Effect of SARS-CoV-2 S protein on the proteolytic cleavage of the epithelial Na+ channel ENaC.

Severe cases of COVID-19 are characterized by development of acute respiratory distress syndrome (ARDS). Water accumulation in the lungs is thought to occur as consequence of an exaggerated inflammatory response. A possible mechanism could involve decreased activity of the epithelial Na+ channel, ENaC, expressed in type II pneumocytes. Reduced transepithelial Na+ reabsorption could contribute to lung edema due to reduced alveolar fluid clearance. This hypothesis is based on the observation of the presence of a novel furin cleavage site in the S protein of SARS-CoV-2 that is identical to the furin cleavage site present in the alpha subunit of ENaC. Proteolytic processing of αENaC by furin-like proteases is essential for channel activity. Thus, competition between S protein and αENaC for furin-mediated cleavage in SARS-CoV-2-infected cells may negatively affect channel activity. Here we present experimental evidence showing that coexpression of the S protein with ENaC in a cellular model reduces channel activity. In addition, we show that bidirectional competition for cleavage by furin-like proteases occurs between 〈ENaC and S protein. In transgenic mice sensitive to lethal SARS-CoV-2, however, a significant decrease in gamma ENaC expression was not observed by immunostaining of lungs infected as shown by SARS-CoV2 nucleoprotein staining.

KS-WNK1 is Required for the Renal Response to Extreme Changes in Potassium Intake.

KS-WNK1 is an isoform of WNK1 kinase that is predominantly found in the distal convoluted tubule of the kidney. The precise physiological function of KS-WNK1 remains unclear. Some studies suggest that it could play a role in regulating potassium renal excretion by modulating the activity of the Na+-Cl- cotransporter (NCC). However, changes in the potassium diet from normal to high failed to reveal a role for KS-WNK1, but under a normal potassium diet, the expression of KS-WNK1 is negligible. It is only detectable when mice are exposed to a low potassium diet. In this study, we investigated the role of KS-WNK1 in regulating potassium excretion under extreme changes in potassium intake. After following a zero-potassium diet (0KD) for 10 days, KS-WNK1-/- mice had lower plasma levels of K+ and Cl-, while exhibiting higher urinary excretion of Na+, Cl-, and K+ compared to KS-WNK1+/+ mice. After 10 days of 0KD or normal-potassium diet (NKD), all mice were challenged with a high-potassium diet (HKD). Plasma K+ levels markedly increased after the HKD challenge only in mice previously fed with 0KD, regardless of genotype. KSWNK1+/+ mice adapt better to HKD-challenge than KS-WNK1-/- mice after a potassium-retaining state. The difference in the pNCC/NCC ratio between KS-WNK1+/+ and KS-WNK1-/- mice after 0KD and HKD indicates a role for KS-WNK1 in both, NCC phosphorylation and dephosphorylation. These observations show that KS-WNK1 helps the DCT to respond to extreme changes in potassium intake, such as those occurring in wildlife.

Arginine vasopressin regulates the renal Na+-Cl- and Na+-K+-Cl- cotransporters through with-no-lysine kinase 4 and inhibitor 1 phosphorylation.

Vasopressin regulates water homeostasis via the V2 receptor in the kidney at least in part through protein kinase A (PKA) activation. Vasopressin, through an unknown pathway, upregulates the activity and phosphorylation of Na+-Cl- cotransporter (NCC) and Na+-K+-2Cl- cotransporter 2 (NKCC2) by Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1), which are regulated by the with-no-lysine kinase (WNK) family. Phosphorylation of WNK4 at PKA consensus motifs may be involved. Inhibitor 1 (I1), a protein phosphatase 1 (PP1) inhibitor, may also play a role. In human embryonic kidney (HEK)-293 cells, we assessed the phosphorylation of WNK4, SPAK, NCC, or NKCC2 in response to forskolin or desmopressin. WNK4 and cotransporter phosphorylation were studied in desmopressin-infused WNK4-/- mice and in tubule suspensions. In HEK-293 cells, only wild-type WNK4 but not WNK1, WNK3, or a WNK4 mutant lacking PKA phosphorylation motifs could upregulate SPAK or cotransporter phosphorylation in response to forskolin or desmopressin. I1 transfection maximized SPAK phosphorylation in response to forskolin in the presence of WNK4 but not of mutant WNK4 lacking PP1 regulation. We observed direct PP1 regulation of NKCC2 dephosphorylation but not of NCC or SPAK in the absence of WNK4. WNK4-/- mice with desmopressin treatment did not increase SPAK/OSR1, NCC, or NKCC2 phosphorylation. In stimulated tubule suspensions from WNK4-/- mice, upregulation of pNKCC2 was reduced, whereas upregulation of SPAK phosphorylation was absent. These findings suggest that WNK4 is a central node in which kinase and phosphatase signaling converge to connect cAMP signaling to the SPAK/OSR1-NCC/NKCC2 pathway.NEW & NOTEWORTHY With-no-lysine kinases regulate the phosphorylation and activity of the Na+-Cl- and Na+-K+-2Cl- cotransporters. This pathway is modulated by arginine vasopressin (AVP). However, the link between AVP and WNK signaling remains unknown. Here, we show that AVP activates WNK4 through increased phosphorylation at putative protein kinase A-regulated sites and decreases its dephosphorylation by protein phosphatase 1. This work increases our understanding of the signaling pathways mediating AVP actions in the kidney.

From Fish Physiology to Human Disease: The Discovery of the NCC, NKCC2, and the Cation-Coupled Chloride Cotransporters.

The renal Na-K-2Cl and Na-Cl cotransporters are the major salt reabsorption pathways in the thick ascending limb of Henle loop and the distal convoluted tubule, respectively. These transporters are the target of the loop and thiazide type diuretics extensively used in the world for the treatment of edematous states and arterial hypertension. The diuretics appeared in the market many years before the salt transport systems were discovered. The evolving of the knowledge and the cloning of the genes encoding the Na-K-2Cl and Na-Cl cotransporters were possible thanks to the study of marine species. This work presents the history of how we came to know the mechanisms for the loop and thiazide type diuretics actions, the use of marine species in the cloning process of these cotransporters and therefore in the whole solute carrier cotransproters 12 (SLC12) family of electroneutral cation chloride cotransporters, and the disease associated with each member of the family.

Dysregulation of the WNK4-SPAK/OSR1 pathway has a minor effect on baseline NKCC2 phosphorylation.

The with-no-lysine kinase 4 (WNK4)-sterile 20/SPS-1-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) pathway mediates activating phosphorylation of the furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) and the thiazide-sensitive NaCl cotransporter (NCC). The commonly used pT96/pT101-pNKCC2 antibody cross-reacts with pT53-NCC in mice on the C57BL/6 background due to a five amino acid deletion. We generated a new C57BL/6-specific pNKCC2 antibody (anti-pT96-NKCC2) and tested the hypothesis that the WNK4-SPAK/OSR1 pathway strongly regulates the phosphorylation of NCC but not NKCC2. In C57BL/6 mice, anti-pT96-NKCC2 detected pNKCC2 and did not cross-react with NCC. Abundances of pT96-NKCC2 and pT53-NCC were evaluated in Wnk4-/-, Osr1-/-, Spak-/-, and Osr1-/-/Spak-/- mice and in several models of the disease familial hyperkalemic hypertension (FHHt) in which the CUL3-KLHL3 ubiquitin ligase complex that promotes WNK4 degradation is dysregulated (Cul3+/-/Δ9, Klhl3-/-, and Klhl3R528H/R528H). All mice were on the C57BL/6 background. In Wnk4-/- mice, pT53-NCC was almost absent but pT96-NKCC2 was only slightly lower. pT53-NCC was almost absent in Spak-/- and Osr1-/-/Spak-/- mice, but pT96-NKCC2 abundance did not differ from controls. pT96-NKCC2/total NKCC2 was slightly lower in Osr1-/- and Osr1-/-/Spak-/- mice. WNK4 expression colocalized not only with NCC but also with NKCC2 in Klhl3-/- mice, but pT96-NKCC2 abundance was unchanged. Consistent with this, furosemide-induced urinary Na+ excretion following thiazide treatment was similar between Klhl3-/- and controls. pT96-NKCC2 abundance was also unchanged in the other FHHt mouse models. Our data show that disruption of the WNK4-SPAK/OSR1 pathway only mildly affects NKCC2 phosphorylation, suggesting a role for other kinases in NKCC2 activation. In FHHt models NKCC2 phosphorylation is unchanged despite higher WNK4 abundance, explaining the thiazide sensitivity of FHHt.NEW & NOTEWORTHY The renal cation cotransporters NCC and NKCC2 are activated following phosphorylation mediated by the WNK4-SPAK/OSR1 pathway. While disruption of this pathway strongly affects NCC activity, effects on NKCC2 activity are unclear since the commonly used phospho-NKCC2 antibody was recently reported to cross-react with phospho-NCC in mice on the C57BL/6 background. Using a new phospho-NKCC2 antibody specific for C57BL/6, we show that inhibition or activation of the WNK4-SPAK/OSR1 pathway in mice only mildly affects NKCC2 phosphorylation.

Regulation of the WNK4-SPAK-NCC pathway by the calcium-sensing receptor.

PURPOSE OF REVIEW: Regulation of the sodium chloride cotransporter (NCC) in the distal convoluted tubule (DCT) plays a crucial role in renal salt handling. The calcium-sensing receptor (CaSR) has been shown to activate NCC through the WNK4-SPAK pathway, which is independent of the Renin-Angiotensin-Aldosterone system. In this review, we examine new information about the mechanism of how the CaSR regulates NCC through the WNK4-SPAK pathway and its physiological and therapeutic implications. RECENT FINDINGS: The activation of CaSR in TALH cells during hypercalcemia inhibits NKCC2 and ROMK activity, reducing paracellular Ca2+ reabsorption but decreasing salt reabsorption. This pathway enables NaCl reabsorption in the DCT while promoting Ca2+ excretion. CaSR activation in the apical DCT stimulates a signaling pathway involving PKC, WNK4, and SPAK, which increases NCC activation to recover the NaCl not reabsorbed in TAHL. Glucose or fructose acting as calcimimetics enhance apical CaSR sensitivity, increasing NCC activity, which contribute to the mechanism of hypertension prevalence in diabetic patients or in those with high fructose consumption. SUMMARY: These findings reveal the importance of the CaSR-mediated activation of the WNK4-SPAK pathway in regulating salt and calcium homeostasis and its potential as a therapeutic target for hypertension and related diseases.

SLC12A cryo-EM: analysis of relevant ion binding sites, structural domains, and amino acids.

The solute carrier family 12A (SLC12A) superfamily of membrane transporters modulates the movement of cations coupled with chloride across the membrane. In doing so, these cotransporters are involved in numerous aspects of human physiology: cell volume regulation, ion homeostasis, blood pressure regulation, and neurological action potential via intracellular chloride concentration modulation. Their physiological characterization has been largely studied; however, understanding the mechanics of their function and the relevance of structural domains or specific amino acids has been a pending task. In recent years, single-particle cryogenic electron microscopy (cryo-EM) has been successfully applied to members of the SLC12A family including all K+:Cl- cotransporters (KCCs), Na+:K+:2Cl- cotransporter NKCC1, and recently Na+:Cl- cotransporter (NCC); revealing structural elements that play key roles in their function. The present review analyzes the data provided by these cryo-EM reports focusing on structural domains and specific amino acids involved in ion binding, domain interactions, and other important SCL12A structural elements. A comparison of cryo-EM data from NKCC1 and KCCs is presented in the light of the two recent NCC cryo-EM studies, to propose insight into structural elements that might also be found in NCC and are necessary for its proper function. In the final sections, the importance of key coordination residues for substrate specificity and their implication on various pathophysiological conditions and genetic disorders is reviewed, as this could provide the basis to correlate structural elements with the development of novel and selective treatments, as well as mechanistic insight into the function and regulation of cation-coupled chloride cotransporters (CCCs).

Thirty years of the NaCl cotransporter: from cloning to physiology and structure.

The primary structure of the thiazide-sensitive NaCl cotransporter (NCC) was resolved 30 years ago by the molecular identification of the cDNA encoding this cotransporter, from the winter's flounder urinary bladder, following a functional expression strategy. This review outlines some aspects of how the knowledge about thiazide diuretics and NCC evolved, the history of the cloning process, and the expansion of the SLC12 family of electroneutral cotransporters. The diseases associated with activation or inactivation of NCC are discussed, as well as the molecular model by which the activity of NCC is regulated. The controversies in the field are discussed as well as recent publication of the three-dimensional model of NCC obtained by cryo-electron microscopy, revealing not only the amino acid residues critical for Na+ and Cl- translocation but also the residues critical for polythiazide binding to the transporter, opening the possibility for a new era in thiazide diuretic therapy.

SerpinA3K Deficiency Reduces Oxidative Stress in Acute Kidney Injury.

We previously showed that SerpinA3K is present in urine from rats and humans with acute kidney injury (AKI) and chronic kidney disease (CKD). However, the specific role of SerpinA3K during renal pathophysiology is unknown. To begin to understand the role of SerpinA3K on AKI, SerpinA3K-deficient (KOSA3) mice were studied 24 h after inducing ischemia/reperfusion (I/R) and compared to wild type (WT) mice. Four groups were studied: WT+S, WT+IR, KOSA3+S, and KOSA3+IR. As expected, I/R increased serum creatinine and BUN, with a GFR reduction in both genotypes; however, renal dysfunction was ameliorated in the KOSA3+IR group. Interestingly, the increase in UH2O2 induced by I/R was not equally seen in the KOSA3+IR group, an effect that was associated with the preservation of antioxidant enzymes' mRNA levels. Additionally, FOXO3 expression was initially greater in the KOSA3 than in the WT group. Moreover, the increase in BAX protein level and the decrease in Hif1a and Vegfa induced by I/R were not observed in the KOSA3+IR group, suggesting that these animals have better cellular responses to hypoxic injury. Our findings suggest that SerpinA3K is involved in the renal oxidant response, HIF1α/VEGF pathway, and cell apoptosis.

The serine-threonine protein phosphatases that regulate the thiazide-sensitive NaCl cotransporter.

The activity of the Na+-Cl- cotransporter (NCC) in the distal convoluted tubule (DCT) is finely tuned by phosphorylation networks involving serine/threonine kinases and phosphatases. While much attention has been paid to the With-No-lysine (K) kinase (WNK)- STE20-related Proline Alanine rich Kinase (SPAK)/Oxidative Stress Responsive kinase 1 (OSR1) signaling pathway, there remain many unanswered questions regarding phosphatase-mediated modulation of NCC and its interactors. The phosphatases shown to regulate NCC's activity, directly or indirectly, are protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), calcineurin (CN), and protein phosphatase 4 (PP4). PP1 has been suggested to directly dephosphorylate WNK4, SPAK, and NCC. This phosphatase increases its abundance and activity when extracellular K+ is increased, which leads to distinct inhibitory mechanisms towards NCC. Inhibitor-1 (I1), oppositely, inhibits PP1 when phosphorylated by protein kinase A (PKA). CN inhibitors, like tacrolimus and cyclosporin A, increase NCC phosphorylation, giving an explanation to the Familial Hyperkalemic Hypertension-like syndrome that affects some patients treated with these drugs. CN inhibitors can prevent high K+-induced dephosphorylation of NCC. CN can also dephosphorylate and activate Kelch-like protein 3 (KLHL3), thus decreasing WNK abundance. PP2A and PP4 have been shown in in vitro models to regulate NCC or its upstream activators. However, no studies in native kidneys or tubules have been performed to test their physiological role in NCC regulation. This review focuses on these dephosphorylation mediators and the transduction mechanisms possibly involved in physiological states that require of the modulation of the dephosphorylation rate of NCC.

Structure-function relationships in the sodium chloride cotransporter.

The thiazide sensitive Na+:Cl- cotransporter (NCC) is the principal via for salt reabsorption in the apical membrane of the distal convoluted tubule (DCT) in mammals and plays a fundamental role in managing blood pressure. The cotransporter is targeted by thiazide diuretics, a highly prescribed medication that is effective in treating arterial hypertension and edema. NCC was the first member of the electroneutral cation-coupled chloride cotransporter family to be identified at a molecular level. It was cloned from the urinary bladder of the Pseudopleuronectes americanus (winter flounder) 30 years ago. The structural topology, kinetic and pharmacology properties of NCC have been extensively studied, determining that the transmembrane domain (TM) coordinates ion and thiazide binding. Functional and mutational studies have discovered residues involved in the phosphorylation and glycosylation of NCC, particularly on the N-terminal domain, as well as the extracellular loop connected to TM7-8 (EL7-8). In the last decade, single-particle cryogenic electron microscopy (cryo-EM) has permitted the visualization of structures at high atomic resolution for six members of the SLC12 family (NCC, NKCC1, KCC1-KCC4). Cryo-EM insights of NCC confirm an inverted conformation of the TM1-5 and TM6-10 regions, a characteristic also found in the amino acid-polyamine-organocation (APC) superfamily, in which TM1 and TM6 clearly coordinate ion binding. The high-resolution structure also displays two glycosylation sites (N-406 and N-426) in EL7-8 that are essential for NCC expression and function. In this review, we briefly describe the studies related to the structure-function relationship of NCC, beginning with the first biochemical/functional studies up to the recent cryo-EM structure obtained, to acquire an overall view enriched with the structural and functional aspects of the cotransporter.

Glucose/Fructose Delivery to the Distal Nephron Activates the Sodium-Chloride Cotransporter via the Calcium-Sensing Receptor.

BACKGROUND: The calcium-sensing receptor (CaSR) in the distal convoluted tubule (DCT) activates the NaCl cotransporter (NCC). Glucose acts as a positive allosteric modulator of the CaSR. Under physiologic conditions, no glucose is delivered to the DCT, and fructose delivery depends on consumption. We hypothesized that glucose/fructose delivery to the DCT modulates the CaSR in a positive allosteric way, activating the WNK4-SPAK-NCC pathway and thus increasing salt retention. METHODS: We evaluated the effect of glucose/fructose arrival to the distal nephron on the CaSR-WNK4-SPAK-NCC pathway using HEK-293 cells, C57BL/6 and WNK4-knockout mice, ex vivo perfused kidneys, and healthy humans. RESULTS: HEK-293 cells exposed to glucose/fructose increased SPAK phosphorylation in a WNK4- and CaSR-dependent manner. C57BL/6 mice exposed to fructose or a single dose of dapagliflozin to induce transient glycosuria showed increased activity of the WNK4-SPAK-NCC pathway. The calcilytic NPS2143 ameliorated this effect, which was not observed in WNK4-KO mice. C57BL/6 mice treated with fructose or dapagliflozin showed markedly increased natriuresis after thiazide challenge. Ex vivo rat kidney perfused with glucose above the physiologic threshold levels for proximal reabsorption showed increased NCC and SPAK phosphorylation. NPS2143 prevented this effect. In healthy volunteers, cinacalcet administration, fructose intake, or a single dose of dapagliflozin increased SPAK and NCC phosphorylation in urinary extracellular vesicles. CONCLUSIONS: Glycosuria or fructosuria was associated with increased NCC, SPAK, and WNK4 phosphorylation in a CaSR-dependent manner.

The burden of chronic kidney disease in Mexico: data analysis based on the Global Burden of Disease 2021 study.

BACKGROUND: Chronic kidney disease (CKD) represents a substantial global burden of disease due to a lack of universal tests and misinterpretation of biomarkers. OBJECTIVE: To analyze CKD epidemiology in Mexico and guide public policies. MATERIAL AND METHODS: Data from the Global Burden of Disease (GBD) 2021 study were used to describe CKD prevalence and mortality in Mexico for the 1990-2021 period, stratifying by gender and age groups. RESULTS: The prevalence of CKD in Mexico in 2021 was 9,184.9 per 100,000 population. Diabetes was the most common cause of CKD, and CKD-related mortality was high, with an increase in 2019 and 2021, possibly as a consequence of the COVID-19 pandemic. CONCLUSIONS: CKD in Mexico entails a high burden of mortality and years of life lost, but it barely contributes to disability. It is essential to improve CKD early detection, access to treatments and coding of the causes of the disease. Moreover, investigating the causes of CKD of unknown etiology, including genetic factors, is crucial in order for specific treatments to be developed in the future.

ANTECEDENTES: La enfermedad renal crónica (ERC) representa una elevada carga global de enfermedad debido a la falta de pruebas universales y a la interpretación errónea de biomarcadores. OBJETIVO: Analizar la epidemiología de la ERC en México y orientar las políticas públicas. MATERIAL Y MÉTODOS: Se utilizaron los datos del estudio Global Burden of Disease (GBD) 2021 para describir la prevalencia y mortalidad de la ERC en México durante el periodo de 1990 a 2021, estratificando por sexo y grupos de edad. RESULTADOS: La prevalencia de la ERC en México en 2021 fue de 9184.9 por 100 000 habitantes. La diabetes constituyó la causa más común de ERC y la mortalidad por ERC fue elevada, se incrementó en 2019 y 2021, posiblemente debido a la pandemia de COVID-19. CONCLUSIONES: La ERC en México presenta una alta carga de mortalidad y años de vida perdidos, pero contribuye poco a la discapacidad. Es esencial mejorar la detección temprana de la ERC, el acceso a tratamientos y la codificación de las causas de la enfermedad. Además, investigar las causas de la ERC de etiología desconocida, incluidos factores genéticos, es crucial para desarrollar tratamientos específicos en el futuro.

Towards the development of an epitope-focused vaccine for SARS-CoV-2.

The rapid spread of COVID-19 on all continents and the mortality induced by SARS-CoV-2 virus, the cause of the pandemic coronavirus disease 2019 (COVID-19) has motivated an unprecedented effort for vaccine development. Inactivated viruses as well as vaccines focused on the partial or total sequence of the Spike protein using different novel platforms such us RNA, DNA, proteins, and non-replicating viral vectors have been developed. The high global need for vaccines, now and in the future, and the emergence of new variants of concern still requires development of accessible vaccines that can be adapted according to the most prevalent variants in the respective regions. Here, we describe the immunogenic properties of a group of theoretically predicted RBD peptides to be used as the first step towards the development of an effective, safe and low-cost epitope-focused vaccine. One of the tested peptides named P5, proved to be safe and immunogenic. Subcutaneous administration of the peptide, formulated with alumina, induced high levels of specific IgG antibodies in mice and hamsters, as well as an increase of IFN-γ expression by CD8+ T cells in C57 and BALB/c mice upon in vitro stimulation with P5. Neutralizing titers of anti-P5 antibodies, however, were disappointingly low, a deficiency that we will attempt to resolve by the inclusion of additional immunogenic epitopes to P5. The safety and immunogenicity data reported in this study support the use of this peptide as a starting point for the design of an epitope restricted vaccine.

Arterial Blood Pressure, Neuronal Excitability, Mineral Metabolism and Cell Volume Regulation Mechanisms Revealed by Xenopus laevis oocytes.

Xenopus laevis oocytes have been an invaluable tool to discover and explore the molecular mechanisms and characteristics of many proteins, in particular integral membrane proteins. The oocytes were fundamental in many projects designed to identify the cDNA encoding a diversity of membrane proteins including receptors, transporters, channels and pores. In addition to being a powerful tool for cloning, oocytes were later used to experiment with the functional characterization of many of the identified proteins. In this review I present an overview of my personal 30-year experience using Xenopus laevis oocytes and the impact this had on a variety of fields such as arterial blood pressure, neuronal excitability, mineral metabolism and cell volume regulation.

An RBD-Based Diagnostic Method Useful for the Surveillance of Protective Immunity against SARS-CoV-2 in the Population.

After more than two years, the COVID-19 pandemic is still ongoing and evolving all over the world; human herd immunity against SARS-CoV-2 increases either by infection or by unprecedented mass vaccination. A substantial change in population immunity is expected to contribute to the control of transmission. It is essential to monitor the extension and duration of the population's immunity to support the decisions of health authorities in each region and country, directed to chart the progressive return to normality. For this purpose, the availability of simple and cheap methods to monitor the levels of relevant antibodies in the population is a widespread necessity. Here, we describe the development of an RBD-based ELISA for the detection of specific antibodies in large numbers of samples. The recombinant expression of an RBD-poly-His fragment was carried out using either bacterial or eukaryotic cells in in vitro culture. After affinity chromatography purification, the performance of both recombinant products was compared by ELISA in similar trials. Our results showed that eukaryotic RBD increased the sensitivity of the assay. Interestingly, our results also support a correlation of the eukaryotic RBD-based ELISA with other assays aimed to test for neutralizing antibodies, which suggests that it provides an indication of protective immunity against SARS-CoV-2.