Sph2(176–191) and Sph2(446–459): Identification of B-Cell Linear Epitopes in Sphingomyelinase 2 (Sph2), Naturally Recognized by Patients Infected by Pathogenic Leptospires

Sphingomyelin is a major constituent of eukaryotic cell membranes, and if degraded by bacteria sphingomyelinases may contribute to the pathogenesis of infection. Among Leptospira spp., there are five sphingomyelinases exclusively expressed by pathogenic leptospires, in which Sph2 is expressed during natural infections, cytotoxic, and implicated in the leptospirosis hemorrhagic complications. Considering this and the lack of information about associations between Sph2 and leptospirosis severity, we use a combination of immunoinformatics approaches to identify its B-cell epitopes, evaluate their reactivity against samples from leptospirosis patients, and investigate the role of antibodies anti-Sph2 in protection against severe leptospirosis. Two B-cell epitopes, Sph2(176-191) and Sph2(446-459), were predicted in Sph2 from L. interrogans serovar Lai, presenting different levels of identity when compared with other pathogenic leptospires. These epitopes were recognized by about 40% of studied patients with a prevalence of IgG antibodies against both Sph2(176-191) and Sph2(446-459). Remarkably, just individuals with low reactivity to Sph2(176-191) presented clinical complications, while high responders had only mild symptoms. Therefore, we identified two B-cell linear epitopes, recognized by antibodies of patients with leptospirosis, that could be further explored in the development of multi-epitope vaccines against leptospirosis.


Introduction
Leptospirosis is a tropical and neglected emerging zoonotic disease that afflicts humans and other animals [1]. It is considered a global public health problem, with an estimated one million new leptospirosis cases reported annually and a mortality rate of about 60,000 [2]. Although the disease occurs worldwide, it is most prevalent in tropical countries, where conditions for transmission are most favorable [3]. In Brazil, leptospirosis is a serious public health problem because over 3500 cases are reported annually, leading to an up to 75% hospitalization rate and resulting in the death of about 10% of patients [4,5].
The disease is caused by pathogenic spirochetes from the genus Leptospira, which can infect humans and almost all mammals, as well as reptiles and amphibians [6]. The transmission occurs when bacteria from contaminated soil or water come into contact with
Regarding the used algorithms, Bepipred combines the hidden Markov model with the propensity scale by Parker et al. to predict linear B-cell epitopes through the sequence of the protein in FASTA format [32]. Emini Surface Accessibility prediction indicates the probability of a peptide being found on the surface of a protein by calculating the surface accessibility of hexapeptides [33]. ABCpred is a server that allows the prediction of continuous B-cell epitopes in a protein through the amino acid sequence with 65.93% accuracy according to its validation tests [34]. ElliPro is a web tool that allows the prediction of antibody epitopes in a protein amino acid sequence or structure with the implementation of a modified version of Thornton's method, residue clustering algorithm, and the MODELLER program [35]. BCePred allows users to predict B-cell epitopes using physicochemical properties (hydrophilicity, flexibility/mobility, accessibility, polarity, exposed surface, and turns) or a combination of properties [36]. LBtope is a web server that predicts linear B-cell epitopes through SVM-based models using dipeptide composition generated from the query sequence(s). The overall accuracy of this server is approximately 81% [37]. COBEpro is a two-step system for predicting continuous B-cell epitopes, that first uses a support vector machine to make predictions on short peptide fragments within the query antigen sequence and then calculates an epitopic propensity score for each residue based on the fragment predictions. Secondary structure and solvent accessibility information (either predicted or exact) can be incorporated to improve performance. COBEpro is incorporated into the SCRATCH prediction suite at http://scratch.proteomics.ics.uci.edu [38]. All the mentioned algorithms were used considering their default thresholds. Sequences with at least 10 amino acids, predicted by at least five algorithms, were considered predicted B-cell linear epitopes.

Antigenicity Analysis
Each predicted epitope was evaluated for antigenicity by the VaxiJen algorithm http: //www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html (accessed on 12 June 2020) with the default threshold (0.4). VaxiJen is a protective antigen prediction server that allows classification based only on the physical-chemical properties of the protein of interest [39].

Conservation Analysis
Sequence alignment and identification of conserved patterns among sphingomyelinases from L. interrogans serovar Lai [Sph2 (Uniprot ID: P59116), Sph1 (Uniprot ID: P59115), Sph3 (Uniprot ID: A0A0E2DC81), Sph4 (Uniprot ID: A0A0E2DCF7), and SphH (Uniprot ID: O34095)] and other bacteria including Listeria ivanovii (SmcLUniprot ID: Q9RLV9), Bacillus cereus (BC SMase, Uniprot ID: P09599), Staphylococcus aureus (Uniprot ID: A0A7U4AUV1), and Pseudomonas spp. strain TK4 (Uniprot ID: Q93HR5) were conducted by MAFFT [40], using the software MegAlign pro. In the same way, to verify the conservation degree of Sph2 among pathogenic Leptospira spp., the reference Sph2 was aligned and compared with all other 17 Sph2 described in Table 1. To investigate the conservation degree of predicted epitopes among pathogenic leptospires, each predicted sequence was aligned and compared with all Sph2 sequences described in Table 1. Values of identity (%) represent the percentage of equal amino acids aligned. Moreover, we compared the conservation of amino acids present in the catalytic sites, and metal-binding sites were compared among Sph2 from pathogenic leptospires and other sphingomyelinases, based on the study of Narayanavari S.A. and collaborators [18].

Peptide Synthesis
Sequences predicted as antigenic linear B-cell epitopes were synthesized using fluorenylmethoxycarbonyl (F-moc) solid-phase chemistry [41,42] (WatsonBio, Houston, TX, USA). Analytical chromatography of the peptides demonstrated a purity degree higher than 95%, and mass spectrometry analysis of the peptides indicated estimated masses corresponding to the molecular masses of the peptides.

Studied Population
In this study, 87 serum samples of Brazilian patients were provided by the National Reference Laboratory for Leptospirosis -Fiocruz-RJ. All samples were previously tested by microscopic agglutination test (MAT), resulting in 51 leptospirosis patients (MAT positive), reactive in MAT, and 36 negative controls (MAT negative). The study was reviewed and approved by the Oswaldo Cruz Foundation Ethical Committee and the National Ethical Committee of Brazil (number CAAE: 31405820.8.0000.5262).

Evaluation of Natural Immunogenicity of Predicted Epitopes
Samples of confirmed leptospirosis cases and the control group were screened for the presence of naturally acquired antibodies against the synthetic peptides via ELISA as previously described [24].
Briefly, MaxiSorp 96-well plates (Nunc, Rochester, NY, USA) were coated with 100 µg/mL of a peptide. After overnight incubation at 4 • C, plates were washed with phosphatebuffered saline (PBS) and blocked with PBS-containing 5% non-fat dry milk (PBS-M) for 1 h at 37 • C. Individual serum samples diluted 1:100 on PBS-M were added in duplicate wells, and the plates were incubated at 37 • C for 1.5 h. After three washes with PBS-Tween20 (0.05%), bound antibodies were detected with peroxidase-conjugated goat antihuman IgG (SouthernBiotech, catalog number: 2048-05) or goat anti-human IgM (catalog number: 2020-05), diluted at 1:1000 (in PBS-M), and incubated for 1 h at 37 • C, followed by TMB (3,3 ,5,5 -tetramethylbenzidine). The reaction was stopped by the addition of HCl (1N), and the absorbance was read at 450 nm using an xMark™ microplate absorbance spectrophotometer (Bio-Rad, Hercules, CA, USA). The results for total IgG and IgM were expressed as the reactivity index (RI)-the ratio between the mean optical density (OD) of tested samples and the mean OD of 44 control group samples plus 2.5 standard deviations (SD). Subjects were considered IgG responders to a particular antigen if the RI was higher than 1.

Statistical Analysis of Data
The obtained data were analyzed using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA). First, to determine if a variable was normally distributed, the one-sample Kolmogorov-Smirnoff test was used. Differences in frequencies of IgG and IgM responders to synthetic peptides were evaluated using Fisher's exact test, while the reactivity indices against synthetic peptides between responders to each epitope were compared using the Mann-Whitney test. A two-sided p-value < 0.05 was considered significant.

Epitopes Location and Sph2 Active Sites
In 2012, Narayanavari and collaborators described the main amino acids that compose the SPh2 catalytic and metal-binding sites [18]. Based on their study, we investigated the conservation of both catalytic and metal-bind sites among SPh2 of studied pathogenic leptospires. As shown in Table 4, there are four main amino acids described in the L. interrogans serovar Lai Sph2 catalytic site (H293, D393, Y394, and H433) and five in the central metal-binding site (N161, E200, D341, N343, and D432). Considering this, when we compared aligned amino acids in Sph2 from L. interrogans serovar Lai with other pathogenic leptospires, the histidine at position 293 (H293) was the unique amino acid conserved in all studied leptospires. Moreover, the number of modified amino acids in the catalytic or metal-binding sites ranged from one to six among the studied Sph2. In this context, while Sph2 from L. interrogans serovar Bataviae; L. alstonii serovars Pingchang and Sichuan; L. borgpetersenii serovars Pomona and Hardjo-bovis; L. noguchii serovars Panama and Autumnalis; and L. santarosai serovar Arenal presented no modifications in amino acid residues in the catalytic or metal-binding sites, L. interrogans serovar Australis presented six (67%) different residues. Additionally, there were eight surface-exposed amino acids described as being associated with the interaction with the host cell membrane (W172, Y242, W274, F275, Y382, Y383, Y384, and Y425). Interestingly, these amino acids seemed to be less conserved among pathogenic leptospires, only one out of the three known serovars (L. interrogans serovar Bataviae, and L. noguchii serovars Panama and Autumnalis) presented no changed amino acids in the alignment, while the other studied proteins presented from three to six changed amino acids. Additionally, when compared with sphingomyelinases from other bacteria, Sph2 presented differences only in amino acids involved with the interaction with host cells, while all amino acids of the catalytic site and metal-binding sites remained unaltered (Table 4). Table 4. Amino acids in pathogenic leptospires Sph2 catalytic sites, metal-binding sites, and amino acids involved in host membrane interactions.

Specie (Serovar)-Protein
Catalytic Site Amino Acids in the Central Metal-Binding Site Surface-Exposed Amino Acids Involved with the Host Membrane Interaction Non-conserved amino acids with L. interrogans serovar Lai are indicated by gray cells.
Regarding the location of Sph2 epitopes, only the epitope Sph2(176-191) was inserted in Sphingomyelinase C domain (Sph2(155-440)). Finally, aiming to allow the visualization of predicted epitopes, catalytic and metal-binding sites, and amino acids associated with the interaction with the host cell membrane in the Sph2 3D structure, we highlight these structures in the Alphafold predicted model (ID: AF-P59116-F1) (Figure 1).

Studied Population
The studied population was composed of 87 Brazilian febrile suspected leptospirosis cases based on their contact with rodents, floods, or other risk factors. Among them, 51 patients were reactive to Leptospira spp. (MAT positive), presenting antibody titers on MAT ranging from 1:800 to 1:12800 (mean: 1:2839 ± 2407), and were grouped as reactive against Leptospira spp. (RL), while 36 patients were non-reactive against Leptospira spp. (NRL).
The studied population presented was 35.7 (±17.5) mean years old, and clinical and epidemiological data were obtained between 1 and 74 days after the beginning of symptoms (mean 12.5 ± 12). Our studied group was mostly composed of men (85%), but we did not observe statistical differences in the frequency of men in RL (90%) and NRL (78%) (p = 0.134). Moreover, in both groups, RL and NRL, about 11.5% of deaths cases were reported. As shown in Table 5, these groups were also similar in age and days of symptoms.

Studied Population
The studied population was composed of 87 Brazilian febrile suspected leptospirosis cases based on their contact with rodents, floods, or other risk factors. Among them, 51 patients were reactive to Leptospira spp. (MAT positive), presenting antibody titers on MAT ranging from 1:800 to 1:12800 (mean: 1:2839 ± 2407), and were grouped as reactive against Leptospira spp. (RL), while 36 patients were non-reactive against Leptospira spp. (NRL).
The studied population presented was 35.7 (±17.5) mean years old, and clinical and epidemiological data were obtained between 1 and 74 days after the beginning of symptoms (mean 12.5 ± 12). Our studied group was mostly composed of men (85%), but we did not observe statistical differences in the frequency of men in RL (90%) and NRL (78%) (p = 0.134). Moreover, in both groups, RL and NRL, about 11.5% of deaths cases were reported. As shown in Table 5, these groups were also similar in age and days of symptoms. Regarding reported symptoms, fever (84%), myalgia (77%), and headache (63%) were the most prevalent symptoms among studied patients. However, comparing the frequencies of symptoms among groups, only jaundice (RL = 68.6% and NRL = 41.7%; p = 0.0159), calf pain (RL = 60.8% and NRL = 41.7%, p = 0.0257), renal insufficiency (RL = 39.2% and NRL = 13.9%, p = 0.0463), and pulmonary hemorrhage (RL = 13.7% and NRL = 0%, p = 0.0382) were statistically more frequent in RL patients.
In the RL group, only one Leptospira serovar was detected by MAT using serum samples of 35 patients (69%), while the samples of 14 individuals (27%) cross-reacted with two serovars, one individual sample (2%) cross-reacted with serovars Australis, Hebdomadis, and Autumnalis, and another (2%) recognized the serovars Copenhageni, Canicola, Icterohaemorrhagiae, and Tarassovi (Table 5). Regarding diagnosed serovars in studied patients, Tarassovi and Copenhageni were the most prevalent serovars, each of them diagnosed in about 47% of studied patients, singly reported in 31.4% and 23.5%, respectively, and in 15.7% and 23.5% of patients whose sera recognized two or more serovars.

Discussion
Sphingomyelin (SM) is a major constituent of eukaryotic cell membranes and the ability to degrade this phospholipid by bacteria may consequently contribute to the pathogenesis of infection. Sphingomyelinases are a group of hemolysins, present in both eukaryotes and prokaryotes, which are related to phospholipid metabolism in the former and that frequently act as toxins in the latter [43], that are absent in non-pathogenic Leptospira spp. [17,44]. In pathogenic Leptospira, sphingomyelinases play an important role in their survival in the mammalian host by mediating the lysis and release of essential nutrients from the host cells [18], but are implicated in the hemorrhagic complications associated with leptospirosis [45,46]. Among sphingomyelinases described in L. interrogans

Discussion
Sphingomyelin (SM) is a major constituent of eukaryotic cell membranes and the ability to degrade this phospholipid by bacteria may consequently contribute to the pathogenesis of infection. Sphingomyelinases are a group of hemolysins, present in both eukaryotes and prokaryotes, which are related to phospholipid metabolism in the former and that frequently act as toxins in the latter [43], that are absent in non-pathogenic Leptospira spp. [17,44]. In pathogenic Leptospira, sphingomyelinases play an important role in their survival in the mammalian host by mediating the lysis and release of essential nutrients from the host cells [18], but are implicated in the hemorrhagic complications associated with leptospirosis [45,46]. Among sphingomyelinases described in L. interrogans serovar Lai, Sph2 and SphH are proven to have cytotoxic properties and are expressed during infection [19,47]. However, studies focused on the associations of sphingomyelinases with leptospirosis severity, their potential as protective antigens, and the identification of their B-cell epitopes, remain scarce. To the best of our knowledge, this was the first study aiming to identify linear B-cell epitopes in L. interrogans serovar Lai Sph2 and to explore their association with the clinical data of naturally infected patients.
First, we used a combination of algorithms to predict linear B-cell epitopes in L. interrogans serovar Lai Sph2. This approach had been used and improved by our group in recent years to predict epitopes in viruses [25,26], bacteria [24], and protozoans [28,48,49], resulting in prediction accuracy up to 90% in the most recent studies. Here, we predicted two sequences as antigenic and linear B-cell epitopes: GHDERAKRISKSDYVK (Sph2 (176)(177)(178)(179)(180)(181)(182)(183)(184)(185)(186)(187)(188)(189)(190)(191) ) and TPTKSGHKKKYDQV (Sph2 (446-459) ), which did not present similar epitopes described in BLASTP, in the Immune Epitope Database http://www.iedb.org/home_v3.php (accessed on 20 June 2020) (data not shown), and also did not present similar sequences described in humans and mice, or in other databases from PeptideAtlas http://www.peptideatlas. org/map/ (accessed on 20 June 2020). Therefore, predicted epitopes were considered nonconserved among hosts and other bacteria, suggesting them as antibody targets specific against Leptospira spp. In this context, when compared with other sphingomyelinases from L. interrogans serovar Lai, we observed that Sph2 is highly similar to Sph4, presenting more than 99% of identity, while other sphingomyelinases presented identities from 53% to 66.4% (Table 3). Moreover, Sph2 is highly variable among pathogenic leptospires, with identities that ranged from 55% to 90%. Despite the similarity observed between L. interrogans serovar Lai and serovar Bataviae (identity of 90%), Sph2 from serovar Lai was more similar to L. noguchii serovars Panama and Autumnalis (identities of 89.6%) than to other serovars of L. interrogans (Lora, Zanoni, and Pyrogenes), corroborating studies that recognized the great numbers of serovars as an obstacle that has hampered the development of a universal vaccine for leptospirosis [50].
Based on these data and aiming for the future constructions of multi-epitope vaccines, we explored the conservation of predicted epitopes among other sphingomyelinases from L. interrogans serovar Lai and Sph2 from other pathogenic leptospires. Remarkably, the epitope Sph2 (446-459) was highly conserved among Sph2 from pathogenic leptospires and among sphingomyelinase from L. interrogans serovar Lai, presenting more than 71.4% of identity when compared with studied sequences. However, Sph2 (176)(177)(178)(179)(180)(181)(182)(183)(184)(185)(186)(187)(188)(189)(190)(191) was highly conserved in only three studied Sph2 (L. interrogans serovar Bataviae and L. noguchii serovars Panama and Autumnalis) and in Sph4, presenting 100% of identity with these proteins, while presenting less than 56.3% of identity when compared with other studied proteins. From our point of view, the high conservation of Sph2 (446-459) among pathogenic leptospires suggests that this epitope may be inserted in a region under low selective pressure by the host immune response. In line with this assumption, according to the prediction of algorithms InterPro family, TIGRFAMs, and nSMase (data not shown), this epitope is located out of the Sph2 Sphingomyelinase C domain (Sph2 (155-440) ), supporting the hypothesis of low selective pressure given that all amino acids involved in Sph2 activities are located on the Sphingomyelinase C domain [18], a common exo-endo-phosphatase domain that classifies sphingomyelinases in the DNase I superfamily, which differs in structure and substrate specificity [51].
In the same way, we evaluated the variability of amino acids in the catalytic site, metal-binding site, and the region of interaction with host membranes (Table 5). First, we highlighted that Sph2 from L. interrogans serovar Lai presents all amino acids described in the catalytic and metal-binding sites highly conserved (100% of identity) when compared with sphingomyelinases from other bacteria, such as Listeria ivanovii and Bacillus cerus, which are considered membrane-damaging virulence factors that induce hemolysis, a reduction in phagocytosis [52], and escape from the phagocytic vacuole [53]. Additionally, our data showed high variability in amino acids associated with the host membrane interaction when compared with other bacterial sphingomyelinases and when compared with Sph2 from other leptospires. These data suggest that Sph2 from different serovars may play a role in host tropism and the pathogenicity in different hosts, corroborating the hypothesis of Gonzáles-Zorn and collaborators, to scmL [53], which is structurally close to Sph2 from L. interrogans serovar Lai [18], and propose an additional explanation for the inability of Sph2 from L. interrogans serovar Copenhageni Fiocruz L1-130 to lyse sheep erythrocytes [20]. Based on these data, we explored the location of predicted epitopes in the Sph2 3D structure (Figure 3), observing that epitope Sph2 (176)(177)(178)(179)(180)(181)(182)(183)(184)(185)(186)(187)(188)(189)(190)(191) was closely located to the catalytic site, metal-binding site, and the region related to interaction with the host membrane. This finding suggests that antibodies against Sph2 (176)(177)(178)(179)(180)(181)(182)(183)(184)(185)(186)(187)(188)(189)(190)(191) could hamper the functionality of Sph2 by blocking the interaction with the host, or even the ligation to cofactor or substrate. However, studies aiming to identify the active sites of Sph2 from Leptospira spp. and to evaluate their activity and the role of antibodies against their epitopes remain a lack in the literature.
Until now, antibodies against Sph2 were detected in the blood of mares following leptospiral abortion, but not in horses immunized with bacterins [21], and in serum from leptospirosis convalescent patients [20]. These studies confirmed the protein expression during leptospirosis but lacked the investigation of associations between the presence of antibodies and clinical data. To the best of our knowledge, this was the first study aiming to identify Sph2 B-cell epitopes and investigate their association with clinical data. Here, we evaluated the presence of antibodies against epitopes Sph2 (176)(177)(178)(179)(180)(181)(182)(183)(184)(185)(186)(187)(188)(189)(190)(191) and Sph2 (446-459) in blood samples from 51 Brazilian patients with leptospirosis, which presented reactivity (or cross-reactivity) to 14 Leptospira spp. serovars, with the prevalence of Tarassovi and Copenhageni. Interestingly, this observation is in disagreement with the recent revision of Browne and collaborators, which reported the serovars Icterohaemorrhagiae, Canicola, and Pomona as the most prevalent in human leptospirosis in the Americas from 1930 to 2017, with Pomona and Canicola being the most prevalent in Brazil [54]. From our point of view, this controversy reinforces the necessity of improved surveillance of leptospirosis cases, aiming to know what are the most important pathogens to focus on in vaccine development.
Moreover, the protective potential of antibodies against Sph2 is still unknown. Regarding its potential as a vaccine antigen, hamsters immunized with the recombinant Sph2 from L. interrogans serovar Copenhageni did not present protection when challenged with a virulent strain of L. interrogans serovar Pomona; however, the authors propose that this can be related to a lack of correct folding of the recombinant proteins [20]. Unfortunately, due to the limited number of patients involved in this study, we believe that our data are not sufficient to confirm or discard the protective role of antibodies against Sph2 epitopes. Here, we observed a higher frequency of cases of pulmonary hemorrhage among responders to Sph2 (176)(177)(178)(179)(180)(181)(182)(183)(184)(185)(186)(187)(188)(189)(190)(191) (Figure 2). We believe that this finding could be indicative of the association of Sph2 with hemorrhages in leptospirosis since only individuals with low reactivity (R.I. < 2) to Sph2 epitopes presented clinical complications (pulmonary hemorrhage, renal insufficiency, or death), while high responders to Sph2 (176)(177)(178)(179)(180)(181)(182)(183)(184)(185)(186)(187)(188)(189)(190)(191) presented only mild symptoms. Based on this data and on the position of Sph2 (176)(177)(178)(179)(180)(181)(182)(183)(184)(185)(186)(187)(188)(189)(190)(191) close to the catalytic site and the region of interaction of Sph2 with the host membrane, we conjecture that this epitope should be better explored as a target of protective antibodies against leptospirosis; however, more studies are necessary to prove this hypothesis. On the other hand, we also hypothesized that different levels of Sph2 expression related to Leptospira species and host factors can also be related to reactivity against Sph2 epitopes and could be associated with leptospirosis hemorrhagic symptoms, reinforcing the necessity of more studies to prove Sph2 and its epitopes as vaccine candidates.
Up to now, the main vaccines proposed against Leptospira elicit a serovar-dependent immunity [50]. Though, considering that there are more than 300 classified serovars, the development of a universal vaccine for leptospirosis persists as a great challenge [10]. Moreover, inactivated vaccines do not promote long-term protection, and some side effects have been reported [56], supporting the necessity of novel strategies for vaccine development, such as multi-epitope vaccines. This approach is based on the rational combination of epitopes from proteins previously classified as vaccine candidates and has been used in an increasing number of studies aiming to propose protective vaccines for leptospirosis [57][58][59][60][61]. However, the number of epitopes from Leptospira antigens experimentally validated remains scarce, and justifies our study, since the identification of protective epitopes is the key to the design of an effective and universal multi-epitope vaccine for human leptospirosis.

Institutional Review Board Statement:
This study was conducted in accordance with the Declaration of Helsinki, and was approved by the Institutional Ethics Committee of the Oswaldo Cruz Foundation (CAAE: 31405820.8.0000.5262, approved on 14 July 2021).

Informed Consent Statement:
Patient consent was waived due to only serum samples from the National Reference Diagnostic Center being used in this study, with high privacy of patients' personal information. The study authors did not have access to the personal information of donors, ensuring the confidentiality of this information, as approved by the Ethics Committee.
Data Availability Statement: Data are available on request due to restrictions of privacy or ethical. The data presented in this study are available on request from the corresponding author. The data are not publicly available due to confidential information related to the personal information of donors, in accordance with the Institutional Ethics Committee of the Oswaldo Cruz Foundation.