantly elevated in ALS patients compared to multiple sclerosis (SM), Alzheimer’s disease (AD), and healthy control subjects, both in serum and cerebrospinal fluid (CSF), suggesting HERV-K-env-su(20–38) as a possible early novel biomarker [It is noteworthy that HERV-K expression is regulated by TDP-43, which can bind to the long terminal repeat region (LTR) of the virus. Human neurons transfected with TDP-43 showed an increase in HERV-K expression, while knockdown of endogenous TDP-43 resulted in a decrease in HERV-K expression . Furtheriomarker . Specifiiomarker . Li et aiomarker  demonstriomarker .TDP-43 is a multifunctional protein associated with several biological functions , includiTo date, the ALS diagnostic process requires approximately one year, since only the progression of symptoms and the presence of signs of both upper and lower motor neuron involvement can confirm the ALS diagnosis. The diagnosis is based on clinical examination, electrophysiological findings, medical history, and exclusion of confounding disorders . UnfortuWe evaluated an ALS group of 45 patients , and an age- and gender-matched healthy control group (HCs) collected from the Blood Transfusion Centre of Sassari . Data are summarized in The ALS population, sampled from January 2016 to December 2019, comprised newly diagnosed (ND) ALS patients , hospitalized at the Neurology Unit Clinic of the University Hospital of Sassari and long-survivor (LS) ALS patients  reported by primary doctors and doctors in the Sassari local district.Peripheral venous blood samples were collected in a K2-EDTA tube. Whole blood was gently layered over an equal volume of Ficoll  in a 15 mL tube and centrifuged for 20 min at 1800 RPM without brake. The plasma contained in the uppermost layer was collected by pipetting and tested for the presence of Abs against TDP-43- and HERV-K-env-derived epitopes. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of ALS 1 Sassari (2149/CE).(20–38) , TDP-43(258–271), TDP-43(398–411), and TDP-43(398–411)P  were designed using the Immune Epitope Database and analysis resource (IEDB) and synthesized at > 95% purity . The IEDB software predicts regions of proteins that are likely to be recognized as epitopes in the context of a B-cell response. All peptides were dissolved in DMSO and stored at −80 °C in single-use aliquots (10 mM) (The peptides HERV-K-env-su (10 mM) .(20–38), TDP-43(258–271), TDP-43(398–411), and TDP-43(398–411)P epitopes. Ninety-six-well Nunc immuno-plates were incubated overnight at 4 °C with a solution 0.05 M of carbonate-bicarbonate, pH 9.5  and the respective peptides at 10 µg/mL. The plates were incubated for 1 h at room temperature in a blocking solution with 5% non-fat dried milk  and phosphate-buffered saline (PBS) and washed twice in a solution with 0.05% Tween-20 and PBS (PBS-T). The plasma samples were added at 1:100 concentration and incubated for 2 h. After this, each plate was washed five times in PBS-T and incubated for 1 h at room temperature (RT) with 100 µL of PBS and alkaline phosphate-conjugated goat anti-human IgG polyclonal antibody . After another washing step in PBS-T, plates were incubated in a dark environment for 8 to 10 min in milli-Q water and p-nitrophenyl phosphate , and an absorbance of 405 nm was recorded using a SpectraMax Plus 384 microplate reader . Each sample was run in duplicate, and normalization was performed with the positive control included in each assay. Background activity was calculated as the mean signal of an immobilized peptide with secondary Ab. Results are expressed as the means of duplicates of 405 nm optical density (OD) values.Indirect ELISA was performed to detect specific Abs against HERV-K-env-sup < 0.05. Receiver-operating characteristic (ROC) was used to choose the cut-off value to assess the sample positivity, which was consequently tested through Fisher’s exact test. A Spearman correlation test was performed among levels of antibodies to HERV-K env- and TDP-43-derived peptides.All data were analyzed using GraphPad Prism 8.2.0 software . A Mann–Whitney U test was used to analyze non-parametric data and compare differences among two groups. Kruskal–Wallis and Dunn’s post hoc tests were carried out to compare differences among three groups. Statistically significant difference was set with a We enrolled 45 ALS patients and 45 matched HCs in our study. Details of the ALS patients’ clinical information are shown in (20–38) and three selected epitopes of TDP-43 was evaluated in the plasma of the ALS and HCs groups. A Mann–Whitney U test was performed to compare quantitative values of the antibodies’ OD among groups. Cut-off values were selected by ROC analysis, and the comparison between the percentage of positive and negative samples was performed using Fisher’s exact test. Briefly, Abs against HERV-K-env-su(20–38) were present in 40% of the ALS population, versus 8.89% in the healthy control group , whereas only 4.44% in the healthy group showed a humoral response against the same peptide  epitope . (398–411)P; only 4.44% of healthy subjects were positive, whereas in ALS patients, we observed an increased percentage of up to 20.00% .The humoral response against HERV-K-env-suol group A: Mann–W peptide B: Mann–W epitope C, with aIn order to investigate a potential difference in the humoral response in the plasma of ALS-ND and ALS-LS patients, the Kruskal–Wallis test and Dunn’s post hoc analysis were performed. The limitation of this analysis is that the three groups  are not matched by sex due to the small ALS population; it would be very interesting to more deeply understand if statistical differences correlated with sex exist in a larger population. The obtained results highlight a significant difference in HCs compared to ALS-ND and ALS-LS for the peptides derived from TDP-43 and HERV-K envelope protein, while no difference was observed in the human response between ALS-ND and ALS-LS .(20–38) and TDP-43(258–271)  ((20–38), and TDP-43(398–411)  ((20–38) and TDP-43(398–411)P  ((20–38) and TDP-43(258–271) showed r and p values, respectively, of −0.138 and ns ((20–38), and TDP-43(398–411) showed an r = −0.173 and p = ns, ((20–38) and TDP-43(398–411)P showed an r = −0.099 and p = ns  A, HERV-K= 0.001) C, and fi= 0.003) E. The sa8 and ns B, while  p = ns, D, and fid p = ns F.(20–38) and TDP-43(258–271)  ((20–38) and TDP-43(398–411)  ((20–38) and TDP-43(398–411)P  ((20–38) and TDP-43(258–271)  ((20–38) and TDP-43(398–411)  ((20–38) and TDP-43(398–411)P   A, HERV-K= 0.018) C, and la p = ns) E. We obs p = ns) B, HERV-K= 0.013) D, and fi= 0.047) F.In this study, we investigated the humoral response directed against specific epitopes of the HERV-K envelope and TDP-43 proteins in human ALS plasma samples. Autoantibodies were found to be significantly higher in ALS patients compared with healthy control samples. These results confirm our previous data and highlight the reactivation of the human endogenous retrovirus in ALS pathology .Likewise, Conti et al., documented increased levels of both anti-TDP-43 Abs and TDP-43 protein in ALS-serum patients compared with healthy controls, motor neuron disease mimics, and Alzheimer’s and frontotemporal lobar degeneration patients, but autoantibodies and protein serum levels failed to correlate . HoweverSeveral studies ,39 suggeThe hypothesis regarding the possible physiological role of autoantibodies in the prevention of excessive accumulation of neuronal inclusions is in agreement with the data reported .Otherwise, the literature has quite clearly proven that the cytoplasmic aggregation of phosphorylated, ubiquitinated, and truncated TDP-43 represents a pathologic hallmark across the clinical spectrum of ALS . The C-tIn order to investigate the role of autoantibodies as a novel biomarker in ALS, we searched for the presence of different autoantibodies in both ALS-ND and ALS-LS patients . The humpol transcripts in post-mortem brain samples of ALS patients has been confirmed compared to patients with other nervous system diseases and healthy subjects [Different studies have documented the implication of HERV-K in ALS. An altered expression of HERV-K subjects . HERV-K subjects .pol-gene expression was found to correlate with TDP-43 mRNA in post-mortem brain tissue from patients with ALS [(20–38) and TDP-43 epitopes.Furthermore, HERV-K with ALS , and we Interestingly, no correlations have arisen in HCs. This endorses the results obtained in ALS patients and the specific relationship between HERV-K and TDP-43 in pathological conditions . AdditioCurrently, the regulatory role of TDP-43 in the HERV-K expression is well known , but furOur investigation concludes that the epitopes of TDP-43 and HERV-K envelope identified by the Immune Epitope Database (IEDB) are highly immunogenic and recognized by the humoral response found in ALS patients in comparison with the weak response of the healthy control subjects analyzed. We are currently enrolling for a larger study to deepen our hypothesis. In particular, clinical data regarding ALS are going to be collected, in order to point out any putative relationship. It would be interesting to conduct a prospective study to better understand if our results are the consequences of the pathology, or if they are the result of a connection that might exist between TDP-43 and HERV-K env.Currently, no specific biomarkers are available for the differential diagnosis of ALS, thus diagnostic and prognostic biomarkers for ALS remain a major unmet clinical need, and the results obtained in this work suggest a need for further study in this direction with a larger number of patients and controls.