Flexible active-site loops fine-tune substrate specificity of hyperthermophilic metallo-oxidases.

Hyperthermophilic ('superheat-loving') archaea found in high-temperature environments such as Pyrobaculum aerophilum contain multicopper oxidases (MCOs) with remarkable efficiency for oxidizing cuprous and ferrous ions. In this work, directed evolution was used to expand the substrate specificity of P. aerophilum McoP for organic substrates. Six rounds of error-prone PCR and DNA shuffling followed by high-throughput screening lead to the identification of a hit variant with a 220-fold increased efficiency (kcat/Km) than the wild-type for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) without compromising its intrinsic activity for metal ions. The analysis of the X-ray crystal structure reveals four proximal mutations close to the T1Cu active site. One of these mutations is within the 23-residues loop that occludes this site, a distinctive feature of prokaryotic MCOs. The increased flexibility of this loop results in an enlarged tunnel and one additional pocket that facilitates bulky substrate-enzyme interactions. These findings underscore the synergy between mutations that modulate the dynamics of the active-site loop enabling enhanced catalytic function. This study highlights the potential of targeting loops close to the T1Cu for engineering improvements suitable for biotechnological applications.

Interferon regulatory factor-5-dependent CD11c+ macrophages contribute to the formation of rupture-prone atherosclerotic plaques.

AIMS: Inflammation is a key factor in atherosclerosis. The transcription factor interferon regulatory factor-5 (IRF5) drives macrophages towards a pro-inflammatory state. We investigated the role of IRF5 in human atherosclerosis and plaque stability. METHODS AND RESULTS: Bulk RNA sequencing from the Carotid Plaque Imaging Project biobank were used to mine associations between major macrophage associated genes and transcription factors and human symptomatic carotid disease. Immunohistochemistry, proximity extension assays, and Helios cytometry by time of flight (CyTOF) were used for validation. The effect of IRF5 deficiency on carotid plaque phenotype and rupture in ApoE-/- mice was studied in an inducible model of plaque rupture. Interferon regulatory factor-5 and ITGAX/CD11c were identified as the macrophage associated genes with the strongest associations with symptomatic carotid disease. Expression of IRF5 and ITGAX/CD11c correlated with the vulnerability index, pro-inflammatory plaque cytokine levels, necrotic core area, and with each other. Macrophages were the predominant CD11c-expressing immune cells in the plaque by CyTOF and immunohistochemistry. Interferon regulatory factor-5 immunopositive areas were predominantly found within CD11c+ areas with a predilection for the shoulder region, the area of the human plaque most prone to rupture. Accordingly, an inducible plaque rupture model of ApoE-/-Irf5-/- mice had significantly lower frequencies of carotid plaque ruptures, smaller necrotic cores, and less CD11c+ macrophages than their IRF5-competent counterparts. CONCLUSION: Using complementary evidence from data from human carotid endarterectomies and a murine model of inducible rupture of carotid artery plaque in IRF5-deficient mice, we demonstrate a mechanistic link between the pro-inflammatory transcription factor IRF5, macrophage phenotype, plaque inflammation, and its vulnerability to rupture.

Disentangling Unusual Catalytic Properties and the Role of the [4Fe-4S] Cluster of Three Endonuclease III from the Extremophile D. radiodurans.

Endonuclease III (EndoIII) is a bifunctional DNA glycosylase with specificity for a broad range of oxidized DNA lesions. The genome of an extremely radiation- and desiccation-resistant bacterium, Deinococcus radiodurans, possesses three genes encoding for EndoIII-like enzymes (DrEndoIII1, DrEndoIII2 and DrEndoIII3), which reveal different types of catalytic activities. DrEndoIII2 acts as the main EndoIII in this organism, while DrEndoIII1 and 3 demonstrate unusual and no EndoIII activity, respectively. In order to understand the role of DrEndoIII1 and DrEndoIII3 in D. radiodurans, we have generated mutants which target non-conserved residues in positions considered essential for classic EndoIII activity. In parallel, we have substituted residues coordinating the iron atoms in the [4Fe-4S] cluster in DrEndoIII2, aiming at elucidating the role of the cluster in these enzymes. Our results demonstrate that the amino acid substitutions in DrEndoIII1 reduce the enzyme activity without altering the overall structure, revealing that the residues found in the wild-type enzyme are essential for its unusual activity. The attempt to generate catalytic activity of DrEndoIII3 by re-designing its catalytic pocket was unsuccessful. A mutation of the iron-coordinating cysteine 199 in DrEndoIII2 appears to compromise the structural integrity and induce the formation of a [3Fe-4S] cluster, but apparently without affecting the activity. Taken together, we provide important structural and mechanistic insights into the three EndoIIIs, which will help us disentangle the open questions related to their presence in D. radiodurans and their particularities.

Loops around the Heme Pocket Have a Critical Role in the Function and Stability of BsDyP from Bacillus subtilis.

Bacillus subtilis BsDyP belongs to class I of the dye-decolorizing peroxidase (DyP) family of enzymes and is an interesting biocatalyst due to its high redox potential, broad substrate spectrum and thermostability. This work reports the optimization of BsDyP using directed evolution for improved oxidation of 2,6-dimethoxyphenol, a model lignin-derived phenolic. After three rounds of evolution, one variant was identified displaying 7-fold higher catalytic rates and higher production yields as compared to the wild-type enzyme. The analysis of X-ray structures of the wild type and the evolved variant showed that the heme pocket is delimited by three long conserved loop regions and a small α helix where, incidentally, the mutations were inserted in the course of evolution. One loop in the proximal side of the heme pocket becomes more flexible in the evolved variant and the size of the active site cavity is increased, as well as the width of its mouth, resulting in an enhanced exposure of the heme to solvent. These conformational changes have a positive functional role in facilitating electron transfer from the substrate to the enzyme. However, they concomitantly resulted in decreasing the enzyme's overall stability by 2 kcal mol-1, indicating a trade-off between functionality and stability. Furthermore, the evolved variant exhibited slightly reduced thermal stability compared to the wild type. The obtained data indicate that understanding the role of loops close to the heme pocket in the catalysis and stability of DyPs is critical for the development of new and more powerful biocatalysts: loops can be modulated for tuning important DyP properties such as activity, specificity and stability.

GLP-1 improves adipose tissue glyoxalase activity and capillarization improving insulin sensitivity in type 2 diabetes.

Methylglyoxal was shown to impair adipose tissue capillarization and insulin sensitivity in obese models. We hypothesized that glyoxalase-1 (GLO-1) activity could be diminished in the adipose tissue of type 2 diabetic obese patients. Moreover, we assessed whether such activity could be increased by GLP-1-based therapies in order to improve adipose tissue capillarization and insulin sensitivity. GLO-1 activity was assessed in visceral adipose tissue of a cohort of obese patients. The role of GLP-1 in modulating GLO-1 was assessed in type 2 diabetic GK rats submitted to sleeve gastrectomy or Liraglutide treatment, in the adipose tissue angiogenesis assay and in the HUVEC cell line. Glyoxalase-1 activity was decreased in visceral adipose tissue of pre-diabetic and diabetic obese patients, together with other markers of adipose tissue dysfunction and correlated with increased HbA1c levels. Decreased adipose tissue GLO-1 levels in GK rats were increased by sleeve gastrectomy and Liraglutide, being associated with overexpression of angiogenic and vasoactive factors, as well as insulin receptor phosphorylation (Tyr1161). Moreover, GLP-1 increased adipose tissue capillarization and HUVEC proliferation in a glyoxalase-dependent manner. Lower adipose tissue GLO-1 activity was observed in dysmetabolic patients, being a target for GLP-1 in improving adipose tissue capillarization and insulin sensitivity.

Analysis of a new flavodiiron core structural arrangement in Flv1-ΔFlR protein from Synechocystis sp. PCC6803.

Flavodiiron proteins (FDPs) play key roles in biological response mechanisms against oxygen and/or nitric oxide; in particular they are present in oxygenic phototrophs (including cyanobacteria and gymnosperms). Two conserved domains define the core of this family of proteins: a N-terminal metallo-ß-lactamase-like domain followed by a C-terminal flavodoxin-like one, containing the catalytic diiron centre and a FMN cofactor, respectively. Members of the FDP family may present extra modules in the C-terminus, and were classified into several classes according to their distribution and composition. The cyanobacterium Synechocystis sp. PCC6803 contains four Class C FDPs (Flv1-4) that include at the C-terminus an additional NAD(P)H:flavin oxidoreductase (FlR) domain. Two of them (Flv3 and Flv4) have the canonical diiron ligands (Class C, Type 1), while the other two (Flv1 and Flv2) present different residues in that region (Class C, Type 2). Most phototrophs, either Bacterial or Eukaryal, contain at least two FDP genes, each encoding for one of those two types. Crystals of the Flv1 two core domains (Flv1-ΔFlR), without the C-terminal NAD(P)H:flavin oxidoreductase extension, were obtained and the structure was determined. Its pseudo diiron site contains non-canonical basic and neutral residues, and showed anion moieties, instead. The presented structure revealed for the first time the structure of the two-domain core of a Class C-Type 2 FDP.

The dual function of flavodiiron proteins: oxygen and/or nitric oxide reductases.

Flavodiiron proteins have emerged in the last two decades as a newly discovered family of oxygen and/or nitric oxide reductases widespread in the three life domains, and present in both aerobic and anaerobic organisms. Herein we present the main features of these fascinating enzymes, with a particular emphasis on the metal sites, as more appropriate for this special issue in memory of the exceptional bioinorganic scientist R. J. P. Williams who pioneered the notion of (metal) element availability-driven evolution. We also compare the flavodiiron proteins with the other oxygen and nitric oxide reductases known until now, highlighting how throughout evolution Nature arrived at different solutions for similar functions, in some cases adding extra features, such as energy conservation. These enzymes are an example of the (bioinorganic) unpredictable diversity of the living world.

Biocatalysis for biorefineries: The case of dye-decolorizing peroxidases.

Dye-decolorizing Peroxidases (DyPs) are heme-containing enzymes in fungi and bacteria that catalyze the reduction of hydrogen peroxide to water with concomitant oxidation of various substrates, including anthraquinone dyes, lignin-related phenolic and non-phenolic compounds, and metal ions. Investigation of DyPs has shed new light on peroxidases, one of the most extensively studied families of oxidoreductases; still, details of their microbial physiological role and catalytic mechanisms remain to be fully disclosed. They display a distinctive ferredoxin-like fold encompassing anti-parallel ß-sheets and α-helices, and long conserved loops surround the heme pocket with a role in catalysis and stability. A tunnel routes H2O2 to the heme pocket, whereas binding sites for the reducing substrates are in cavities near the heme or close to distal aromatic residues at the surface. Variations in reactions, the role of catalytic residues, and mechanisms were observed among different classes of DyP. They were hypothetically related to the presence or absence of distal H2O molecules in the heme pocket. The engineering of DyPs for improved properties directed their biotechnological applications, primarily centered on treating textile effluents and degradation of other hazardous pollutants, to fields such as biosensors and valorization of lignin, the most abundant renewable aromatic polymer. In this review, we track recent research contributions that furthered our understanding of the activity, stability, and structural properties of DyPs and their biotechnological applications. Overall, the study of DyP-type peroxidases has significant implications for environmental sustainability and the development of new bio-based products and materials with improved end-of-life options via biodegradation and chemical recyclability, fostering the transition to a sustainable bio-based industry in the circular economy realm.

Corrigendum to "Unveiling molecular details behind improved activity at neutral to alkaline pH of an engineered DyP-type peroxidase" Comput Struct Biotechnol J, 20 (2022), 3899-3910.

[This corrects the article DOI: 10.1016/j.csbj.2022.07.032.].

Mechanistic insights into glycoside 3-oxidases involved in C-glycoside metabolism in soil microorganisms.

C-glycosides are natural products with important biological activities but are recalcitrant to degradation. Glycoside 3-oxidases (G3Oxs) are recently identified bacterial flavo-oxidases from the glucose-methanol-coline (GMC) superfamily that catalyze the oxidation of C-glycosides with the concomitant reduction of O2 to H2O2. This oxidation is followed by C-C acid/base-assisted bond cleavage in two-step C-deglycosylation pathways. Soil and gut microorganisms have different oxidative enzymes, but the details of their catalytic mechanisms are largely unknown. Here, we report that PsG3Ox oxidizes at 50,000-fold higher specificity (kcat/Km) the glucose moiety of mangiferin to 3-keto-mangiferin than free D-glucose to 2-keto-glucose. Analysis of PsG3Ox X-ray crystal structures and PsG3Ox in complex with glucose and mangiferin, combined with mutagenesis and molecular dynamics simulations, reveal distinctive features in the topology surrounding the active site that favor catalytically competent conformational states suitable for recognition, stabilization, and oxidation of the glucose moiety of mangiferin. Furthermore, their distinction to pyranose 2-oxidases (P2Oxs) involved in wood decay and recycling is discussed from an evolutionary, structural, and functional viewpoint.

Effects of the Whole-Body Vibration Exercise on Sleep Disorders, Body Temperature, Body Composition, Tone, and Clinical Parameters in a Child with Down Syndrome Who Underwent Total Atrioventricular Septal Defect Surgery: A Case-Report.

BACKGROUND: The health and developmental issues of people with Down syndrome (DS) are complex and are associated with many medical, psychological, and social problems from childhood through into adulthood. DS children have an increased risk of multiorgan comorbidities, including congenital heart disease. Atrioventricular septal defect (AVSD) is a congenital heart malformation that often occurs in DS people. AIM: Physical activity and exercise are recommended for patients with cardiovascular disease and are considered to be the gold standard of cardiac rehabilitation. Whole-body vibration exercise (WBVE) is considered a form of exercises. The aim of this case report is to show the effects of WBVE on sleep disturbances, body temperature, body composition, tone, and clinical parameters in a child with DS with corrected total AVSD. The subject is a 10-year-old girl, with free-type DS, who underwent surgery to correct a total AVSD at 6 months. She underwent periodic cardiological monitoring and was released to perform any type of physical exercise, including WBVE. WBVE improved sleep quality and body composition. CONCLUSION: WBVE leads to physiological effects that benefit the DS child.

Therapeutic Approach of Whole-Body Vibration Exercise on Wound Healing in Animal Models: A Systematic Review.

Human skin wounds pose a gathering threat to the public health, carrying an immense epidemiologic and financial burden. Pharmacological and non-pharmacological (NP) treatments have been proposed to the management of wound healing. Physical exercise is a strong NP intervention considered for patients in wound healing. Particularly, a type of exercise intervention known as whole-body vibration (WBV) exercise has gained increasing interest. WBV exercise is generated due to the transmission of mechanical vibrations, produced by a vibrating platform, to the body. The aim of this review was to summarize studies in experimental animal models using WBV exercise in wound healing. Searches were performed in EMBASE, PubMed, Scopus and Web of Science including publications on 21 November 2022 using the string "whole body vibration" AND "wound healing" (animal or mice or mouse or rat or rodent). The SYRCLE tool was used to assess the risk of bias (RoB). From 48 studies, five studies met the inclusion criteria. RoB indicated that none of the studies fulfilled all methodological analyzed criteria, resulting in possible biases. The studies were homogeneous, and results suggest beneficial effects of WBV exercise in wound healing, mainly related to enhancing angiogenesis, granulation tissue formation, reducing the blood glucose level and enhancing blood microcirculation, by increasing myofiber growth and rapid re-epithelialization. In conclusion, the various biological effects of the response to the WBV exercise indicate the relevance of this intervention in wound healing in animals. Moreover, considering the translation approach, it is possible to speculate that the beneficial effects of this non-pharmacological therapy might justify clinical trials for wound healing also in humans, after criterion evaluation.

Imaging articular cartilage in osteoarthritis using targeted peptide radiocontrast agents.

BACKGROUND: Established MRI and emerging X-ray contrast agents for non-invasive imaging of articular cartilage rely on non-selective electrostatic interactions with negatively charged proteoglycans. These contrast agents have limited prognostic utility in diseases such as osteoarthritis (OA) due to the characteristic high turnover of proteoglycans. To overcome this limitation, we developed a radiocontrast agent that targets the type II collagen macromolecule in cartilage and used it to monitor disease progression in a murine model of OA. METHODS: To confer radiopacity to cartilage contrast agents, the naturally occurring tyrosine derivative 3,5-diiodo-L-tyrosine (DIT) was introduced into a selective peptide for type II collagen. Synthetic DIT peptide derivatives were synthesised by Fmoc-based solid-phase peptide synthesis and binding to ex vivo mouse tibial cartilage evaluated by high-resolution micro-CT. Di-Iodotyrosinated Peptide Imaging of Cartilage (DIPIC) was performed ex vivo and in vivo 4, 8 and 12 weeks in mice after induction of OA by destabilisation of the medial meniscus (DMM). Finally, human osteochondral plugs were imaged ex vivo using DIPIC. RESULTS: Fifteen DIT peptides were synthesised and tested, yielding seven leads with varying cartilage binding strengths. DIPIC visualised ex vivo murine articular cartilage comparably to the ex vivo contrast agent phosphotungstic acid. Intra-articular injection of contrast agent followed by in vivo DIPIC enabled delineation of damaged murine articular cartilage. Finally, the translational potential of the contrast agent was confirmed by visualisation of ex vivo human cartilage explants. CONCLUSION: DIPIC has reduction and refinement implications in OA animal research and potential clinical translation to imaging human disease.

Distal Mutations Shape Substrate-Binding Sites during Evolution of a Metallo-Oxidase into a Laccase.

Laccases are in increasing demand as innovative solutions in the biorefinery fields. Here, we combine mutagenesis with structural, kinetic, and in silico analyses to characterize the molecular features that cause the evolution of a hyperthermostable metallo-oxidase from the multicopper oxidase family into a laccase (k cat 273 s-1 for a bulky aromatic substrate). We show that six mutations scattered across the enzyme collectively modulate dynamics to improve the binding and catalysis of a bulky aromatic substrate. The replacement of residues during the early stages of evolution is a stepping stone for altering the shape and size of substrate-binding sites. Binding sites are then fine-tuned through high-order epistasis interactions by inserting distal mutations during later stages of evolution. Allosterically coupled, long-range dynamic networks favor catalytically competent conformational states that are more suitable for recognizing and stabilizing the aromatic substrate. This work provides mechanistic insight into enzymatic and evolutionary molecular mechanisms and spots the importance of iterative experimental and computational analyses to understand local-to-global changes.

Unveiling molecular details behind improved activity at neutral to alkaline pH of an engineered DyP-type peroxidase.

DyP-type peroxidases (DyPs) are microbial enzymes that catalyze the oxidation of a wide range of substrates, including synthetic dyes, lignin-derived compounds, and metals, such as Mn2+ and Fe2+, and have enormous biotechnological potential in biorefineries. However, many questions on the molecular basis of enzyme function and stability remain unanswered. In this work, high-resolution structures of PpDyP wild-type and two engineered variants (6E10 and 29E4) generated by directed evolution were obtained. The X-ray crystal structures revealed the typical ferredoxin-like folds, with three heme access pathways, two tunnels, and one cavity, limited by three long loops including catalytic residues. Variant 6E10 displays significantly increased loops' flexibility that favors function over stability: despite the considerably higher catalytic efficiency, this variant shows poorer protein stability compared to wild-type and 29E4 variants. Constant-pH MD simulations revealed a more positively charged microenvironment near the heme pocket of variant 6E10, particularly in the neutral to alkaline pH range. This microenvironment affects enzyme activity by modulating the pK a of essential residues in the heme vicinity and should account for variant 6E10 improved activity at pH 7-8 compared to the wild-type and 29E4 that show optimal enzymatic activity close to pH 4. Our findings shed light on the structure-function relationships of DyPs at the molecular level, including their pH-dependent conformational plasticity. These are essential for understanding and engineering the catalytic properties of DyPs for future biotechnological applications.

Negative impact of SARS-CoV-2 infection in acute coronary syndrome mortality in a Latin American cohort study.

Purpose: COVID-19 infection has been associated with a high risk of complications and death among patients with acute coronary syndrome (ACS). However, there is little information on the simultaneous involvement in Latin American countries. Methods: In the period between May 2020 and February 2021, an observational, longitudinal, prospective cohort study with two parallel branches was conducted in private and public hospitals in Brasilia, Brazil, including patients with ACS with and without a positive SARS-CoV-2 test result during hospitalization. Results: A total of 149 patients with ACS were included (75 with COVID-19 and 74 controls). Patients with COVID-19 exhibited an average of 62 years of age, 57% men, 40% diabetics, 67% hypertensive, 48% had an ACS with ST-segment elevation, Killip I was predominant, a low Syntax Score in 72%, with an average Grace Score of 117, and a length of hospitalization of 43 days in average. The control branch was similar in clinical characteristics, except for a lower proportion of ST-segment elevation ACS (16%, p < 0.01) and a higher incidence of arrhythmias (8 vs. 20 %, p = 0.03). Using the Cox regression method of analysis of covariates collected in the study, it was identified that patients with COVID-19 had a risk of death 2.34 times higher than patients without COVID-19 (p = 0.049). Conclusion: In this study conducted in a Latin American capital, SARS-CoV-2 infection predicted a higher chance of death in patients admitted with ACS, which is a finding that reinforces the need for greater care when diseases develop in overlapping ways.

Population homogeneity for the antibody response to COVID-19 BNT162b2/Comirnaty vaccine is only reached after the second dose across all adult age ranges.

While mRNA vaccines are administrated worldwide in an effort to contain the COVID-19 pandemic, the heterogeneity of the humoral immune response they induce at the population scale remains unclear. Here, in a prospective, longitudinal, cohort-study, including 1245 hospital care workers and 146 nursing home residents scheduled for BNT162b2 vaccination, together covering adult ages from 19 to 99 years, we analyse seroconversion to SARS-CoV-2 spike protein and amount of spike-specific IgG, IgM and IgA before vaccination, and 3-5 weeks after each dose. We show that immunogenicity after a single vaccine dose is biased to IgG, heterogeneous and reduced with increasing age. The second vaccine dose normalizes IgG seroconversion in all age strata. These findings indicate two dose mRNA vaccines is required to reach population scale humoral immunity. The results advocate for the interval between the two doses not to be extended, and for serological monitoring of elderly and immunosuppressed vaccinees.

Structure of Burkholderia cepacia UDP-glucose dehydrogenase (UGD) BceC and role of Tyr10 in final hydrolysis of UGD thioester intermediate.

Members of the Burkholderia cepacia complex (BCC) are serious respiratory pathogens in immunocompromised individuals and in patients with cystic fibrosis (CF). They are exceptionally resistant to many antimicrobial agents and have the capacity to spread between patients, leading to a decline in lung function and necrotizing pneumonia. BCC members often express a mucoid phenotype associated with the secretion of the exopolysaccharide (EPS) cepacian. There is much evidence supporting the fact that cepacian is a major virulence factor of BCC. UDP-glucose dehydrogenase (UGD) is responsible for the NAD-dependent 2-fold oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronic acid (UDP-GlcA), which is a key step in cepacian biosynthesis. Here, we report the structure of BceC, determined at 1.75-Å resolution. Mutagenic studies were performed on the active sites of UGDs, and together with the crystallographic structures, they elucidate the molecular mechanism of this family of sugar nucleotide-modifying enzymes. Superposition with the structures of human and other bacterial UGDs showed an active site with high structural homology. This family contains a strictly conserved tyrosine residue (Y10 in BceC; shown in italics) within the glycine-rich motif (GXGYXG) of its N-terminal Rossmann-like domain. We constructed several BceC Y10 mutants, revealing only residual dehydrogenase activity and thus highlighting the importance of this conserved residue in the catalytic activity of BceC. Based on the literature of the UGD/GMD nucleotide sugar 6-dehydrogenase family and the kinetic and structural data we obtained for BceC, we determined Y10 as a key catalytic residue in a UGD rate-determining step, the final hydrolysis of the enzymatic thioester intermediate.

Longitudinal Analysis of Antibody Responses to the mRNA BNT162b2 Vaccine in Patients Undergoing Maintenance Hemodialysis: A 6-Month Follow-Up.

Background: Patients on hemodialysis (HD) are at higher risk for COVID-19, overall are poor responders to vaccines, and were prioritized in the Portuguese vaccination campaign. Objective: This work aimed at evaluating in HD patients the immunogenicity of BTN162b2 after the two doses induction phase, the persistence of specific antibodies along time, and factors predicting these outcomes. Methods: We performed a prospective, 6-month long longitudinal cohort analysis of 156 HD patients scheduled to receive BTN162b2. ELISA quantified anti-spike IgG, IgM, and IgA levels in sera were collected every 3 weeks during the induction phase (t0 before vaccine; t1, d21 post first dose; and t2 d21 post second dose), and every 3-4 months during the waning phase (t3, d140, and t4, d180 post first dose). The age-matched control cohort was similarly analyzed from t0 to t2. Results: Upon exclusion of participants identified as previously exposed to SARS-CoV-2, seroconversion at t1 was lower in patients than controls (29 and 50%, respectively, p = 0.0014), while the second vaccine dose served as a boost in both cohorts (91 and 95% positivity, respectively, at t2, p = 0.2463). Lower response in patients than controls at t1 was a singularity of the participants ≤ 70 years (p = 2.01 × 10-05), associated with immunosuppressive therapies (p = 0.013), but not with lack of responsiveness to hepatitis B. Anti-spike IgG, IgM, and IgA levels decreased at t3, with IgG levels further waning at t4 and resulting in >30% seronegativity. Anti-spike IgG levels at t1 and t4 were correlated (ρ = 0.65, p < 2.2 × 10-16). Conclusions: While most HD patients seroconvert upon 2 doses of BNT162b2 vaccination, anti-spike antibodies levels wane over the following 4 months, leading to early seroreversion in a sizeable fraction of the patients. These findings warrant close monitoring of COVID-19 infection in vaccinated HD patients, and advocate for further studies following reinforced vaccination schedules.