A Report on the Safety of Acitretin Use in Renal Failure Patients on Hemodialysis.

Acitretin, commonly used for severe psoriasis and keratinocyte carcinoma chemoprevention in high-risk patients, is contraindicated in patients with end stage renal disease on hemodialysis. However, these patients often lack medication choices and in certain clinical scenarios, the benefits of acitretin may outweigh the potential risks. We identified 24 end stage renal disease patients on HD taking acitretin from Duke and Vanderbilt University Medical Centers. While adverse effects were common, patients did not frequently discontinue the medication due to them. We also found no association between acitretin with hospital admissions or mortality. We lastly found statistically significant increases in ALP and total bilirubin when on acitretin and dialysis compared to baseline. However, there was no dose-dependency or temporal association with acitretin or hemodialysis initiation. Based off these preliminary findings, we find that acitretin may safely be used in patients receiving HD with close monitoring of ALP and bilirubin.

Structural and computational design of a SARS-CoV-2 spike antigen with improved expression and immunogenicity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern challenge the efficacy of approved vaccines, emphasizing the need for updated spike antigens. Here, we use an evolutionary-based design aimed at boosting protein expression levels of S-2P and improving immunogenic outcomes in mice. Thirty-six prototype antigens were generated in silico and 15 were produced for biochemical analysis. S2D14, which contains 20 computationally designed mutations within the S2 domain and a rationally engineered D614G mutation in the SD2 domain, has an ~11-fold increase in protein yield and retains RBD antigenicity. Cryo-electron microscopy structures reveal a mixture of populations in various RBD conformational states. Vaccination of mice with adjuvanted S2D14 elicited higher cross-neutralizing antibody titers than adjuvanted S-2P against the SARS-CoV-2 Wuhan strain and four variants of concern. S2D14 may be a useful scaffold or tool for the design of future coronavirus vaccines, and the approaches used for the design of S2D14 may be broadly applicable to streamline vaccine discovery.

Immune resilience despite inflammatory stress promotes longevity and favorable health outcomes including resistance to infection.

Some people remain healthier throughout life than others but the underlying reasons are poorly understood. Here we hypothesize this advantage is attributable in part to optimal immune resilience (IR), defined as the capacity to preserve and/or rapidly restore immune functions that promote disease resistance (immunocompetence) and control inflammation in infectious diseases as well as other causes of inflammatory stress. We gauge IR levels with two distinct peripheral blood metrics that quantify the balance between (i) CD8+ and CD4+ T-cell levels and (ii) gene expression signatures tracking longevity-associated immunocompetence and mortality-associated inflammation. Profiles of IR metrics in ~48,500 individuals collectively indicate that some persons resist degradation of IR both during aging and when challenged with varied inflammatory stressors. With this resistance, preservation of optimal IR tracked (i) a lower risk of HIV acquisition, AIDS development, symptomatic influenza infection, and recurrent skin cancer; (ii) survival during COVID-19 and sepsis; and (iii) longevity. IR degradation is potentially reversible by decreasing inflammatory stress. Overall, we show that optimal IR is a trait observed across the age spectrum, more common in females, and aligned with a specific immunocompetence-inflammation balance linked to favorable immunity-dependent health outcomes. IR metrics and mechanisms have utility both as biomarkers for measuring immune health and for improving health outcomes.

Effective Multivalent Oriented Presentation of Meningococcal NadA Antigen Trimers by Self-Assembling Ferritin Nanoparticles.

The presentation of viral antigens on nanoparticles in multivalent arrays has emerged as a valuable technology for vaccines. On the nanoparticle surface, highly ordered, repetitive arrays of antigens can mimic their geometric arrangement on virion surfaces and elicit stronger humoral responses than soluble viral antigens. More recently, bacterial antigens have been presented on self-assembling protein nanoparticles and have elicited protective antibody and effective T-helper responses, further supporting the nanoparticle platform as a universal approach for stimulating potent immunogenicity. Here, we present the rational design, structural analysis, and immunogenicity of self-assembling ferritin nanoparticles displaying eight copies of the Neisseria meningitidis trimeric adhesin NadA. We engineered constructs consisting of two different NadA fragments, head only and head with stalk, that we fused to ferritin and expressed in Escherichia coli. Both fusion constructs self-assembled into the expected nanoparticles as determined by Cryo electron microscopy. In mice, the two nanoparticles elicited comparable NadA antibody levels that were 10- to 100-fold higher than those elicited by the corresponding NadA trimer subunits. Further, the NadAferritin nanoparticles potently induced complement-mediated serum bactericidal activity. These findings confirm the value of self-assembling nanoparticles for optimizing the immunogenicity of bacterial antigens and support the broad applicability of the approach to vaccine programs, especially for the presentation of trimeric antigens.

In Vivo Gene Expression Profiling of Staphylococcus aureus during Infection Informs Design of Stemless Leukocidins LukE and -D as Detoxified Vaccine Candidates.

Staphylococcus aureus is a clinically important bacterial pathogen that has become resistant to treatment with most routinely used antibiotics. Alternative strategies, such as vaccination and phage therapy, are therefore actively being investigated to prevent or combat staphylococcal infections. Vaccination requires that vaccine targets are expressed at sufficient quantities during infection so that they can be targeted by the host's immune system. While our knowledge of in vitro expression levels of putative vaccine candidates is comprehensive, crucial in vivo expression data are scarce and promising vaccine candidates during in vitro assessment often prove ineffective in preventing S. aureus infection. Here, we show how a newly developed high-throughput quantitative reverse transcription-PCR (qRT-PCR) assay monitoring the expression of 84 staphylococcal genes encoding mostly virulence factors can inform the selection and design of effective vaccine candidates against staphylococcal infections. We show that this assay can accurately quantify mRNA expression levels of these genes in several host organs relying only on very limited amounts of bacterial mRNA in each sample. We selected two highly expressed genes, lukE and lukD, encoding pore-forming leukotoxins, to inform the design of detoxified recombinant proteins and showed that immunization with recombinant genetically detoxified LukED antigens conferred protection against staphylococcal skin infection in mice. Consequently, knowledge of in vivo-expressed virulence determinants can be successfully deployed to identify and select promising candidates for optimized design of effective vaccine antigens against S. aureus. Notably, this approach should be broadly applicable to numerous other pathogens. IMPORTANCE Vaccination is an attractive strategy for preventing bacterial infections in an age of increased antimicrobial resistance. However, vaccine development frequently suffers significant setbacks when candidate antigens that show promising results in in vitro experimentation fail to protect from disease. An alluring strategy is to focus resources on developing bacterial virulence factors that are expressed during disease establishment or maintenance and are critical for bacterial in-host survival as vaccine targets. While expression profiles of many virulence factors have been characterized in detail in vitro, our knowledge of their in vivo expression profiles is still scarce. Here, using a high-throughput qRT-PCR approach, we identified two highly expressed leukotoxins in a murine infection model and showed that genetically detoxified derivatives of these elicited a protective immune response in a murine skin infection model. Therefore, in vivo gene expression can inform the selection of promising candidates for the design of effective vaccine antigens.

Interventions After First Post-Transplant Cutaneous Squamous Cell Carcinoma: A Proposed Decision Framework.

Cutaneous squamous cell carcinoma (CSCC) is a major cause of morbidity and mortality after organ transplant. Many patients subsequently develop multiple CSCC following a first CSCC, and the risk of metastasis and death is significantly increased compared to the general population. Post-transplant CSCC represents a disease at the interface of dermatology and transplant medicine. Both systemic chemoprevention and modulation of immunosuppression are frequently employed in patients with multiple CSCC, yet there is little consensus on their use after first CSCC to reduce risk of subsequent tumors. While relatively few controlled trials have been undertaken, extrapolation of observational data suggests the most effective interventions may be at the time of first CSCC. We review the need for intervention after a first post-transplant CSCC and evidence for use of various approaches as secondary prevention, before discussing barriers preventing engagement with this approach and finally highlight areas for future research. Close collaboration between specialties to ensure prompt deployment of these interventions after a first CSCC may improve patient outcomes.

Research engagement by British early-career practitioners in nephrology: a multidisciplinary survey.

OBJECTIVES: To establish barriers and motivators underlying research engagement among early-career practitioners in nephrology across the UK, in order to guide potential interventions to enhance research involvement in renal units. DESIGN: Cross-sectional online survey employing a range of free-text, Likert scale and binomial/multiple-choice responses, distributed via mailing lists and social media. Topics covered research experience, research involvement and barriers, impact of COVID-19 and strategies to improve research engagement. Thematic analysis was used to assess free-text responses. SETTING: Renal units throughout the UK. PARTICIPANTS: Non-consultant healthcare staff self-identifying as working in nephrology were included (n=211), with responses from non-UK respondents or consultant nephrologists excluded (n=12). RESULTS: Responses were received from across the multidisciplinary team (physicians (n=83) and nurses (n=83)) and other allied health professionals (n=45). Most were aware of ongoing local research, but under half of them were actively involved. Multivariate analysis indicated employment as a physician, protected time for research activity and provision of appropriate training were associated with greater research experience and output. There was general enthusiasm to undertake research, but perceived barriers included insufficient staffing, lack of time, funding and encouragement. COVID-19 was felt to have further impacted negatively upon opportunities. Among the suggested strategies to promote engagement, mentorship and an online research resource were felt to be of most interest. CONCLUSIONS: In the first survey of this type in nephrology, we demonstrate differences across the multidisciplinary spectrum in perceived research experience and accessibility, which have been worsened by COVID-19. Our findings will guide strategies to broaden engagement in early-career practitioners and serve as a baseline to assess the impact of these interventions.

Self-assembling protein nanoparticles and virus like particles correctly display ß-barrel from meningococcal factor H-binding protein through genetic fusion.

Recombinant protein-based vaccines are a valid and safer alternative to traditional vaccines based on live-attenuated or killed pathogens. However, the immune response of subunit vaccines is generally lower compared to that elicited by traditional vaccines and usually requires the use of adjuvants. The use of self-assembling protein nanoparticles, as a platform for vaccine antigen presentation, is emerging as a promising approach to enhance the production of protective and functional antibodies. In this work we demonstrated the successful repetitive antigen display of the C-terminal ß-barrel domain of factor H binding protein, derived from serogroup B Meningococcus on the surface of different self-assembling nanoparticles using genetic fusion. Six nanoparticle scaffolds were tested, including virus-like particles with different sizes, geometries, and physicochemical properties. Combining computational and structure-based rational design we were able generate antigen-fused scaffolds that closely aligned with three-dimensional structure predictions. The chimeric nanoparticles were produced as recombinant proteins in Escherichia coli and evaluated for solubility, stability, self-assembly, and antigen accessibility using a variety of biophysical methods. Several scaffolds were identified as being suitable for genetic fusion with the ß-barrel from fHbp, including ferritin, a de novo designed aldolase from Thermotoga maritima, encapsulin, CP3 phage coat protein, and the Hepatitis B core antigen. In conclusion, a systematic screening of self-assembling nanoparticles has been applied for the repetitive surface display of a vaccine antigen. This work demonstrates the capacity of rational structure-based design to develop new chimeric nanoparticles and describes a strategy that can be utilized to discover new nanoparticle-based approaches in the search for vaccines against bacterial pathogens.

Dampened Inflammatory Signalling and Myeloid-Derived Suppressor-Like Cell Accumulation Reduces Circulating Monocytic HLA-DR Density and May Associate With Malignancy Risk in Long-Term Renal Transplant Recipients.

Background: Malignancy is a major cause of morbidity and mortality in transplant recipients. Identification of those at highest risk could facilitate pre-emptive intervention such as reduction of immunosuppression. Reduced circulating monocytic HLA-DR density is a marker of immune depression in the general population and associates with poorer outcome in critical illness. It has recently been used as a safety marker in adoptive cell therapy trials in renal transplantation. Despite its potential as a marker of dampened immune responses, factors that impact upon monocytic HLA-DR density and the long-term clinical sequelae of this have not been assessed in transplant recipients. Methods: A cohort study of stable long-term renal transplant recipients was undertaken. Serial circulating monocytic HLA-DR density and other leucocyte populations were quantified by flow cytometry. Gene expression of monocytes was performed using the Nanostring nCounter platform, and 13-plex cytokine bead array used to quantify serum concentrations. The primary outcome was malignancy development during one-year follow-up. Risk of malignancy was calculated by univariate and multivariate proportionate hazards modelling with and without adjustment for competing risks. Results: Monocytic HLA-DR density was stable in long-term renal transplant recipients (n=135) and similar to non-immunosuppressed controls (n=29), though was suppressed in recipients receiving prednisolone. Decreased mHLA-DRd was associated with accumulation of CD14+CD11b+CD33+HLA-DRlo monocytic myeloid-derived suppressor-like cells. Pathway analysis revealed downregulation of pathways relating to cytokine and chemokine signalling in monocytes with low HLA-DR density; however serum concentrations of major cytokines did not differ between these groups. There was an independent increase in malignancy risk during follow-up with decreased HLA-DR density. Conclusions: Dampened chemokine and cytokine signalling drives a stable reduction in monocytic HLA-DR density in long-term transplant recipients and associates with subsequent malignancy risk. This may function as a novel marker of excess immunosuppression. Further study is needed to understand the mechanism behind this association.

The COVID-19 Pandemic Is Associated with Reduced Survival after Pancreatic Ductal Adenocarcinoma Diagnosis: A Single-Centre Retrospective Analysis.

The COVID-19 pandemic has hugely disrupted healthcare provision, including oncology services. To evaluate the effects of the pandemic on referral routes leading to diagnosis, treatments, and prognosis in patients with pancreatic ductal adenocarcinoma, we performed a retrospective cohort study at a single tertiary centre in the UK. The patients were identified from the weekly hepatopancreatobiliary multidisciplinary team meetings between February 2018 and March 2021. The demographic, referral, and treatment data for each patient and date of death, where applicable, were extracted from the electronic patient record. The patients (n = 203) were divided into "pre-pandemic" and "pandemic" cohorts based on a referral date cut-off of 23rd March 2020. The median survival was 7.4 months [4.9-9.3] in the "pre-pandemic" cohort (n = 125), halving to 3.3 months [2.2-6.0], (p = 0.015) in the "pandemic" cohort (n = 78). There was no significant difference in patient characteristics between the two cohorts. There was a trend toward increased emergency presentations at diagnosis and reduced use of surgical resection in the "pandemic" cohort. This small-scale study suggested that the COVID-19 pandemic is associated with a halving of median survival in pancreatic ductal adenocarcinoma. Urgent further studies are required to confirm these findings and examine corresponding effects in other cancer types.

Structural characterization of a cross-protective natural chimera of factor H binding protein from meningococcal serogroup B strain NL096.

Invasive meningococcal disease can cause fatal sepsis and meningitis and is a global health threat. Factor H binding protein (fHbp) is a protective antigen included in the two currently available vaccines against serogroup B meningococcus (MenB). FHbp is a remarkably variable surface-exposed meningococcal virulence factor with over 1300 different amino acid sequences identified so far. Based on this variability, fHbp has been classified into three variants, two subfamilies or nine modular groups, with low degrees of cross-protective activity. Here, we report the crystal structure of a natural fHbp cross-variant chimera, named variant1-2,3.x expressed by the MenB clinical isolate NL096, at 1.2 Å resolution, the highest resolution of any fHbp structure reported to date. We combined biochemical, site-directed mutagenesis and computational biophysics studies to deeply characterize this rare chimera. We determined the structure to be composed of two adjacent domains deriving from the three variants and determined the molecular basis of its stability, ability to bind Factor H and to adopt the canonical three-dimensional fHbp structure. These studies guided the design of loss-of-function mutations with potential for even greater immunogenicity. Moreover, this study represents a further step in the understanding of the fHbp biological and immunological evolution in nature. The chimeric variant1-2,3.x fHbp protein emerges as an intriguing cross-protective immunogen and suggests that identification of such naturally occurring hybrid proteins may result in stable and cross-protective immunogens when seeking to design and develop vaccines against highly variable pathogens.

Transplantation Without Overimmunosuppression (TWO) study protocol: a phase 2b randomised controlled single-centre trial of regulatory T cell therapy to facilitate immunosuppression reduction in living donor kidney transplant recipients.

INTRODUCTION: Regulatory T cell (Treg) therapy has been demonstrated to facilitate long-term allograft survival in preclinical models of transplantation and may permit reduction of immunosuppression and its associated complications in the clinical setting. Phase 1 clinical trials have shown Treg therapy to be safe and feasible in clinical practice. Here we describe a protocol for the TWO study, a phase 2b randomised control trial of Treg therapy in living donor kidney transplant recipients that will confirm safety and explore efficacy of this novel treatment strategy. METHODS AND ANALYSIS: 60 patients will be randomised on a 1:1 basis to Treg therapy (TR001) or standard clinical care (control). Patients in the TR001 arm will receive an infusion of autologous polyclonal ex vivo expanded Tregs 5 days after transplantation instead of standard monoclonal antibody induction. Maintenance immunosuppression will be reduced over the course of the post-transplant period to low-dose tacrolimus monotherapy. Control participants will receive a standard basiliximab-based immunosuppression regimen with long-term tacrolimus and mycophenolate mofetil immunosuppression. The primary endpoint is biopsy proven acute rejection over 18 months; secondary endpoints include immunosuppression burden, chronic graft dysfunction and drug-related complications. ETHICS AND DISSEMINATION: Ethical approval has been provided by the National Health Service Health Research Authority South Central-Oxford A Research Ethics Committee (reference 18/SC/0054). The study also received authorisation from the UK Medicines and Healthcare products Regulatory Agency and is being run in accordance with the principles of Good Clinical Practice, in collaboration with the registered trials unit Oxford Clinical Trials Research Unit. Results from the TWO study will be published in peer-reviewed scientific/medical journals and presented at scientific/clinical symposia and congresses. TRIAL REGISTRATION NUMBER: ISRCTN: 11038572; Pre-results.

Towards regulatory cellular therapies in solid organ transplantation.

Organ transplantation is a modern medical success story. However, since its inception it has been limited by the need for pharmacological immunosuppression. Regulatory cellular therapies offer an attractive solution to these challenges by controlling transplant alloresponses through multiple parallel suppressive mechanisms. A number of cell types have seen an accelerated development into human trials and are now on the threshold of a long-awaited breakthrough in personalized transplant therapeutics. Here we assess recent developments with a focus on the most likely candidates, some of which have already facilitated successful immunosuppression withdrawal in early clinical trials. We propose that this may constitute a promising approach in clinical transplantation but also evaluate outstanding issues in the field, providing cause for cautious optimism.

Improved epitope resolution of the prefusion trimer-specific antibody AM14 bound to the RSV F glycoprotein.

Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infections resulting in medical intervention and hospitalizations during infancy and early childhood, and vaccination against RSV remains a public health priority. The RSV F glycoprotein is a major target of neutralizing antibodies, and the prefusion stabilized form of F (DS-Cav1) is under investigation as a vaccine antigen. AM14 is a human monoclonal antibody with the exclusive capacity of binding an epitope on prefusion F (PreF), which spans two F protomers. The quality of recognizing a trimer-specific epitope makes AM14 valuable for probing PreF-based immunogen conformation and functionality during vaccine production. Currently, only a low-resolution (5.5 Å) X-ray structure is available of the PreF-AM14 complex, revealing few reliable details of the interface. Here, we perform complementary structural studies using X-ray crystallography and cryo-electron microscopy (cryo-EM) to provide improved resolution structures at 3.6 Å and 3.4 Å resolutions, respectively. Both X-ray and cryo-EM structures provide clear side-chain densities, which allow for accurate mapping of the AM14 epitope on DS-Cav1. The structures help rationalize the molecular basis for AM14 loss of binding to RSV F monoclonal antibody-resistant mutants and reveal flexibility for the side chain of a key antigenic residue on PreF. This work provides the basis for a comprehensive understanding of RSV F trimer specificity with implications in vaccine design and quality assessment of PreF-based immunogens.

The respiratory syncytial virus (RSV) prefusion F-protein functional antibody repertoire in adult healthy donors.

Respiratory syncytial virus (RSV) is the leading cause of death from lower respiratory tract infection in infants and children, and is responsible for considerable morbidity and mortality in older adults. Vaccines for pregnant women and elderly which are in phase III clinical studies target people with pre-existing natural immunity against RSV. To investigate the background immunity which will be impacted by vaccination, we single cell-sorted human memory B cells and dissected functional and genetic features of neutralizing antibodies (nAbs) induced by natural infection. Most nAbs recognized both the prefusion and postfusion conformations of the RSV F-protein (cross-binders) while a smaller fraction bound exclusively to the prefusion conformation. Cross-binder nAbs used a wide array of gene rearrangements, while preF-binder nAbs derived mostly from the expansion of B-cell clonotypes from the IGHV1 germline. This latter class of nAbs recognizes an epitope located between Site Ø, Site II, and Site V on the F-protein, identifying an important site of pathogen vulnerability.

Convergent structural features of respiratory syncytial virus neutralizing antibodies and plasticity of the site V epitope on prefusion F.

Respiratory syncytial virus (RSV) is a global public health burden for which no licensed vaccine exists. To aid vaccine development via increased understanding of the protective antibody response to RSV prefusion glycoprotein F (PreF), we performed structural and functional studies using the human neutralizing antibody (nAb) RSB1. The crystal structure of PreF complexed with RSB1 reveals a conformational, pre-fusion specific site V epitope with a unique cross-protomer binding mechanism. We identify shared structural features between nAbs RSB1 and CR9501, elucidating for the first time how diverse germlines obtained from different subjects can develop convergent molecular mechanisms for recognition of the same PreF site of vulnerability. Importantly, RSB1-like nAbs were induced upon immunization with PreF in naturally-primed cattle. Together, this work reveals new details underlying the immunogenicity of site V and further supports PreF-based vaccine development efforts.

4CMenB vaccine induces elite cross-protective human antibodies that compete with human factor H for binding to meningococcal fHbp.

Neisseria meningitidis serogroup B (MenB) is the leading cause of meningococcal meningitis and sepsis in industrialized countries, with the highest incidence in infants and adolescents. Two recombinant protein vaccines that protect against MenB are now available (i.e. 4CMenB and MenB-fHbp). Both vaccines contain the Factor H Binding Protein (fHbp) antigen, which can bind the Human Factor H (fH), the main negative regulator of the alternative complement pathway, thus enabling bacterial survival in the blood. fHbp is present in meningococcal strains as three main variants which are immunologically distinct. Here we sought to obtain detailed information about the epitopes targeted by anti-fHbp antibodies induced by immunization with the 4CMenB multicomponent vaccine. Thirteen anti-fHbp human monoclonal antibodies (mAbs) were identified in a library of over 100 antibody fragments (Fabs) obtained from three healthy adult volunteers immunized with 4CMenB. Herein, the key cross-reactive mAbs were further characterized for antigen binding affinity, complement-mediated serum bactericidal activity (SBA) and the ability to inhibit binding of fH to live bacteria. For the first time, we identified a subset of anti-fHbp mAbs able to elicit human SBA against strains with all three variants and able to compete with human fH for fHbp binding. We present the crystal structure of fHbp v1.1 complexed with human antibody 4B3. The structure, combined with mutagenesis and binding studies, revealed the critical cross-reactive epitope. The structure also provided the molecular basis of competition for fH binding. These data suggest that the fH binding site on fHbp v1.1 can be accessible to the human immune system upon immunization, enabling elicitation of human mAbs broadly protective against MenB. The novel structural, biochemical and functional data are of great significance because the human vaccine-elicited mAbs are the first reported to inhibit the binding of fH to fHbp, and are bactericidal with human complement. Our studies provide molecular insights into the human immune response to the 4CMenB meningococcal vaccine and fuel the rationale for combined structural, immunological and functional studies when seeking deeper understanding of the mechanisms of action of human vaccines.

Identification, selection, and expansion of non-gene modified alloantigen-reactive Tregs for clinical therapeutic use.

Transplantation is limited by the need for life-long pharmacological immunosuppression, which carries significant morbidity and mortality. Regulatory T cell (Treg) therapy holds significant promise as a strategy to facilitate immunosuppression minimization. Polyclonal Treg therapy has been assessed in a number of Phase I/II clinical trials in both solid organ and hematopoietic transplantation. Attention is now shifting towards the production of alloantigen-reactive Tregs (arTregs) through co-culture with donor antigen. These allospecific cells harbour potent suppressive function and yet their specificity implies a theoretical reduction in off-target effects. This review will cover the progress in the development of arTregs including their potential application for clinical use in transplantation, the knowledge gained so far from clinical trials of Tregs in transplant patients, and future directions for Treg therapy.