Arteriovenous grafts are routinely placed to facilitate hemodialysis in patients with end stage renal disease. These grafts are conduits between higher pressure arteries and lower pressure veins. The connection on the vein end of the graft, known as the graft-to-vein anastomosis, fails frequently and chronically due to high rates of stenosis and thrombosis. These failures are widely believed to be associated with pathologically high and low flow shear strain rates at the graft-to-vein anastomosis. We hypothesized that consistent with pipe flow dynamics and prior work exploring vein-to-artery anastomosis angles in arteriovenous fistulas, altering the graft-to-vein anastomosis angle can reduce the incidence of pathological shear rate fields. We tested this via computational fluid dynamic simulations of idealized arteriovenous grafts, using the Bird-Carreau constitutive law for blood. We observed that low graft-to-vein anastomosis angles ( This life-sustaining procedure facilitates removal of circulating metabolic toxins in a patient’s blood stream by diverting arterial flow into a hemodialyzer. The blood is then returned in real-time into the venous circulation1. Arteriovenous grafts are commonly implanted in patients who require hemodialysis to provide direct access to a patient’s arterial and venous blood streams2. Non-autologous polytetrafluoroethylene (PTFE) arteriovenous conduits are most common, and are anastomosed to an artery at one end and to a vein at the other end. The body of the graft is then tunneled subcutaneously to facilitate percutaneous hemodialysis cannulation in the future4.Hemodialysis is provided to more than 300,000 Americans annually who have end stage renal disease3. Accordingly, brachio-axillary arteriovenous grafts , tapered brachio-axillary arteriovenous graft conduits are commonly used in adult patients in clinical practice9. These grafts are typically tapered from 4 mm at the graft-to-artery anastomosis to at least 7 mm at the graft-to-venous anastomosis  and the axillary vein (near the arm pit)fts Fig. a are tun12. Arteriovenous graft thrombosis can be problematic if graft patency is not immediately restored, or alternative arteriovenous access placed to facilitate continued hemodialysis. This leads to a high degree of repeated interventions and hemodialysis access placement, which leads to increased overall disease morbidity13. As many as 80% of arteriovenous grafts develop high grade stenoses at the graft-to-vein thrombosis leading to eventual graft thrombosis and loss of hemodialysis access14. It is postulated that stenosis at the graft-to-vein anastomosis develops due to pathological flow fields that cause intimal damage15, leading to myointimal hyperplasia16, and eventual activation of circulating clotting factors21. Shear strain rates below the threshold of 50 s22. On the other hand, shear rates elevated above 1000 s23. It is therefore recognized that flow rates within a physiological range are necessary to minimize risk of thrombosis at the graft-to-vein anastomosis and sustain graft patency.Despite advances in arteriovenous graft material technology, brachio-axillary grafts failure rates can be as high as 30% within the first 12 months24. In the vasculature, this is especially true, as demonstrated in arteriovenous fistulas 25. In fistulas, the vein-to-artery anastomosis angle can greatly impact pathological flow fields in the vicinity of the arteriovenous anastomosis28. However, a similar dynamic flow analysis of the problematic graft-to-vein anastomosis in arteriovenous grafts has yet to be conducted. We hypothesized that identifying an optimal range for graft-to-vein anasotmosis angles can potentially decrease the risk of pathological flow fields in this segment of interest, and test this using a series of computational dynamics simulations. Results from our study may ultimately improve arteriovenous graft surgical implantation techniques, and help decrease morbidity associated with hemodialysis access.Wall shear stresses at a junction between two connected pipes is strongly impacted by the attachment angleμ = 0.0035 Pau = 1.5 m/s yielded an estimate of We estimated the pulsatile flow fields of blood in a brachio-axillary arteriovenous graft system with a graft in place through pressure-based transient computational fluid dynamics (CFD) simulations. An estimate of the Reynolds number, A series of idealized, straight arteriovenous grafts were constructed in commercial CAD software  based upon the aforementioned data for typical arterial and venous presentations. In each case, the inner diameter of the artery was 4 mm, and the inner diameter of the vein was 8 mm. The arteriovenous graft was tapered from a 4 mm diameter on the arterial end to a 7 mm diameter on the venous side. These dimensions were chosen to fit both within the range of 3–5 mm for the brachial artery, and within the range of 6–10 mm for the axillary vein. The length of the arteriovenous graft was 150 mm ± 2 mm, depending on the arteriovenous graft-to-vein anastomosis angle. To assess the range of venous anastomosis configurations that may be utilized by a surgeon at the time of implantation, we studied the range of anastomosis major axis sizes that are used in our clinical practice, ranging from a circular anastomosis with a diameter of 7 mm, corresponding to a 90° anastomosis angle, down to an elliptical anastomosis with a major axis of 30 mm, corresponding to an anastomosis angle of 13°. The major axis was defined as the maximum length of a line bisecting the ellipse formed when the cylindrical, conical graft intersected the cylindrical vein. The 7 mm, 8 mm, 10 mm, 15 mm, 25 mm, and 30 mm anastomosis major axis lengths corresponded to graft-to-vein anastomosis angles of 90°, 60°, 45°, 30°, 15°, and 13°, respectively.For all simulations, the vein length was 72 mm, with the length distal to the graft held at 25 mm and that proximal to the graft ranging from 40 mm to 17 mm depending on the anastomosis angle. This was done to ensure that the proximal and distal sections of the vein were sufficiently long for flow fields to be fully developed before and after the anastomosis, so that regions over which pathological flow rates were observed were fully contained within the model.29. The Blasius solution . Care was taken to ensure that discretization was not distorted in regions of high curvature and at the edges of the anastomosis. For each simulation, mesh size was refined until convergence was reached. Meshes were refined so as to ensure satisfactory levels of element skewness and orthogonalitytion Eq.  was usedp is the hydrostatic pressure. Because the flow was incompressible, Eq. (R is the artery radius)33:n is a constitutive parameter and 34, in which Blood was modeled as a non-Newtonian fluid following the Navier-Stokes equation and the incompressible mass continuity equation:ble, Eq.  was idenmentclass2pt{minim38, and was reconstructed using Gaussian wave fitting39 converted to a Fourier series:t is the simulation time, and 40. The volumetric flow rate into the arteriovenous graft was measured to be 700 ml/min, which is within the range of healthy flow rate for an arteriovenous graft41. The venous system does not experience significant pulsatile flow42. The flow from the distal side of the vein to the proximal side of the vein was specified as a constant, centerline velocity of 15 cm/s, as appropriate for flow in the axillary vein43.The inlet condition for the artery was uniform flow defined by a centerline velocity function. This velocity waveform was based off ultrasound data from the centerline velocity measured in the brachial artery of patients with dialysis access29, this fails to account for the downstream effects that are present in the circulatory system. A common approach is therefore to use an outflow boundary condition for simulating vascular systems, especially for arteriovenous grafts45. Following National Kidney Foundation KDOQI Clinical Practices41, inflow was distributed so that 90% was sent to the graft and 10% to the distal artery. An outflow boundary condition was also imposed at the proximal end of the vein. No-slip boundary conditions were used along the vessel walls, and vessels were treated as rigid.Outlet boundary conditions are a well known challenge for vascular flow simulation. Although standard practice is for pipe flow to use pressure outlet conditionsEquations were solved using the ANSYS Fluent CFD solver . The semi-implicit method for pressure-linked equations (“SIMPLE”) pressure-velocity coupling method was used with second order spatial discretization and first order transient discretization. The pressure-implicit with splitting-operations (“PISO”) method was also used, with second order spatial discretization and transient discretization, with negligible differences in results but with increased computation time.29. Convergence criteria for the continuity, x-velocity, y-velocity, and z-velocity were all set at an absolute tolerance of 0.001.Hybrid initialization was done by solving Laplace’s equation, We defined metrics to quantify the extent and magnitude of pathological shear stresses experienced by the vein walls during a single cardiac cycle. For low shear strain rates, we defined a metric For shear high strain rates we defined Blood flow entering the axillary vein from a brachio-axillary arteriovenous graft led to perturbations in the laminar venous blood outflow Fig. . To quanColor maps demonstrated that the graft-to-vein anastomosis angle clearly impacted the shear environment of the adjacent vein wall. Additionally, the data demonstrated the extent of how blood entering the vein from the graft was affected by the graft-to-vein anastomosis angle Fig. . For a 9To further understand how the shear environment was affected by the graft-to-vein anastomosis angle, we plotted flow fields over cross-sections of the anastomoses at the peak of high wall shear rate (0.1 s into the 0.3 s heart beat shown) and at the peak of low wall shear rate (0.1 s into the 0.3 s heart beat shown, Fig. These summary metrics suggested that the fraction of vein wall area undergoing pathologically high shear strain rates diminished with decreasing the graft-to-vein anastomosis angle, while that undergoing pathologically low shear rates stay low from 90These data suggest that a 3046. In this study we observed that low graft-to-vein anastomosis angles that are 4. Future research will explore a the range of optimal graft-to-vein anastomosis angles relative to different diameter axillary vein diameters.Here we demonstrate that the graft-to-vein anastomosis of a brachio-axillary arteriovenous graft can develop severe pathological flow fields. It is understood that these flow fields are a major contributor to myointimal hyperplasia at the graft-to-vein anastomosis that ultimately lead to stenosis, graft failure, and thrombosis47. Although fistulas are usually the preferred primary method to facilitate hemodialysis access, there are many patients who do not have suitable superficial venous anatomy for fistula placement48. Accordingly, in an aging population with a rising incidence of end stage renal disease, the use of arteriovenous grafts is anticipated to continue to increase over the coming years4. However, the 1 year primary patency of arteriovenous grafts varies between 40-57%, and the 1 year secondary patency is between 62 and 78%48. Clearly, technical challenges persist in maintaining adequate arteriovenous graft patency, and this is a clear impetus for better characterization of the underlying complicating factors22.For patients who require chronic hemodialysis, placement of an arteriovenous graft can be a life-saving alternative to an arteriovenous fistula4. Depending on the techniques used to subcutaneously tunnel the arteriovenous graft, the patient’s body habitus, and the axillary vein diameter, the graft-to-vein implantation angle can vary significantly from one patient to another. Various studies have explored ways to decrease graft associated thrombotic complications. For example, adjunct therapy with antiplatelet and anticoagulant therapy has been used as means to reduce graft thrombosis, but several studies have that does not provide any clinically meaningful benefits and may actually increase the risk of bleeding51. Other studies have attempted modifications to the graft-to-vein anastomosis to reduce the risk of myointimal hyperplasia and graft thrombosis. For example, a study incorporated a venous cuff at the graft-to-vein anastomosis segment, but this graft modification demonstrated no significant impact on graft patency52. Our study findings suggest that perhaps a fixed and optimized graft-to-vein anastomosis angle may be a novel solution to this ongoing issue. We envision these grafts would be manufactured at pre-set fixed optimized angles, and would be selected and implanted in patients relative to their native axillary vein diameters as a strategy to improve graft primary and secondary patency rates.The graft-to-vein anastomosis angle at which arteriovenous grafts are surgically implanted are entirely up to the discretion of the operating surgeon53. Left unregulated, hypertension would be a consideration for the optimization of anastomosis angle as higher or lower flow rates could shift Fig. 53. Although the size of the vasculature relevant to arteriovenous grafts can vary from patient to patient, the pathologically high and low flow fields were predicted to be localized in all cases to the anastomosis. Because these fields are dominated by the local geometry of the anastomosis rather than by geometry of the vessels, the absolute sizes of the vessels would not be expected to affect the optimization. Lastly, our simulations included blood vessel models that are static. Improving the model to take into account dynamic behaviors of peripheral arteries and veins would further refine our future study findings.We acknowledge there are some limitations associated with our study. While it is a first step in understanding the behavior this fluid system, our simulations were not able to entirely account for all physiological variables that may impact arteriovenous graft flow. Some limitations of this study are that blood flow parameters were held within the physiological range for all simulations, and vein diameters were not varied. Hypertension is a common co-morbidity of diabetesThe graft-to-vein anastomosis angle of brachio-axilllary arteriovenous grafts affects pathological flow fields that arise within the adjacent vein. Optimizing the graft-to-vein anastomosis angle can enhance these flow fields, reduce the incidence of pathological shear strain rates, and may ultimately improve graft patency rates among hemodialysis patients.Supplementary Information.


Humans can regulate ankle moment and stiffness to cope with various surfaces during walking, while the effect of surfaces compliance on ankle moment and stiffness regulations remains unclear. In order to find the underlying mechanism, ten healthy subjects were recruited to walk across surfaces with different levels of compliance. Electromyography (EMG), ground reaction forces (GRFs), and three-dimensional reflective marker trajectories were recorded synchronously. Ankle moment and stiffness were estimated using an EMG-driven musculoskeletal model. Our results showed that the compliance of surfaces can affect both ankle moment and stiffness regulations during walking. When the compliance of surfaces increased, the ankle moment increased to prevent lower limb collapse and the ankle stiffness increased to maintain stability during the mid-stance phase of gait. Our work improved the understanding of gait biomechanics and might be instructive to sports surface design and passive multibody model development. The ankle joint plays several important roles such as shock absorption, stability, and propulsion in different subphases of the gait cycle, which can be realized by the regulations of ankle biomechanics . When enIn order to learn about the ankle moment and stiffness regulations, several methods have been developed to estimate ankle moment and stiffness. Ankle moment can be estimated from muscle forces and their associated moment arms . An invePrevious studies demonstrated that ankle moment and stiffness were regulated according to the subphase of the gait cycle and walking environments. During the early stance phase, the ankle dorsiflexion moment is generated to provide preparation for weight acceptance. During the mid- and late stance phases, the ankle plantar flexion moment generated contributes to support and forward progression . The ankAlthough many factors can affect the ankle moment and stiffness, it remains unclear how humans regulate them while walking on surfaces with different levels of compliance. Our study aimed to determine how humans regulate ankle moment and stiffness while walking on surfaces with different levels of compliance. As each subphase of gait has distinct biomechanical demands, we hypothesized that the compliance of surfaces may affect ankle moment and stiffness regulations in different subphases of a gait cycle. An EMG-driven musculoskeletal model was applied to estimate ankle moment and stiffness while walking on surfaces with different levels of compliance.Three materials were selected to form three surfaces for subjects to walk on. The materials selected were rubber, ethylene–vinyl acetate copolymer (EVA), and expandable polyethylene (EPE). The surface of the force plate made up of aluminum was another surface for subjects to walk on and its elastic modulus was about 70,000 MPa. A universal material testing machine was used to obtain the force deformation data of the samples of the materials. The thickness of the samples was 5 cm. Then the elastic modulus of materials was obtained from the relationship of stress and strain (0–300 kPa). The relationship of stress and strain is shown in f  without lower extremity injury participated in the experiment. All subjects signed the informed consent form before participating in the experiment. This study was approved by the School of Medicine, Sun Yat-sen University Institutional Review Board, on March 1, 2021.Subjects were required to walk barefoot during the experiment. Before starting walking trials on one surface, subjects were required to walk on this surface to adapt to it. One static pose trial was performed before walking trials. Then subjects were required to perform four walking trials on each surface. In walking trials, subjects were required to walk across a walkway of a length of 4.5 m in 4–5 s. The subjects were allowed to take a 1-min rest between each pair of successive walking trials and a 15-min rest when the surfaces for walking trials needed to be changed. One subject was recruited for the pilot study, and the ankle stiffness estimates were obtained using an EMG-driven musculoskeletal model. Effect sizes [Cohen’s f ] of the http://www.seniam.org/) GRF data were recorded at 1,500 Hz using a force plate . Each subject had 30 retroreflective markers placed on their body during the experiment. The retroreflective markers were placed on the trunk, pelvis, and left and right extremities. The placements of retroreflective markers are shown in Recordings of walking trials included the whole stance phase of the subjects’ right leg. EMG data were collected from four ankle muscles: tibialis anterior (TA), soleus (SOL), gastrocnemius lateralis (GAL), and gastrocnemius medialis (GAM). EMG data were recorded at 1,500 Hz using a telemetered EMG system . Electrodes were placed on these muscles using surface EMG for non-invasive assessment of muscles (SENIAM) guidelines. , full-wave–rectified, and low-pass–filtered (6 Hz) using a zero-phase second-order Butterworth filter. For each subject and muscle, the resulting EMG linear envelopes were normalized to the maximum processed values obtained from all recorded trials. The processed and normalized EMG signals would be referred to as excitations. Muscle excitations were subsequently processed using a recursive filter to model the twitch response of the muscle fibers to the excitation onset. The filter used was given by A is the non-linear shape factor.Then a non-linear transfer function was used to account for the non-linearity in the excitation-to-force relationship and obtain the resulting muscle activation :a(t)=eAvia the inverse kinematics (IK) tool. Ground reaction forces (GRFs) and the results of IK were then used to calculate ankle moment via the inverse dynamics (ID) tool. Ankle moment obtained by this way would be referred to as experimental ankle moment.In the inverse kinematics and dynamics block, marker positions recorded from the static pose trials were used to scale a generic model of the human musculoskeletal geometry to match each subject’s anthropometry in OpenSim. Joint angles were calculated using marker trajectories from walking trials The MTU kinematics block received joint angles from the inverse kinematics tool in OpenSim. The MTU lengths and moment arms derived from the scaled model in OpenSim were used to create polynomial fitting functions. These functions described how each MTU length and moment arm change with respect to joint angles . With thThe MTU dynamics block took muscle activation and MTU lengths from previous blocks as input. A hill-type muscle model was used to estimate the instantaneous muscle fiber length and force and series elastic tendon strain and force for each MTU :Fm=Fmaxtionship  and forctionship , respecttionship ; lt isMuscle fiber stiffness The MTU stiffness could be modeled as the muscle fiber stiffness ith MTU, and ith MTU.The joint dynamics block computed ankle moment and stiffness. Ankle moments were calculated as the product of each MTU force and their associated moment arms, as follows:ith MTU, ith MTU, ith MTU.Using the estimated muscle forces and the MTU stiffness, the corresponding ankle stiffness  follows :Kankle=via ID in the inverse kinematics and dynamics block. The optimization formulation is listed as follows:N represented the length of the data used for the calibration. The Nelder–Mead algorithm was used to minimize the objective function C1 and C2 in the muscle activation block, which varied between −1 and 1; A in the muscle activation block, which varied between −3 and 0; The calibration block determined subject-specific parameters for the EMG-driven musculoskeletal model. Some parameters were adjusted through the calibration process within moderate bounds so that joint moments calculated from MTU forces and moment arms in joint dynamics block could be closer to the joint moments calculated via the EMG-driven musculoskeletal model. Similarity between Walking speeds were calculated from the marker placed on the seventh cervical vertebra of subjects. The ankle moment and stiffness were estimated from the recorded GRFs, EMG, and marker trajectories https://spm1d.org/.The presence of significant differences among surfaces in ankle moment, ankle stiffness, GRFs, and muscle excitations was assessed with 1D statistical parametric mapping (SPM). SPM represented the convergence of change distribution analysis and significance probability mapping . One-wayThe mean (standard deviation) speeds of walking trials on force plate, rubber, EVA, and EPE surfaces were 0.90 (0.06), 0.90 (0.05), 0.88 (0.05), and 0.89 (0.05) m/s, respectively. The mean (standard deviation) NRMSE of ten subjects showing similarity between via ID in OpenSim are shown in p < 0.001, effect size f: 0.27–0.48). During the late stance phase, almost no significant difference existed in ankle moments. During the whole stance phase, ankle moments remained almost the same while walking on the force plate and rubber surfaces.The ankle moments calculated p < 0.001, effect size f: 0.22–0.28), while this trend reversed during the 76–85%  and 94–100%  of stance phase. Similar to the pattern of ankle moment, the ankle stiffness on the force plate and rubber surface remained almost the same during the whole stance phase.Results of the ankle stiffness are shown in p < 0.001, effect size f: 0.26–0.34). During the 60–91% of stance phase, vertical GRF decreased as compliance of surfaces increased within the force plate, EVA, and EPE surfaces . As for anterior-posterior GRF, significant difference existed during the 15–25%  and 64–69%  of stance phase. Anterior-posterior GRF on the EPE surface was larger than GRF on the other three surfaces during the 15–25% of stance phase. No obvious trend occurred in anterior–posterior GRF among surfaces during the 64–69% of stance phase. GRFs in both directions remained almost the same on the force plate and rubber surfaces during the stance phase. Excitations of SOL and GAL on EPE surface were significantly larger than other surfaces during the early stance phase . Peak excitations of SOL and GAM tended to decrease as compliance of walking surfaces increased within the force plate, EVA, and EPE surfaces, although no significant difference existed .Results of GRFs and muscle excitations are shown in via an EMG-driven musculoskeletal model. Two main findings in our results were that 1) surface compliance affected the regulations of the ankle moment and stiffness and 2) the effect of the surfaces compliance on ankle moment and stiffness regulations varied in different subphases.In this study, subjects were required to walk on surfaces with four levels of compliance. Time-varying ankle moment and stiffness were estimated from GRFs, EMG, and three-dimensional reflective marker trajectories during the whole stance phase Surface compliance had different effects on ankle moment regulations during the early and mid-stance phases. During the early stance phase, the ankle dorsiflexion moment is a preparation for weight acceptance and provides a deceleration of the foot when landing . ReducedAnkle stiffness regulations in response to surface compliance were different between the mid- and late stance phases. During the mid-stance phase, the ankle stiffness increased with the increase in surfaces compliance, which was consistent with the leg stiffness regulations while running on compliant surfaces . It has Previous studies have shown that alterations in ankle muscle excitations can change ankle stiffness . Our resThe elastic modulus of materials reported in the Materials and Methods was obtained from the relationship of stress and strain (0–300 kPa), and this value was comparable between the EVA and EPE. It should be noticed that these two materials had a non-linear mechanical behavior. If we took only the part of the relationship of stress and strain into account, where stress is below 30 kPa, the obtained elastic modulus (0–30 kPa) of the EVA was more than twice of the EPE . Thus, the compliance of EVA and EPE was quite different during the initial contact with surfaces, probably leading to the difference in ankle moment and stiffness between two surfaces.f of differences in the ankle moment and stiffness were considered medium in this study (Alterations in the ankle moment and stiffness were found in the rubber, EVA, and EPE surfaces, and it is notable that both ankle moments and ankle stiffness remained almost the same while walking on the force plate and rubber surfaces. This might be associated with the mechanical properties of human tissues. The elastic modulus of plantar tissue is about 0.7 MPa , while tis study , which iis study . Only foThe ankle joint plays a key role in adjusting leg mechanics to adapt to alterations in surface properties . Our finOur study provides insights into how humans regulate ankle moment and stiffness during the whole stance phase while walking on surfaces with different levels of compliance. Surfaces with higher levels of compliance increased the ankle plantar flexion moment and stiffness during the mid-stance phase, while decreased the ankle stiffness during the late stance phase. The ankle moment and stiffness regulations in response to surface compliance primarily helped to prevent lower limb collapse and improve stability on surfaces with different compliance. Our work gave a comprehensive understanding about the regulations of ankle biomechanics including ankle moment and ankle stiffness and might be instructive to sports surfaces design and passive multibody model development.


Using whole genome sequencing, we identified three different rgv1 mutants that are allelic to the previously reported early starvation 1 mutant, which is rapidly depleted of starch just before the dawn. ESV1 orthologs are present in starch-producing green organisms, suggesting ESV1 is a functionally conserved protein necessary for the formation of starch granules. Consistent with this, we found that liverwort and rice ESV1 can complement the Arabidopsis ESV1 mutant phenotype for both starch granules and hypocotyl negative gravitropism. To further investigate the function of ESV1 in other plants, we isolated rice ESV1 mutants and found that they show reduced levels of starch in their leaves and loosely packed starch granules in their grains. Both Arabidopsis and rice ESV1 mutants also lack starch granules in root columella and show reduced root gravitropism. Together, these results indicate ESV1 is a functionally conserved protein that promotes gravitropic responses in plants via its role in starch granule formation.Starch granules in the endodermis of plant hypocotyls act as statoliths that promote hypocotyl negative gravitropism—the directional growth of hypocotyls against gravity—in the dark. To identify the molecular components that regulate hypocotyl negative gravitropism, we performed a mutagenesis screen and isolated  We then further characterized rgv1, allelic to ESV1, mutants, which lack starch granules both in the endodermis and the root columella in the dark. By analyzing Arabidopsis and rice ESV1 mutants as well as transgenic plants expressing ESV1 from liverwort, Arabidopsis, and rice, we have identified ESV1 as a functionally conserved protein that promotes gravitropism in land plants via its role in the formation of starch granules.In a mutagenesis screen with PIF1 is sufficient to rescue the endodermal starch granule and hypocotyl negative gravitropism phenotypes of pifQ mutants in the dark . From this screen, we isolated 30 reduced gravitropic (rgv) mutants that show reduced hypocotyl negative gravitropism in the dark. We classified these 30 rgv mutants into three groups based on the status of their endodermal starch granules when stained by Lugol's iodine: a starchless group lacking starch granules, a pifQ-like group with reduced starch granules, and a WT-like group with large starch granules . Both rgv1 and rgv2 mutants lack starch granules not only in the endodermis but also in the root columella, whereas the rgv3 mutant lacks starch granules in the endodermis but has weakly stained granules in the root columella  mutants, like rgv1 and rgv2, lack starch granules in both the hypocotyl endodermis and the root columella. The isoamylase 1 (isa1) and isoamylase 2 (isa2) mutants, like rgv3, lack starch granules in the hypocotyl endodermis but do show stainable granules in the root columella  carry non-sense mutations in AT1G42430, which is previously reported as EARLY STARVATION 1 (ESV1)  in this gene. We found that this T-DNA insertion mutant lacks starch granules in both the endodermis and root columella (ESV1 under the 35S promoter in the ESV1 mutant background (ESV1-OX/ESV1-2) rescues both endodermal starch granules and hypocotyl negative gravitropism (rgv1 mutant is caused by mutations in the ESV1 gene (AT1G42430). It is noticeable, however, that the ESV1 mutant still produces high levels of starch granules in hypocotyl cortex and cotyledons at the end of long day (ZT16), which is different from the pgm mutant that does not produce any starch granule and the phosphoglucoisomerase (pgi) mutant that produces high levels of starch granules in the hypocotyl endodermis but strongly reduced levels in cotyledons at ZT16  that is 44% identical at the amino acid level to ESV1. Unlike ESV1 mutants, however, mutations in LESV do not disrupt endodermal starch granules (LESV under the 35S promoter in the ESV1 mutant background (LESV-OX/ESV1-2) does not rescue the ESV1 mutant endodermal starch granule or hypocotyl negative gravitropism phenotypes (ESV1 and LESV do not have the same function.To identify the gene responsible for the 1 (ESV1)  (Feike e1 (ESV1) . Thus, wolumella  and showolumella . Furtheritropism . These r at ZT16 . At 4 h granules , neithergranules . The ectenotypes , indicatESV1 is reported to be localized in plastids and prevents the hasty depletion of starch reserves during the night  and transgenic lines expressing ESV1 under the control of the epidermis-specific MERISTEM LAYER 1 (ML1) promoter in the same ESV1-2 mutant background (ML1p:ESV1/ESV1-2) , we only observed starch granule formation in the upper hypocotyl endodermis , a PIF direct target gene used as a marker  and rice (Oryza sativa) but not in those of cyanobacteria that do not produce starch. To investigate the relationship between ESV1's conservation and the formation of starch granules, we generated transgenic Arabidopsis seedlings expressing either liverwort ESV1 , 74% amino acid identity to At-ESV1) or rice ESV1 , 88% amino acid identity to At-ESV1) under the control of the endodermis-specific SCR promoter in the ESV1 mutant background . If the ESV1 proteins from these species are functionally conserved, these transgenic seedlings should produce endodermal starch granules. We found that both Mp-ESV1 and Os-ESV1 fully rescue the production of endodermal starch granules  in two different rice cultivars  and ADP-glucose pyrophosphorylase large subunit 2 (osapl2) mutants, which produce shrunken grains due to a lack of starch biosynthesis in the endosperm  mutants, which are male sterile because of a lack of starch reserves in their pollen grains   mutants . Of these, rgv2 and rgv3 are allelic to the pgm and isa1 mutants, respectively   . ESV1 enormation . ESV1 ho1 mutant . This sugranules , and thegranules . This logranules . TogetheESV1 homologs use starch-filled amyloplasts as gravity sensors. Genome sequence information indicates that ESV1 homologs are present not only in starch-producing land plants including liverwort, rice, and Arabidopsis, but also in starch-producing unicellular green algae including Chlamydomonas and starch-producing multicellular green algae including Chara. Land plants sense gravity using starch-filled amyloplasts as gravity sensors and bend toward or against the direction of gravity, displaying either positive or negative gravitropism . Previous studies found that two Arabidopsis GPT genes  are expressed in all organs including cotyledons, hypocotyls, and roots  were grown in a growth room with a 16-h light/8-h dark cycle at 22–24°C for general growth and seed harvesting. T-DNA insertion mutants for Arabidopsis ESV1 and LESV were obtained from the Arabidopsis stock center and homozygous mutants were selected by genotyping. The corresponding stock numbers are as follows: ESV1-2 (GABI_031C11), lesv-2 , lesv-3 , aps1 , isa1 , isa2 . The phot1 phot2 mutant was obtained from Eiji Gotoh at Kyushu University and the pgm-1 mutant from Samuel Zeeman at ETH Zurich.Arabidopsis plants  and introduced into Arabidopsis . At least two independent transgenic lines were generated. Synthesized ESV1 genes from Marchantia polymorpha (Mapoly0189s0007) and Oryza sativa (LOC_Os03g04100) were also used to generate transgenic lines . To visualize endodermal plastids, a DNA fragment corresponding to the Rubisco small subunit (RBCS1A) transit peptide was cloned into a binary vector containing the mCitrine gene under the control of the SCR promoter as previously reported  were grown in a greenhouse with a 14-h light/10-h dark cycle (30/20°C). T-DNA insertion mutants of rice ESV1 were identified from the rice stock center  and osESV1-2 (PFG_1A-07022). Their parental lines are Kitaake and Dongjin, respectively. The primers used for cloning are described in Rice plants (PIF1 under the control of the endodermis-specific SCR promoter (SCRp:PIF1/pifQ) were mutagenized with EMS. M2 seedlings that did not grow upward in the dark were selected and mutants displaying stable phenotypes over generations were selected as reduced gravitropic (rgv) mutants. For complementation grouping, seven starchless rgv mutants were each crossed with one another and the endodermal starch granules of the F1 seedlings were examined with Lugol's iodine. To determine if the starchless mutants are allelic to known starchless biosynthetic mutants, rgv1-1, rgv2-1, and rgv3-1, representing three complementation groups, were crossed with pgm, aps1, isa1, and isa2 mutants and the endodermal starch granules of the resulting F1 seedlings were examined with Lugol's iodine. Simultaneously, the genomic regions of PGM, APS1, and ISA1 were amplified and sequenced from the seven starchless mutants. To identify the mutated loci in the three rgv1 mutant alleles , each mutant was backcrossed to the parental line three times and then used for whole genome sequencing analysis. The whole-genome sequencing was performed by Illumina NextSeq and the results were mapped to the Arabidopsis reference genome . High quality homozygous single nucleotide polymorphisms (SNPs) caused by G to A or C to T substitutions were identified and, by comparing the SNPs in the three rgv1 alleles with the parental line, SNPs were identified in AT1G42430 in all three rgv1 alleles but not in the parental line. To further support the hypothesis that rgv1 mutants are caused by mutations in this gene, a T-DNA insertion line of AT1G42430 was obtained from the Arabidopsis stock center. After confirming that the rgv1 mutants were caused by mutations in AT1G42430, the rgv1 mutant was renamed to the ESV1 mutant.A total of 10,000 seeds from an Arabidopsis transgenic line expressing 2-KI solution  for 1 min. Seedlings were de-stained with a clearing solution (Trichloroacetic acid: phenol: lactate = 2: 1: 1) for 1 min and mounted on slide glass with fresh clearing solution for microscopy. For rice, samples were fixed in 0.05 M sodium-phosphate buffer (pH 7.4) containing 2% paraformaldehyde for 4 h at 4°C. After fixation, the tissues were washed overnight in phosphate-buffered saline  containing 6.8% sucrose, dehydrated in an ethanol gradient series, and embedded in Technovit 8100 resin . Tissues were then sliced into 10 μm sections with a rotary microtome  and stained with 10% diluted I2-KI solution  for 1 min.Lugol's iodine staining was used to visualize starch granules in seedlings. Arabidopsis seedlings were fixed in FAA solution  for 24 h at 4°C. After fixation, the seedlings were washed with 50% ethanol once, and stained with I2s) for 6 h at 22°C to induce germination. Germination-induced seeds were then incubated for 3 days in the dark or in red light (20 μmol/m2s) at 22°C. Seedlings were counted as standing if their cotyledons did not touch the agar surface. Total 40 of seedlings were used for each experiment . Phototropic seedlings were similarly determined after growing them on horizontal plates under blue light (1 μmol/m2s) for 4 days. To determine Arabidopsis root gravitropic responses, seedlings were grown for 5 days on vertical plates under white light (50 μmol/m2s). Then, they were grown for an additional 12 h after rotating the plates by 90 degrees. The angles formed by the bent root were measured and supplementary angles were used to indicate the degree of root gravitropism. To determine rice root gravitropic responses, surface-sterilized seeds were germinated on half MS media  in Magenta vessels  for 7 days in the dark at 25°C until the shoots and roots established a vertical extension of seedling growth. These were then further incubated for 1 day after rotating the vessels by 90 degrees.Hypocotyl negative gravitropism was determined by growing seedlings for 3 days on horizontal plates in different light conditions and counting the number of standing seedlings. For the assay, surface-sterilized seeds were plated on half Murashige and Skoog (MS) media  and stratified for 3 days in the dark at 4°C. After the stratification, the seeds were incubated under white light . After two more washes with PBS buffer, the seedlings were mounted on a slide glass with anti-fade mounting solution (VECTASHIELD Antifade Mounting Medium H-1000). Fluorescence images were taken with a confocal microscope . The relative position of each plastid was calculated by assigning the initial bottom of a cell to 0 and the top of the cell to 100.Seedlings were grown on vertical plates in the dark for 3 days before being inverted. For the 2s) for 3 days and whole seedlings were harvested for RNA or protein expression analysis. For mRNA expression analysis, total RNAs were extracted using the Spectrum plant total RNA kit (Sigma-Aldrich) and converted to cDNA using MMLV-RTase (Promega). Relative transcript levels were determined by quantitative PCR  and normalized to the level of PP2A. All experiments were performed with biological triplicates.After the induction of seed germination, seedlings were grown on half MS media with 1% sucrose either in the dark or in red light , and protease inhibitor cocktail). After removing the debris by centrifugation, the supernatant protein extract was mixed with 5X SDS sample buffer  and boiled at 100°C for 5 min. Proteins were then detected by immunoblot analysis using an anti-GFP antibody for ESV1-GFP and an anti-α-tubulin antibody for α-tubulin. For the quantitative analysis, the intensity of ESV1-GFP was normalized by α-tubulin .For protein expression analysis, harvested seedlings were ground in liquid nitrogen and further homogenized in a denaturing buffer .Approximately 100 mg of leaves were sampled from 6-week-old rice plants grown under a 14/10 light cycle in a greenhouse. Each sample was harvested at zeitgeber (ZT) 0, 8, 16, 18, 20, and 22. Glucose, fructose, sucrose, and starch were measured in the soluble and insoluble fractions of ethanol-water extracts using enzymatic methods  analysis were carried out with Carl Zeiss MERLIN™ FE-SEM operated at low accelerating voltage of 1 kV. The fractured surface morphologies of wild type and mutant rice grains were observed without any coating materials.APS1: AT5G48300, GWD: AT1G10760, IAA19: AT3G15540, ISA1: AT2G39930, ISA2: AT1G03310, ML1: AT4G21750, PGM: AT5G51820, PHOT1: AT3G45780, PHOT2: AT5G58140, PHYA: AT1G09570, PHYB: AT2G18790, PIF1: AT2G20180, PIF3: AT1G09530, PIF4: AT2G43010, PIF5: AT3G59060, PIL1: AT2G46970, PP2A: AT1G13320, RBCS1A: AT1G67090, ESV1: AT1G42430, LESV: AT3G55760, and SCR: AT3G54220.ESV1: LOC_Os03g04100, APS1: LOC_Os09g12660, GWD: LOC_Os06g30310, APS2: LOC_Os08g25734, APL2: LOC_Os01g44220, PGM: LOC_Os10g11140, APL4: LOC_Os07g13980.Os-ESV1: Mapoly0189s0007.Mp-The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.KS, D-WL, JeK, J-SJ, and GC designed the experiments. KS, D-WL, JeK, JaK, HG, and KK performed the experiments. KS, D-WL, J-SJ, and GC wrote the paper. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


CD4+ memory T-helper cells from a previous immunizing event can potentially be reactivated by exposure to HLA mismatches that share T-cell epitopes with the initial immunizing HLA. Consequently, reactivity of CD4+ memory T-helper cells toward T-cell epitopes that are shared between immunizing HLA and donor HLA could increase the risk of alloimmunity following transplantation, thus affecting transplant outcome. In this study, the amount of T-cell epitopes shared between immunizing and donor HLA was used as a surrogate marker to evaluate the effect of donor-reactive CD4+ memory T-helper cells on the 10-year risk of death-censored kidney graft failure in 190 donor/recipient combinations using the PIRCHE-II algorithm. The T-cell epitopes of the initial theoretical immunizing HLA and the donor HLA were estimated and the number of shared PIRCHE-II epitopes was calculated. We show that the natural logarithm-transformed PIRCHE-II overlap score, or Shared T-cell EPitopes (STEP) score, significantly associates with the 10-year risk of death-censored kidney graft failure, suggesting that the presence of pre-transplant donor-reactive CD4+ memory T-helper cells might be a strong indicator for the risk of graft failure following kidney transplantation.CD4 Kidney transplantation is the preferred treatment option for many patients with end-stage kidney disease. The outcome of such a treatment is most optimal when recipient and donor are human leukocyte antigen (HLA) matched , 2. ThroThe degree of HLA matching at the antigen level has been observed to be associated with graft survival . However+ T-helper cells are of significant importance during allograft rejection. During T-cell-mediated rejection, these cells are likely able to stimulate intermediary antigen-presenting cells, which can consequently prime CD8+ cytotoxic T-cells  and the mismatched HLA molecules present in the new transplant (designated as donor HLA). Consequently, the presence of T-cell epitopes that are shared between immunizing and donor HLA could potentially increase the risk of alloimmune reactions following transplantation. These shared T-cell epitopes may explain why recipients with pre-transplant non-donor-specific HLA antibodies experience have a decreased graft survival  assay  according to the manufacturer’s instructions . Raw meaviawww.pirche.com). HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1/DQB1, and -DPA1/DP1 were taken into consideration as presented loci, and HLA-DRB1 was considered as presenting locus. As only HLA typing data at serological level were available in our cohort, the PIRCHE-II peptides and their weights were calculated based on serological typing data as described previously  and the PIRCHE-II peptides originating from the donor’s HLA alleles serving as potential target for the recipient  were identified using the PIRCHE-II algorithm version 3.3.30  with the PIRCHE-II peptides originating from the donor’s HLA typing . First, duplicate PIRCHE-II peptides originating from the donor’s HLA presented by the same HLA-DRB1 molecule were merged and their weights were summed. Subsequently, for each recipient, the PIRCHE-II 9-meric peptides – representing the core peptide presented by HLA class II  – presenhttps://www.pirche.com/pirche/#/pirche/matchmaker/multi/matching/request). The HLA typing of the donor and the HLA typing of the recipient with the non-extrapolated HLA-DRB1 alleles was used as input for the algorithm. HLAMatchmaker scores were determined by calculating the sum of all potential B-cell epitopes mismatches.To investigate the impact of B-cell epitopes on the clinical outcome, B-cell epitope mismatches for HLA-A, -B, -C, -DRB1, and -DQB1 were determined for each donor/recipient combination as described before using the HLAMatchmaker algorithm  . The above-described four approaches to positively select the theoretical immunizing HLA alleles were evaluated by determining the area under the curve  for each method. In addition, the median ln-transformed PIRCHE-II overlap scores (ln(PIRCHE-II overlap score + 1) – or Shared T-cell EPitopes (STEP) scores – of recipients who did and recipients who did not experience graft failure during the 10-year follow-up were compared using Mann-Whitney U tests.The effect of recipient age, recipient sex, donor age, donor sex, year of transplantation, historic peak panel reactive antibodies (hPRA), repeat transplant, type of donation [donation after brain death (DBD)/donation after circulatory death (DCD)], cold ischemic period, number of HLA-A/B/DR mismatches, HLAMatchmaker score, ln(PIRCHE-II score + 1), and ln(PIRCHE-II overlap score + 1) on the 10-year risk of death-censored kidney graft failure were studied using an univariate Cox proportional hazards analysis . In addition, a multivariable Cox proportional hazards analysis with all univariately studied variables was performed using a stepwise variable selection method alternating between forward and backward selection (‘My.stepwise’ package version 0.1.0). Significance Levels for Entry (SLE) and Stay (SLS) were set to p = 0.15. For both univariate and multivariable Cox models, repeat transplant and type of donation were considered as categorical variables . All other variables were treated as continuous variables. The proportional hazards assumption was checked for all variables using the Schoenfeld residuals against the transformed time . When proportional hazards could not be assumed for a variable, a time-dependent covariate was constructed using the time-transforming functionality within the survival package in R.To study the cumulative death-censored graft failure incidence among recipients with a low and high STEP score, the study cohort was separated in two groups. The optimal cut-off value for division was determined by maximizing the Youden Index (J = sensitivity + specificity - 1), using a non-parametric approach based on kernel smoothing (‘cutpointr’ package version 1.1.1). Following division, the baseline characteristics for the two groups were compared using Mann Whitney U tests for continuous variables and chi-square tests for categorical variables. Kaplan Meier curves were constructed to depict the cumulative 10-year death-censored graft failure incidence between the two groups. When the survival graphs of the two groups crossed, an ABS permutation test   per PIRCHE-II were calculated at different virtual time points after transplantation, and among patients who did not experience graft failure before different time points. All covariates implemented in the initial multivariable model were considered when calculating these HRs. Death was considered a censoring event in the analyses. p values <0.05 were considered to be statistically significant.via the regular Eurotransplant Kidney Allocation System , cold ischemic period, number of HLA-A/B/DR mismatches, HLAMatchmaker score, and ln-transformed PIRCHE-II score. The STEP score was shown to be significantly associated with an increased 10-year risk of graft failure censored for death . The HLAMatchmaker score and the ln-converted PIRCHE-II score were associated with death-censored graft failure with an HR of 1.31 (p = 0.033) and an HR of 1.43 (p = 0.082), respectively. All other tested variables were not significantly associated with transplant outcome Table 2.To further investigate these findings and to correct for possible confounders, a multivariable Cox proportional hazard analysis was performed using a stepwise variable selection method. All variables that were univariately analyzed were included in the multivariable analysis as well. Only the STEP score was significantly associated in the multivariable model with death-censored graft failure with a hazard ratio of 1.48 , meaning that with a one-unit increase of the STEP score, the 10-year risk for kidney graft failure increases with 48%. Thus, the STEP score was observed to have a significant effect on the 10-year risk of death-censored kidney graft failure in the univariate as well as in the multivariable Cox proportional hazard analysis.To visualize the effect of shared PIRCHE-II epitopes on the cumulative 10-year risk of death-censored graft failure, the study population was divided into a group with a low and with a high STEP score. Based on the optimal Youden Index, a cut-off value of 0.21 was applied (J = 0.277). Comparison of the baseline characteristics of the two recipient groups showed that the group with a STEP score higher than 0.21 (n = 81) had a significantly higher number of HLA-A/B/DR mismatches (p < 0.001) and a higher HLAMatchmaker (p < 0.001) and ln-converted PIRCHE-II score (p < 0.001) than the group with a STEP score smaller than or equal to 0.21 (n = 109) Table 3.Next, we investigated whether the STEP score had an effect on early or late failure Figure 5To evaluate the influence of the PIRCHE-II overlap score on graft failure independent of the patients with early graft failure, the effect of the STEP score on death-censored kidney graft failure at different time points was calculated among patients who did not develop graft failure before those time points . After ade novo DSA formation as compared to patients without shared T-cell epitopes . Thus, these findings suggest that the difference in cumulative incidence of 10-year graft failure are mainly a consequence of the recall response and not only due to + memory T-helper cells. Our results indicate that the amount of potential T-cell epitopes shared between immunizing HLA and donor HLA might have an effect on graft failure mainly in first year and around 6 years after transplantation, with the biggest effect being present shortly after transplantation , University Medical Center Utrecht. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.Frontiers in Immunology. EP, BM, TT, MN, JD, TK, AZ, KG, and ES were involved in the design of the work and interpretation of the data. IJ, WA, AM, LH, MCB, FR, MV, EK, MS, JS, BH, AL, LB, CR, MT, CV, LW, ED, MG, MC, FI, AN, NL, WS, KP, NW, IB, FB, AV, JF, MGHB, DR, FC, HO, SH, AZ, and ES were involved in the acquisition of the data. All authors were involved in drafting or revising the manuscript and approved the final version. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.All authors met the authorship criteria as described by This study was supported by research funding from the Dutch Kidney Foundation project code CP12.23 "Risk assessment of kidney graft failure by HLA antibody profiling" and project code CP1801 “Prediction of Kidney Graft Survival by Immune Profiling - Towards Clinical Application in Personalized Medicine”, by research funding from EU Horizon 2020 for the ULISES project, code 899708, and by research funding from the International HLA and Immunogenetics Workshop Foundation.The authors of this manuscript have conflicts of interest to disclose. The UMC Utrecht has filed a patent application on the prediction of an alloimmune response against mismatched HLA. ES is listed as inventor on this patent. MN is employed by PIRCHE AG, which publishes the PIRCHE web-portal.The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The results reveal that the DRX behavior of 47Zr-45Ti-5Al-3V alloy strongly depends on hot-working parameters. With rising deformation temperature (T) and decreasing strain rate  behavior of 47Zr-45Ti-5Al-3V alloy was studied by using the experiment and numerical simulation method based on DEFORM-3D software and cellular automata (CA) over a range of deformation temperatures (850 to 1050 °C) and strain rates (10 Zirconium alloys owns low thermal neutron absorption cross-section, superior mechanical properties for long term operations in high pressure and adequate corrosion resistance in contact with high-temperature water, which plays a significant role in structural materials of the aerospace field ,2,3. Witγ′-L12 alloy by the FEM methods, which was discontinuous dynamic recrystallization (DDRX). Geng et al. [Recently, with the rapid increase in computer performance, the DRX kinetics of metals during hot-working has been qualitatively studied by using the software of FEM and CA ,22,23,24u et al.  utilizedu et al.  revealedg et al.  also repg et al.  studied In order to reveal the DRX behavior and construct the DRX kinetics model of 47Zr-45Ti-5Al-3V alloy, in this present work, the CA–FE method is used to study the DRX behavior of 47Zr-45Ti-5Al-3V alloy from the macro- and micro-scales. A developed kinetics model of the DRX for 47Zr-45Ti-5Al-3V alloy was established. The microstructure evolution of the alloy in the process of DRX was analyzed by DEFORM-3D software and CA integrated with the developed kinetics and dislocation models. Additionally, a comparison of the difference between the experimental and simulated results has been carried out to testify the validity of FE–CA simulation.A forged 47Zr-45Ti-5Al-3V alloy (wt.%) was employed in this work. The specific preparation process has been introduced in the previous work . The pha−3 to 100 s−1. The schematic diagram of the experimental tests is shown in Cylindrical samples of ø8 × 12 mm were prepared by the wire cutting. To analyze the DRX behavior and construct the DRX kinetics model of 47Zr-45Ti-5Al-3V alloy, hot compression test was conducted on a Gleeble 3500 at various testing conditions. The deformation temperature was set in the range of 850 to 1050 °C, and the strain rate was set in the range of 103 and 45% H2O (Vol.%). The value of the For optical microstructure observation (OM) by using Leica DMI5000M, the deformed samples were ground and then etched by chemical means. The etching solution is a Kroll reagent of 15% HF, 40% HNO−1, the initial grains of the samples deformed at the temperatures of 850 and 900 °C were elongated perpendicular to the compression direction of deformed samples, indicating that only DRV occurred. When the samples deformed at 950 °C/1 s−1, a few fine grains of DRX formed at the grain boundaries of deformed grains, showing that dynamic recrystallization firstly took place under this deformation condition. As the deformation temperature increases and the −1, the microstructure with uniform equiaxed grains can be achieved, revealing that a full DRX occurred during hot-working. When the T further raised to 1050 °C, there were obvious coarsening for the DRX grains. It is attributed to a fact that high deformation temperature and low According to the flow curves obtained from hot-working, the peak strain ε˙=6.Hence, the relationship between the  formula ,26,27,28Generally, when the strain reaches a critical strain (nglement . The criress (σ) ,31. In tollowing :(3)εc=βεThe DRX behavior consisted of nucleation and growth during hot-working. For the discontinuous recrystallization mechanism, the nucleus of DRX grains generally formed in the grain boundaries, and then grew toward the interior of the grains with a high density nearby the grain boundaries. The equation ,33:(5)XD follows ,35:(6)XDDRX was expressed as follows:The Z parameter was characterized by the follow [The e follow :(9) dDRXZ parameter can be showed as follows:The variation on the Ⅰ, while only a slight deformation was observed in region Ⅱ. Usually, the region II is defined as the free deformation area. It is noted that non-deformation was presented in region Ⅲ, namely “dead zone” [Based on the DRX kinetic equation established above, the isothermal forging process was simulated by the DEFORM-3D software  During the process of isothermal forging simulation, the elastic deformation can be usually ignored. Therefore, the workpiece was treated as an object with plasticity, while the tools were treated as an object with rigidity. For improving the efficiency of FEM calculation and increasing the accuracy of the simulation results, the FE model was established by symmetrical half cylinder. The sample for FEM was segmented in terms of tetrahedral meshes. The numbers of the meshes and the nodes were set to 26,538 and 4726, respectively. The top die was set as movable, while the bottom die was fixed. The coefficient of the friction was set to be 0.3 . For thead zone” ,34. ThisT and high Ⅰ, while dynamic recrystallization cannot occur in region Ⅲ, where the size of grains keeps the original grain size. With the augment of T and the drop of −1, the size of DRX grains increased to approximately 128 μm. It is worth noting that the size of DRX grains in region Ⅲ is higher than that in region Ⅱ. T and high T and the drop of Ⅰ is higher than that in regions Ⅱ and Ⅲ.n of DRX ,38,39,40ferences ,40, the b represents the Burger’s vector; 1k and 2k represent work-hardening and dynamic-softening coefficients, respectively. There is no i and j directions; 0N represents the total number of cells; and i, j). During the hot-working process, as the dislocation density reaches the critical dislocation density  follows :(14)ρ¯=1o the ε˙ ,42:(15)nBased on the initial microstructure of the solution-treated alloy, the initial solid solution structure was simulated by the CA simulation, as shown in (1)T and T and decreasing In this present study, the main softening mechanism of 47Zr-45Ti-5Al-3V alloy was regarded as DRX. The results revealed that the deformation (2)Based on the hot-working test, the (3)The value of (4)The ∆ value of the The DRX behavior of the 47Zr-45Ti-5Al-3V alloy was investigated by using a CA–FE simulation. The main conclusion obtained can be drawn as follows:


Antibody immunodominance refers to the preferential and asymmetric elicitation of antibodies against specific epitopes on a complex protein antigen. Traditional vaccination approaches for rapidly evolving pathogens have had limited success in part because of this phenomenon, as elicited antibodies preferentially target highly variable regions of antigens, and thus do not confer long lasting protection. While antibodies targeting functionally conserved epitopes have the potential to be broadly protective, they often make up a minority of the overall repertoire. Here, we discuss recent protein engineering strategies used to favorably alter patterns of immunodominance, and selectively focus antibody responses toward broadly protective epitopes in the pursuit of next-generation vaccines for rapidly evolving pathogens. Viral antigens like influenza hemagglutinin (HA) or HIV envelope protein (Env), have conserved structural or functional regions. Abs targeting these epitopes are often broadly neutralizing (bnAbs) or protective (bpAbs), the latter through Fc-mediated effector functions3. However, such antibodies are generally immunologically subdominant, and make up a minority of the overall repertoire. Next-generation vaccines for rapidly evolving pathogens aim to alter patterns of dominance to elicit higher levels of broadly neutralizing or protective responses.Antibody (Ab) responses raised against complex protein antigens can preferentially target particular epitopes in a reproducible hierarchy, a phenomenon known as immunodominance. These primary targets of Ab responses are often immunologically dominant, while those engaged by minor portions of the overall response are considered immunologically subdominant. This asymmetry contributes to the host-pathogen ‘arms race’, where particular regions of surface-exposed antigens experience the most immune pressure, and are subsequently key sites of antigenic variation4. (1) Precursor frequency, the number of naïve B cells that engage a specific epitope, is a key limiting factor; if fewer B cells engage an epitope, the greater the likelihood that the subdominant-directed population will be outcompeted by more abundant B cell clones. This is a limiting factor for many bnAb precursors such as VRC01-class Abs targeting the HIV Env CD4 binding site (CD4bs), which are present at very low frequencies in human repertoires5. Precursor frequency may also be influenced by central tolerance as is the case for certain autoreactive HA-stem antibodies; negative selection attempts to remove these autoreactive B cells from the naïve repertoire7. The accessibility of a given epitope can also contribute, as an epitope must be accessible to BCRs in order to trigger an antibody response. (2) Precursor affinity for the antigen drives GC establishment or entry, as high affinity for antigen is linked with increased acquisition of antigen and increased density of surface pMHC8. The relationship between precursor frequency and affinity in GC B cells is nonlinear, but even when precursor frequencies are low, B cells can be recruited to GCs if they have sufficiently high affinity11. (3) The degree of T cell help during the GC reaction is a limiting factor on GC B cell proliferation and maturation13. Increasing the number of T follicular helper (Tfh) cells specific to epitopes on an immunogen may allow B cells into the GC that would otherwise not gain entry14. The modification of even a few helper T cell epitopes to relieve competition between B cell clones can have a marked impact on overall patterns of dominance15.While B cell immunodominance is an incompletely understood phenomenon, there are several key aspects influencing inter-clonal competition in the germinal center (GC) reaction that can be leveraged for rational immunogen design18. While immunodominance hierarchies for antigens such as HA and hepatitis C virus E2 have been experimentally mapped, the importance of epitope structure and how it might impact the trajectory of the affinity maturing B cell repertoire remains relatively undefined20.The structure of a B cell epitope as seen by the BCR likely also plays a role in immunodominance, possibly with subdominant epitopes requiring stringent or stereotyped contacts, but currently there is little experimental evidence directly addressing this topic. Computational analyses of antigen structures has focused primarily on identifying likely B cell epitopes, rather than establishing their relative immunodominanceIn this review, we discuss various protein engineering strategies used to develop immunogens against rapidly evolving pathogens, and how they influence these three rather well-characterized elements of B cell immunodominance to preferentially elicit antibodies to subdominant epitopes.The following sections focus on three general approaches of immunogen design. We discuss recent developments in strategies to (1) magnify the overall humoral response, (2) prevent or reduce the elicitation of ‘off-target’ antibody responses, and (3) specifically amplify responses targeting preferred epitopes. Discussion of these strategies focuses on influenza and HIV viral glycoproteins but extend to other viruses including respiratory syncytial virus (RSV), dengue, and Zika. Importantly, the strategies discussed here are not mutually exclusive, and many immunogens will likely influence immunodominance through multiple mechanisms.21. Historically, multimeric display was accomplished using virus-like particles (VLPs) derived from human, insect, or plant viruses such as hepatitis B, flock house virus, and tobacco mosaic virus28. This multivalent display mimics the natural presentation of viral epitopes, and can elicit protective responses32. However, many of these display platforms are limited to presenting relatively small, linear peptides, and cannot readily present complex conformationally specific epitopes26.The repetitive presentation of viral antigens, sensed by the degree of surface Ig-crosslinking, is a key factor to increasing the robustness of B cell responses33. Importantly, nanoparticle-immunized cohorts had more broadly reactive hemagglutinin inhibition (HAI), neutralization, and higher stem-directed titers, indicating that multimeric display can impact patterns of dominance in favor of cross reactive and subdominant responses34. More recently, the use of genetic fusion-based protein nanoparticle immunogens has extended beyond influenza HA; protein scaffolds displaying prefusion stabilized RSV F or HIV Env glycoproteins elicit robust neutralizing titers underscoring the utility of nanoparticle-based display as a general design strategy36. Next-generation protein-based nanoparticles involve multi-component constructs that allow for stoichiometrically precise display of multiple antigens42. Further computational and rational protein engineering led to the development of nanoparticles with tetrahedral, octahedral, and icosahedral symmetry; allowing for a greater degree of customizable antigen display based on the desired geometry45.In a key study, ferritin-based nanoparticles were engineered at the three-fold axis to display trimeric influenza HA antigens as genetic fusions. These immunogens elicited higher antigen-specific titers with increased breadth and protection relative to recombinant trimeric HAs47. Additionally, recombinantly expressed antigens can be enzymatically ‘clicked’ onto nanoparticle scaffolds  using the sortase transpeptidase from Staphylococcus aureus53. A more recent approach for conjugating antigen to nanoparticle scaffolds involves SpyTag-SpyCatcher54. This technology uses a split fibronectin-binding protein subdomain from Streptococcus pyogenes with a linear peptide tag appended on to the antigen and the remaining protein to the nanoparticle; when combined in vitro a spontaneous covalent linkage occurs via an isopeptide bond54. As a short peptide sequence (13 amino acids), SpyTag is readily appended to nearly any antigen of interest. This platform allows for an easily modifiable ‘plug and display’ approach, where heterologous display can be optimized to present a variety of antigens. Proof of concept studies have used diverse betacoronavirus receptor binding domains (RBDs) as well as influenza HAs58.Additional multivalent assemblies allow for display of antigens when genetic fusions are not possible or if mixed species are required. Lipid nanoparticles or synthetic liposomes, for example, can be engineered to present multiple antigens, resulting in similar amplification of serum titers relative to recombinant protein59.Despite the significant benefits of these multivalent display strategies, they are not without drawbacks. A caveat of protein-based nanoparticles and assembly domains like SpyCatcher-SpyTag is the lack of precise stoichiometric control of multiple antigens and their own innate immunogenicity. Their use introduces additional epitopes and expands the degree of interclonal competition, potentially skewing the immunodominance hierarchy away from subdominant epitopes of interest. Masking  of such ‘scaffold’ epitopes may be necessary to limit off-target reactivity. Furthermore, multivalent antigen display on a nanoparticle may result in steric occlusion of desired epitopes due to antigen spacing and overall density; this may ultimately restrict access of potential BCRs from engaging these epitopes leading to a reduction in response despite being presented in greater number than soluble wild-type antigen61. In addition to magnifying the overall humoral response, immunogen design approaches are leveraged to modulate responses to target different epitopes on a given antigen. The many epitopes present on a single immunogen result in inter-clonal competition between B cells targeting both conserved epitopes of interest, and those engaging ‘off-target’ epitopes. Decreasing the size of the competing B cell pool can influence patterns of immunodominance and is accomplished in two major ways: the outright removal or steric occlusion of undesired epitopes.Complex protein antigens elicit diverse GCs containing B cells that recognize a range of epitopes62. However, many viral proteins such as influenza HA, RSV F protein, or HIV Env can be truncated into domain-based constructs in order to minimize the inclusion of off-target epitopes while still maintaining the integrity of the target epitope72. Subsequent iterations of these constructs, such as the headless, stem-only domain ‘mini-HAs’, or the ‘engineered outer domain’ (eOD) Env constructs continue to expand the available repertoire of truncated immunogens74.The physical removal of undesired epitopes precludes the elicitation of responses against them, in effect modulating precursor frequency by reducing the overall number of competing B cells Fig. . Many co78. Recent refinement of this approach has both decreased the size of the shielding to a single chain variable fragment (scFv), or generated covalently stabilized complexes through chemical cross-linking or expression of an antigen-antibody genetic fusion81. Analogous to epitope removal, the steric shielding of off-target epitopes reduces the size of the precursor B cell pool able to bind the antigen, thus reducing the pool of potential competitors.Complementary to removing undesired epitopes is the steric occlusion of off-target ones, which can be done in a reversible or irreversible manner. The formation of immune complexes between antibodies and an antigen can mask epitopes; subsequent immunization with these immune complexes can sterically shield epitopes and thus bias antibody responses. This was shown for flavivirus, influenza, and HIV glycoproteins90. Overall glycosylation patterns can affect antigen processing, delivery into GCs, and breadth of elicited responses101. The variation in these natural glycosylation patterns in response to immune pressure provides a guide to introduce glycans on rationally designed immunogens103.A commonly used method to restrict off-target responses is to introduce novel predicted N-linked glycosylation (PNG) sites on viral surface proteins to “shield” or occlude undesired epitopes. Glycans are naturally present on viral envelope proteins and play key roles in stability, pathogenicity, and immunogenicity, as well as escape from immune surveillance107. For flavivirus-based immunogens , glycan shielding is critical to reduce the likelihood of antibody-dependent enhancement (ADE) mediated by cross-reactive yet non-neutralizing antibodies104. For influenza HA and HIV Env, the glycosylated immunogens focus responses to broadly cross reactive, yet relatively subdominant epitopes such as the HA head interface, the HA stem region, and the Env CD4bs107. This serum focusing is likely due to an increased proportion of the serum repertoire targeting the epitope of interest; hyperglycosylation generally does not impact overall serum titer, even for heavily glycosylated immunogens108. More recently, the combination of hyperglycosylation and covalent stabilization of HA trimers, where only the RBS epitope remains exposed, decreased overall serum titers without focusing to the subdominant RBS108. Thus, while hyperglycosylation can enrich antibody responses against desired epitopes, it is not always successful in altering immunodominance.Recent work has underscored the effectiveness of glycan masking to restrict off-target responses across a variety of viral glycoproteins including Zika and dengue E, hepatitis C E2, influenza HA, HIV Env, and RSV F proteinIn contrast to the occlusion or removal of off-target epitopes, alternative immunogen design strategies aim to expand responses towards desired epitopes. These approaches manipulate both precursor frequency and affinity, as well as epitope accessibility to preferentially target broadly conserved or protective epitopes of interest. General approaches involve the stabilization of preferred antigen conformations, glycan ‘unmasking’ of shielded epitopes, computationally inferred consensus antigens, germline targeting, and epitope resurfacing.110. Subsequent application of these principles for HIV Env led to the development of stabilized ‘SOSIP’ trimers through inter-subunit disulfides and conformation-locking isoleucine to proline mutations112. The initial successes with this stabilization approach has resulted in broadly applicable guidelines and even automated the design of prefusion stabilized Env antigens across viral clades115. These stabilized constructs have been instrumental in the study of HIV, and form the basis of many current Env immunogens in clinical trials118. The general strategy of prefusion stabilization has been widely implemented for other viral antigens and has informed diverse vaccine development efforts120. Specifically, the introduction of stabilizing proline residues was effective for RSV F, hMPV F, Lassa virus glycoprotein complex, ebola and Marburg glycoproteins, and several coronavirus spike proteins127. For the latter, several new iterations including ‘S-2P’ and ‘HexaPro’ in SARS-CoV-2 spike have improved efficacy and form the basis of the currently approved vaccines127. Such stabilization approaches help limit the overall size of the precursor pool by ensuring the prefusion conformation is dominant, and limit responses against epitopes displayed less frequently on the circulating virus.Viral envelope glycoproteins undergo significant conformational rearrangements between their prefusion and post-fusion states. To elicit immune responses that specifically recognize the prefusion state present on the invading virus, the glycoproteins are stabilized to prevent spontaneous structural rearrangement. This general concept was first demonstrated for influenza HA to characterize its conformational rearrangements necessary for membrane fusion either through introducing prolines or non-native cysteines to stabilize or covalently “staple” the prefusion state Fig. 109,110. 129. However, in some cases, glycan removal to expose neoepitopes was not sufficient to elicit broader responses, indicating that other factors influencing immunodominance are present130. Even if a glycan is not sterically occluding the epitope itself, it can impact the elicitation of antibodies against adjacent epitopes, as demonstrated by the increased breadth of serum responses raised against selectively de-glycosylated Env and HA antigens131.The removal of naturally occurring glycans introduces new targets for antibody responses Fig. . For HIV132. Iterative rounds of consensus sequence generation within, and then between, clades yielded HA immunogens that conferred higher HAI titers and protection against diverse H5 isolates even relative to cocktail immunizations of members from multiple H5 clades134. The COBRA approach translates to other human , avian , and swine  HAs, as well as other viral antigens such as dengue virus E protein. Importantly these immunogens work in both naïve animal models and in the setting of preexisting immunity147. The exact mechanism of enhanced breadth elicited by COBRA immunogens is unknown; one possibility is increased recruitment of B cells against antigenic sites with greater cross-reactive potential leads to the enhanced diversity and breadth of responses149.While many immunogen design strategies are designed to target a single epitope, computationally optimized broadly reactive antigens (COBRAs) use a consensus sequence-based approach to optimize the entire antigen to simultaneously present multiple cross-reactive epitopes Fig. . First dH1–69, but VH6-1, VH1–18, and VH3–30 have also been observed; sialic acid-mimicking HA receptor binding site (RBS)-directed Abs are enriched for JH6 genes158. Similar gene enrichment is seen in responses to other viral glycoproteins, including Zika E , hepatitis C virus E2 , and SARS-CoV-2 spike 163. While germline-encoded affinity can dramatically alter patterns of dominance if the precursor B cells are present with high enough frequency, this natural advantage is often insufficient in the setting of rare precursors164. For example, high-affinity VRC01-class precursors that target the HIV Env CD4bs are predicted to have a frequency of 1 in 0.9 million naïve B cells, and despite nanomolar affinity for rationally designed immunogens targeting these precursors, they remained minor components of the overall repertoire when present in animal models at these levels5.Germline biases for specific epitopes may be observed if the amino acids encoded by the naïve sequence has a threshold affinity for the antigen such that it results in a competitive advantage. For influenza HA, stem-binding antibodies are predominantly enriched for V74. Studies using VRC01 germline knock-in mice showed that elicited antibodies have VRC01-class characteristics, with varying affinity and breadth for native HIV Envs166. Subsequent generations of VRC01-class germline-targeting nanoparticles allowed for isolation and characterization of human VRC01 precursors present in naïve individuals167. Applying similar principles led to the development of immunogens able to engage germline-precursors for HIV Env targeting PGT121- and CH235-like broadly neutralizing antibodies, as well as those from cross-group stem-reactive influenza HA antibodies170.Germline-targeting immunogens are used to preferentially bias responses towards these rare high-affinity clones Fig. . In init171. This represents a conceptual shift in the approach: rather than attempting to engage the exact precursors within the repertoire, immunogens now engage a wider set of precursors that have the potential to mature breadth over the course of multiple immunizations. However, key questions remain about how to ‘guide’ BCR maturation along the desired evolutionary path, and what are the key structural characteristics of immunogens necessary for eliciting antibodies with the desired breadth173. Performing these studies in the context of adoptively transferred germline-presenting B cells, rather than a complete knock-in model, will also be necessary to understand the effects of inter-clonal competition on the ability of a rationally designed immunogen to selectively enrich for desired responses174.Recently, germline targeting designs have been generalized to target larger pools of B cell precursors making CDRH3-mediated contacts with a given epitope175. These resurfaced constructs are thought to increase responses towards the grafted epitope due to preferential recall of memory responses against the graft versus de novo responses against the scaffold. Recently the H1 HA RBS, a complex conformationally specific epitope, was grafted onto the non-circulating avian H4 and H14 HAs. Binding to a panel of H1 RBS-directed antibodies confirmed the successful recapitulation of the grafted epitope175. Immunization with the resurfaced HA in mice primed with H1 HA increased the breadth of RBS-directed B cells against historical H1 isolates175.For antigenically diverse, yet structurally similar antigens, epitope ‘grafting’ or ‘resurfacing’ can be used to target subdominant responses from immunologic memory. Desired epitopes can be transplanted onto antigenically distinct scaffolds Fig. 175. Thes176. Prime-boost immunizations with these cHAs presenting head domains to which there is no preexisting immunity, but with conserved stem domains, elicited cross-reactive stem-directed antibodies capable of protecting via Fc-effector functions184. In clinical trials, cHAs show evidence of eliciting cross-reactive serum antibodies that target the HA stem in human subjects185. More recently, an analogous approach was used to design chimeric coronavirus spike immunogens186.A similar grafting approach was used for the HA stem region to create chimeric HAs (cHAs) whereby antigenically distinct and often non-circulating HA heads have been swapped onto stems of circulating HA subtypes Fig. 176. Prim189. For example, resurfaced dengue E domain III (EDIII) immunogens preserved the broadly neutralizing 4E11 epitope while peripheral epitopes were mutated to disrupt recall responses against these regions189. Similar to masking off-target EDIII epitopes with glycans, resurfacing off-target epitopes may help prevent antibody-dependent enhancement as cross-reactive yet non-neutralizing responses are limited.A complementary type of epitope resurfacing involves mutating ‘off-target’ regions to disrupt recognition of undesired epitopes. This approach has been used as a screening tool to isolate HIV Env CD4bs-directed antibodies, and flavivirus E immunogens with lower cross-reactive but non-neutralizing titers191. While the difficulty in designing orthogonal scaffolds significantly increases with the complexity of the target epitope, this approach completely avoids eliciting antigen-specific off-target responses.A further extension of this approach is the grafting of desired epitopes onto orthogonal protein scaffolds that lack structural homology with the wild-type antigen. In initial studies, multiple neutralizing epitopes of RSV F protein were transplanted onto orthogonal protein scaffolds. The antibody responses elicited by orthogonally-scaffolded immunogens were specific to the grafted epitopes, and had increased titers against the grafted sites despite lower titers to RSV F protein overall176. Likewise, while orthogonal grafting does not present any epitopes shared by that class of antigens , it still presents neoepitopes which will have their own intrinsic immunodominance hierarchy. Overall, while resurfaced immunogens can elicit antibodies against desired epitopes with increased frequency or breadth, current iterations do not optimally alter patterns of dominance. The combination of epitope grafting with other design strategies is likely needed to fully realize the epitope immune-focusing potential of such constructs.A primary drawback of epitope resurfacing is the introduction of a significant number of neoepitopes. For example, grafting the H1 HA RBS onto an H4 full length soluble ectodomain (FLsE) HA scaffold, or grafting an antigenically distinct HA head onto a conserved HA stem domain, expands inter-clonal competition via the introduction of novel immunodominant HA head epitopes192. Additional steps are therefore needed to ensure broad reactivity even if elicited antibodies engage the desired epitope. Immunogen design strategies to increase the breadth of responses can be broadly classified as single- or multi-step approaches.The development of next-generation vaccines is further complicated by the fact that engagement of a conserved epitope does not guarantee breadth194. Inferred germline antibodies or unmutated common ancestors (UCAs) of broadly neutralizing influenza HA antibodies isolated from human donors show high affinity for strains circulating during the donor’s early childhood, suggesting that early exposures imprint responses that are subsequently matured and refined upon re-exposure197. Repeated exposure in the form of seasonal vaccination against influenza does not appear to alter the patterns of dominance established towards particular HA subtypes200. However, recall and maturation of HA stem-directed responses has been observed following immunization with divergent wild-type or chimeric strains, where the stem region is the conserved epitope between exposures205. Even if overall patterns of dominance are not altered, subdominant antibodies can show increased breadth upon re-exposure to antigenically similar epitopes175.Initial exposures to a pathogen can influence subsequent responses, a phenomenon termed “original antigenic sin”209. Too little antigenic distance, and memory responses against variable epitopes will dominate the repertoire; too great a distance, and de novo responses against variable epitopes dominate. Understanding how antigenic distance impacts preferential recall of imprinted responses will be critical for designing optimized immunogens capable of expanding subdominant populations from within the memory compartment.Computational studies suggest that there are optimal antigenic distances between primary and secondary immunogens that will help direct responses toward conserved and broadly protective epitopes34. However, the breadth of these serum responses is due to the summation of individual strain-specific responses rather than broad reactivity from specific antibodies. The simultaneous presentation of multiple antigens within a single immunogen, however, elicits broader antibodies, and in certain cases focuses antibody responses on conserved epitopes56. While the precise mechanism of how heterologous display focuses responses to cross-reactive epitopes has not yet been determined, evidence suggests that such an immunogen confers a competitive avidity advantage to cross-reactive B cells41.Immunization with a cocktail of HA homotypic nanoparticles has been shown to elicit broader serum titers than individual nanoparticlesHowever, with heterologous display, especially when presenting an array of wild-type antigens, there is often a gradient of epitope conservation that ranges from unique, to partially conserved, to fully conserved across the set of antigens being displayed. Thus, there appears to be a ‘sliding scale’ of benefit to all antigen-specific B cells that is combined with the preexisting immunodominance hierarchy to determine the overall epitope distribution Fig. . If a go212.If immunodominance hierarchies are altered by next-generation vaccines, will previously conserved subdominant epitope(s) nevertheless remain conserved in response to significant immune pressure? In other words, by focusing immune responses to a conserved epitope, will escape  occur? While many conserved regions on viral glycoproteins play important functional roles , key residues are often a fraction of the entire eptiopeCan B cell populations targeting a particular conserved epitope be guided towards breadth through serial immunizations with rationally designed constructs, or is the potential for breadth intrinsic to a specific subset of clones? This has significant implications for pathogens such as HIV, where many bnAbs have structural features  that greatly restrict the size of precursor populations.154. In contrast, an HA lateral patch-binding antibody makes contacts with a mostly continuous hydrophilic surface, where the similar CDR mutations are less likely to disrupt binding213. In effect, the affinity maturing antibodies may follow evolutionary paths, determined by the targeted epitope. Over time, the relative frequency of beneficial, neutral, or deleterious mutations impact the size of competing clonal B cell pools, and thus the trajectory of inter-clonal competition.Are there structural, epitope-specific factors that influence immunodominance hierarchies? The major components of precursor frequency and affinity, T cell help, and epitope accessibility do not account for the impact of the epitope itself as a substrate during affinity maturation. For example, an HA receptor-mimicking RBS-directed antibody makes contacts within the sialic acid binding pocket, a recessed epitope surrounded by a highly variable periphery, where acquired mutations in CDRs can affect or completely abrogate bindingDespite significant advancements in recent years, understanding immunodominance and what is required to create a vaccine with long-term efficacy against a rapidly evolving pathogen is necessary. Some key fundamental questions that remain include, but are not limited to:Current protein engineering strategies, like those described above, allow tailoring of an immunogen to elicit the desired antibody response. Importantly, many of the described approaches take orthogonal and therefore complementary approaches to immune-focusing. For example, a germline-targeting immunogen, with hyperglycosylated periphery, displayed on a nanoparticle leverages three different strategies: (1) the multimeric display, (2) steric shielding of ‘off-target’ and nanoparticle scaffold epitopes, and (3) epitope optimization to engage a set of precursor B cells. Multimeric display increases the overall size of the precursor B cell pool and the avidity of antigen interactions, steric shielding of undesired responses leads to the reduction of inter-clonal competition from undesired clones, and germline targeting ensures a select set of precursors have increased affinity and thus a competitive edge in the GC reaction. While the intrinsic immunodominance hierarchies for specific antigens will likely determine the optimal approach needed to expand broadly protective responses, the above strategies allow for selective manipulation of the major contributing factors to immunodominance.These protein engineering approaches can be similarly used to generate probes to deconvolute the GC reaction. Selective resurfacing or occlusion of epitopes, the modulation of endogenous/exogenous T cell epitopes, display on oligomeric scaffolds, and other rational design strategies, provide an opportunity to perform mechanistic experiments investigating underlying B-cell biology. Simultaneous basic and translational advancements will allow for iterative improvement of pre-clinical vaccine candidates in the pursuit of next-generation viral vaccines for rapidly evolving pathogens.