Biochemical and Immunological Insights into Capsular Polysaccharide of Streptococcus pneumoniae Serotype 38 (American type 71)- An emerging non-vaccine serotype

Biochemical and Immunological Insights into Capsular Polysaccharide of Streptococcus pneumoniae Serotype 38 (American type 71)- An emerging non-vaccine serotype | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Biochemical and Immunological Insights into Capsular Polysaccharide of Streptococcus pneumoniae Serotype 38 (American type 71)- An emerging non-vaccine serotype M V N Janardhan Reddy, Yogeshwar Devarakonda, Burki Rajendar, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5880261/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Invasive pneumococcal disease presents a threat to humankind predominantly affecting children and the elderly. Despite the availability of high-valency pneumococcal polysaccharide vaccine of PPSV23 (PNEUMOVAX® 23) and conjugate vaccines such as VAXNEUVANCE and PREVNAR 20®, non-vaccine serotypes continue to contribute to higher mortality rates. The characterization of non-vaccine serotypes is becoming increasingly crucial considering an increase in their prevalence. In this study, biochemical characteristics, immunological properties, and critical quality attributes of the capsular polysaccharide isolated from prevalent non-vaccine serotype 38 (American type 71) have been examined. Advanced analytical techniques, including multi-angle light scattering (MALS), ion chromatography, dynamic light scattering in addition to conventional biochemical methods and SLOTBLOT analysis were employed. We observed that serotype 38 capsular polysaccharide has a molar mass of 768 kDa with a distribution of 1.451 (± 4.460%) and a z-average radius of gyration ( R g ) is 90 nm. The polysaccharide composition included approximately 72% galactose, 9.78% N-acetylglucosamine, and 8.05% galacturonic acid, while the unknown peak accounted for approximately 7.83% of the total peak area of the chromatogram. The O-acetyl content of polysaccharide was determined to be nearly 6% and it lacked methyl pentoses (rhamnose). Zeta potential measurements revealed its zwitterionic state which suggested its potential to trigger T cell-dependent B cell-mediated immunological response. Serotype 38 polysaccharide showed immunological cross-reactivity with serotype 5 and serotype 1 polyclonal sera, likely due to a shared common epitope region having an unknown sugar component (Sug p ) in their polysaccharide repeating units and zwitterionic properties. The findings highlight novel features of serotype 38 polysaccharide, including its amino acid content and zwitterionic nature, which may contribute to the development of new therapeutics and improved vaccines. Biological sciences/Biochemistry Biological sciences/Chemical biology Biological sciences/Immunology Biological sciences/Microbiology Streptococcus pneumoniae non-vaccine serotype 38 Capsular Polysaccharides Zwitterionic Polysaccharide Pneumococcal vaccines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Streptococcus pneumoniae- a Gram-positive bacterium with a polysaccharide capsule, can potentially cause both non-invasive and invasive pneumococcal disease (IPD) involving respiratory system predominantly in children under five and the elderly 1 – 3 . Though more than 100 serotypes of S. pneumoniae are known 4 , a relatively small subset (less than 30) is responsible for the majority of pneumococcal infections in humans 1 , 5 . The diversity of pneumococcal serotypes is primarily determined by variations in the chemical structure of the bacterial capsule's polysaccharides such as differences in the oligosaccharide units or attached side groups 5 . The capsular polysaccharide acts as a virulence factor for S. pneumoniae aiding its survival inside host. Vaccines mediated immune response against capsular polysaccharides involves protective antibodies with opsonophagocytic activity (OPA). Such antibodies facilitate the complement-mediated uptake and killing of pneumococci by human phagocytic cells 5 – 9 . Evidently, the introduction of the pneumococcal capsular polysaccharide conjugate vaccine (PCV7) has reduced IPD-related deaths among children under five in the United States from 2000 to 2007 10 . The PPSV 23 (PNEUMOVAX® 23) is a widely used pneumococcal polysaccharide-based vaccine for adults (fifty years old and above) whereas the PCV13 conjugate vaccine is preferred for children. The PCV13 was further improvised to PCV20 (PREVNAR 20®), which could protect against seven additional serotypes including 8, 10A, 11A, 12F, 15B, 22F, and 33F 11 , 12 . The new serotypes that have not been characterized continues to pose challenges due to their unknown capsular polysaccharide structures 5 . The characterization of such new serotypes of S. pneumoniae involving examination the biological and serological characteristics of capsular polysaccharides may facilitate their inclusion in the vaccine designs. The coverage of existing PCV 10, 13, 20, and PPSV 23 vaccines has been observed at 16%, 24%, 48%, and 66%, respectively 13 . Moreover, approximately 30% of the total S. pneumoniae isolates belong to non-vaccine serotypes (NVTs), highlighting the need to include these serotypes in vaccine coverage to improve population protection 13 , 14 . The prevalence of pneumonia caused by non-vaccine serotypes varies globally 4 , 14 , 15 . In 2019 alone, an estimated one million deaths among children under five were attributed to pneumonia 16 . In India, around 21% of the IPD cases in 2016 were attributed to non-vaccine serotypes 14 . The non-vaccine S. pneumoniae serotypes are continuously emerging as evident by their increased incidence rates and spread 15 . The most common serotypes that infect children include 10A/F, 7C, 35A/B, 16F, 19A, 3, and 38 15,17 . Surveillance data from countries such as the USA, Australia, Finland, France, Norway, Canada, and Ethiopia indicate that serotype 38 has become more prevalent causing IPD across all age groups 15 , 17 – 19 . In India, Manoharan et al. identified serotype 38 in more than 1% of the S. pneumoniae clinical isolates 20 . Together, the understanding of its CPS biochemical characteristics, immunological properties, and critical quality attributes (CQAs) has become vital considering its widespread distribution, high incident rates, and identification in clinical isolates. Earlier, the structure of the type 38 polysaccharide was studied by NMR analysis, however, it did not analyze the zwitterionic state of the polysaccharide, phosphorus content, and the cross-reactivity with other serotypes 21 . In his study, in addition to biochemical characterization, zwitterionic state was determined, phosphorus content was estimated, and immunological cross reactivity with selected serotypes was assessed thereby providing essential insights for designing effective therapeutic strategies against IPD caused by serotype 38. Materials and Methods Materials Purified pneumococcal polysaccharide serotype 38 was procured from the American Type Culture Collection (ATCC 543-X). Glucose (Glc), Galactose (Gal), Mannose (Man), Fucose (Fuc), Rhamnose (Rha), Glucuronic acid (GlcA), Galacturonic acid (GalA), Glucosamine (GlcN), Galactosamine (GalN), N-Acetylglucosamine (GlcNAc), and N-Acetyl galactosamine (GalNAc) were purchased from Sigma-Aldrich Co. (St. Louis, USA). N-Acetyl Fucosamine (FucNAc), N-acetyl-Pneumosamine (PneNAc) were purchased from Omicron Biochemicals Inc. USA. Trifluoroacetic acid (TFA) and hydrofluoric acid (HF) obtained from Merck, India. Sodium acetate and sodium hydroxide 50% solution were purchased from Sigma-Aldrich Co. (St. Louis, USA). Mouse Monoclonal Antibodies of serotype 1 and serotype 5 were procured from AbMax-China, and Rabbit polyclonal antisera and capsular polysaccharide of serotype 1 and serotype 5 were procured from Statens Serum Institute Diagnostica, Denmark. Secondary antibodies of Goat Anti-mouse IgG peroxidase and Anti Rabbit IgG peroxidase purchased from Bio-Rad laboratories-India. All the chemicals were ACS reagent-grade and IC-grade with a purity specification of ≥ 90–99% (Fluka, Sigma–Aldrich). Methods Preparation of CPS of serotype 38 and the sugar standards The stock solution of purified polysaccharide of serotype 38 was prepared by dissolving it in ASTM Type-II water at a concentration of 2 mg/mL (w/v). All other sugar master stocks were prepared as 1mg/mL (w/v) in ASTM Type-II water which were subsequently diluted to the final concentration of 10µg/mL (for each sugar). The buffered serotype stock, originally had sodium chloride in its lyophilized form, has been subjected to desalting procedure wherever needed. SEC-UV-MALS-RI The pneumococcal polysaccharide (PnPS) physical parameters such as molar mass was determined by SEC-UV-MALS-RI. It is crucial to maintain the molecular size (MS) of PnPS within the specified range for the design of conjugate vaccines either through physical or acid hydrolysis before use in the conjugation process 22 , 23 . The purified polysaccharide obtained from ATCC was initially dissolved in ASTM Type-II water to achieve a concentration of 2 mg/mL (w/v). Subsequently, a 100 µL aliquot of this solution was injected into a size exclusion chromatography (SEC) column that was connected to UV-MALS-RI detectors and are calibrated using a BSA monomer standard of 2 mg/mL. The HPLC system utilized for the analysis consisted of SHODEX SB 806HQ and SHODEX SB 803HQ connected in series with 100mM sodium phosphate and 0.05% sodium azide pH 7.2 serving as the mobile phase. The sample separation was performed by pumping the mobile phase at a flow rate of 0.5mL/min through the connected analytical columns. The Agilent 1260 series HPLC, equipped with miniDAWN® TREOS® and Optilab® T-rEX RI detectors from Wyatt Technology-USA, was used to carry out the analysis. The molar mass and size distribution pattern was determined using the refractive index as a concentration source-detector by inputting the dn/dc of 0.133 mL/g 24,25 to the ASTRA software. HPAEC-PAD The 10 µg/mL of polysaccharide treated with 10M TFA with the ratio of 4:1 (v/v) into a glass vial, sealed with caps equipped with Teflon faced rubber insert, and was subjected to incubation at 121°C for 2 hours in the digital dry bath with safety lid. Post incubation, the sample was subjected to nitrogen evaporation set at 45°C for 30min to evaporate the TFA. The polysaccharide smear in the vial was then dissolved in 1mL of ASTM type-II water and then filtered through 0.22µm syringe filter into the HPLC vial. In parallel set, 10 µg/mL of polysaccharide was first treated with HF followed by the TFA as per the referred protocol 26 . The 20 µL of depolymerized polysaccharide and sugar standards were injected onto Carbopac PA10 (4.6X250mm) column and guard (4.6X50mm) connected to the ICS-5000 (Thermo Fisher Scientific, Sunnyvale, CA, USA) system equipped with Electrochemical detector that includes an Ag/AgCl reference electrode and a disposable gold working electrode. The mobile phase of A; 18mM NaOH, B; 100mM NaOH and C;1M Sodium acetate in 100mm NaOH were pumped into the column as a gradient program with the flow rate of 1mL/min. The temperature of column and autosampler (ASAP) were set at 30°C and 15°C, respectively. The carbohydrate (Standard quad) waveform was applied to the ECD. All the data was processed using Chromeleon™ software. HPAEC-CD The O-acetyl content determines the immunogenic response of the polysaccharides as reported 27 , 28 . The O- acetyl content of the capsular polysaccharides was examined. The polysaccharide at a concentration of 200 µg/mL was treated with 0.1 N NaOH and incubated at 37°C for 2 and 4 hours. Following the incubation, the mixture was passed through 10kDa nanosep centrifugal filters that had been pre-rinsed three times with ASTM Type-II water at 10000 rpm for 15 minutes. To avoid any dilution of the sample filtrate with droplets from the filter holder used for the pre-rinse process, the filtrate was collected into a fresh 10kDa filter holder. A working standard was devised using Sodium acetate (as acetate source) in a concentration range of 0.625 to 40 µg/mL. The standard was diluted with ASTM Type-II water and the sample filtrate was analyzed as described elsewhere 29 . The total nitrogen (contribution from hexosamines and residual protein impurity) content was estimated with in the polysaccharide as per the referred protocol 30 . The nitrogen content is not solely attributed to hexosamines but is also influenced by any protein content present in the capsular polysaccharides therefore may help in estimating protein impurities. In addition to amines, phosphates may also exist as sugar derivative components, such as ribitol phosphate and ribose phosphate, within these polysaccharides. Moreover, nucleic acid impurities in the purified capsular polysaccharides may contribute significantly to the overall nitrogen and phosphorus levels. Therefore, it is essential to quantify these components within the polysaccharides, in order to provide indirect insights into the impurity levels of residual proteins and nucleic acids, serving as an orthogonal verification method alongside conventional OD 260/280 measurements. In this study, 200 µg/mL of serotype 38 polysaccharide was subjected to digestion using 0.5 M potassium persulfate in 0.5 M NaOH at 100°C for 16 hours. Following digestion, the sample was diluted before the analysis by HPAEC-CD. The analysis was performed using a Thermo ICS-5000 system, equipped with an IonPac AS15 guard column (50 × 4 mm) and an IonPac AS15 analytical column (250 × 4 mm), to separate and estimate the nitrogen and phosphorus content as previously reported 30 . Orthogonal verification of sugar composition and its derivatives The bacterial capsular polysaccharide structure comprises common sugar derivatives like methyl pentoses and uronic acids, along with the common sugar forms of hexoses and pentoses. The sugar composition obtained by HPAEC-PAD was verified by the conventional colorimetric methods such as Cysteine-HCl method for methyl pentoses 31 and Carbazole method for uronic acids 32 , 33 . As reported, rhamnose was used as a standard for methyl pentose assay, and glucuronic acid was used as a standard for uronic acid assay. Briefly, for the methyl pentose assay, 100 µL of rhamnose standard ranging from 2 to 40µg/mL was prepared alongside the diluted sample. Then, 500 µL of reagent A (1 in 6 v/v Water/ H 2 SO 4 ) was added to the standard solutions and the diluted sample followed by incubation at 90˚C for 7 min in water bath. Then 40 µL of reagent B (3% w/v Cysteine HCl) was added to all the tubes and incubated at 37˚C for 30 min. After incubation, 200 µL of each standard and sample were transferred into 96 well plate and the absorbance was measured at 396nm and 430 nm. As previously reported, the Carbazole method was used for estimating the uronic acid content. Briefly, the 100 µL of glucuronic acid standard range from 5 to 50µg/mL was prepared, and the 100 µL of diluted sample was added to 500 µL of reagent A (0.030M Borate buffer / H 2 SO 4 ) and incubated at 90˚C for 15 min in water bath. After the addition of 20 µL of reagent B (0.125% Carbazole in ethanol), all tubes were incubated at 90˚C for 15 min. After incubation, 200 µL of each standard and sample were transferred to 96 well plate and the absorbance was measured at 530nm and 660 nm 32 , 33 . Dynamic Light Scattering (DLS) The zeta potential was measured in series mode using Omega Cuvettes with the light scattering instrument of Litesizer-500 (Anton Paar, India) by Kalliope software, and the default measurement parameters were set 34 . The lyophilized polysaccharide powder of serotype 38 was received from ATCC and it had high sodium chloride content (6M) which interfered with the charge measurement. Therefore, the polysaccharide powder was dissolved in ASTM Type-II water and desalted by passing it through a 10kDa cutoff centrifugal filter to remove the sodium chloride content. Sodium chloride hinders the zeta potential measurement due to its high conductivity and may lead to charring of the electrode in cuvette. The buffer exchanged sample is further diluted to 200µg/mL with ASTM Type-II water to meet the instrument measurement specifications of the sample such as accepted filter optical density and sufficient mean intensity (> 20 kcounts/s) values. Before analyzing the sample, the instrument was calibrated with the known zeta potential standards. SLOTBLOT The capsular polysaccharide of serotype 38 consisted of an unknown sugar in its repeating unit identified as "Sug p " as described in the reference and we also could not decipher this unknown sugar 21 . However, similar sugar (2-acetamido-2,6-dideoxy-D-xylo-hexos-4-ulose) was also present in another pneumococcal serotype i.e. PnPS serotype 5 35 . Additionally, PnPS serotype 1 also featured a unique and rare amino sugar moiety, AAT sugar (2-acetamido-4-amino-2,4,6-trideoxy-D-galactose), which was previously noted for its immunogenicity 36 , 37 . Therefore, we tested if the serotype 38 polysaccharide may elicit antibodies functionally similar to those elicited by the polysaccharides derived from serotypes 1 and 5. Such antibody response is expected only if the unknown sugar acted as an antigenic epitope in the serotype 38 polysaccharide. We performed a SLOTBLOT assay to test our hypothesis 38 with an aim to evaluate the cross-reactivity of serotype 38 against antibodies specific to serotypes 1 and 5. Briefly, PnPS serotypes 1, 5, and 38 were adhered to a nitrocellulose membrane at two different concentrations in four sets. The 1% skim milk powder solution in PBS was used as a blocking reagent over the membrane. Following blocking, the membrane was washed three times with PBST (PBS with 0.05% Tween 20). With the optimized dilutions of Monoclonal and polyclonal antibodies specific to serotypes 1 and 5 were added to designated blots and incubated for one hour. After incubation, the membrane was washed again with PBST and incubated with respective secondary antibodies labeled with horseradish peroxidase. The reaction was developed using a TMB substrate to visualize the antibody binding on the membrane concerning the lanes of the loaded antigens. Critical quality attributes of capsular polysaccharide Capsular polysaccharides content should meet stringent standards for identity, purity, potency, and safety set by regulatory authorities like the FDA, EMA, and WHO for inclusion in vaccine design 39 . The vaccine's reproducibility depends on various factors including surface charge that affects stability and particle distribution. Based on the regulatory guidelines, monitoring and adhering to critical quality attribute (CQA) is essential for characterization of antigenic content and demonstration of their immunogenic properties in order to ensure optimal efficacy and protection against the corresponding pneumococcal serotype. The chemical composition, including sugars and functional groups, must be precise to maintain the immunogenic properties and mimic the native bacterial surface. Establishing specific percentage limits for each functional group within the polysaccharides is necessary to evaluate the integrity of the immunogenic functional groups and confirm the preservation of the vaccine's immunogenic characteristics. The CQAs for these polysaccharides include several factors that can significantly impact the performance and reliability of the final vaccine product. The most important attributes to assess the PnPS quality are; molecular size, purity, sugar composition, and surface charge. The polysaccharides that are too small may be ineffective, while those too large can complicate manufacturing due to excessive viscosity. Consistent molar mass and size distribution are also necessary for uniform vaccine quality 22 , 23 . Impurities such as proteins, nucleic acids, and endotoxins may reduce vaccine effectiveness and safety. Rigorous purification is essential to minimize these contaminants. Ensuring these CQAs are met is crucial for producing safe and effective vaccines. Hence we attempted to propose the limits of CQAs with the ATCC-purified polysaccharide as it is used as a reference standard by the manufacturers to develop immunological-related methods. Results and Discussion Size and Molar mass by SEC-MALS The molar mass and size distribution pattern of purified CPS are essential quality attributes for glycoconjugate vaccine manufacturing, as they indicate the consistency of upstream and downstream processes. In this study, a sample of purified Spn type 38 CPS (2 mg/mL) was injected into size exclusion columns connected to an Agilent HPLC system equipped with a UV-MALS-RI detector. The normalization was carried out with the BSA monomer prior to analysis. The refractive index increment, dn/dc of the polysaccharide (0.133 mL/g), was used to determine the molar mass, rms radius moments (rz), and size distribution pattern using ASTRA software. The polysaccharide peak was observed between 22 and 30 minutes, with the size distribution graph showing a range of 1.0x107 to 1.0x105 g/mol (Fig. 1 ). The average molar mass of the purified CPS of Spn type 38 was determined to be 7.684x105 g/mol (± 3.72%) with a polydispersity (Mw/Mn) value of 1.451 (± 4.460%) and z-average radius of gyration (Rz) value of 90 nm. These results can be used as a reference value for assessing the consistency of the purification process, both in terms of fermentation and downstream purification, for purified CPS of Spn type 38. Monosaccharide composition by HPAEC-PAD As described in the method section, the depolymerized samples were analyzed in ion- chromatography system using a CarboPac PA10 column coupled with electrochemical detection (ECD). The monosaccharide components identified in the CPS included N-acetylglucosamine, galactose, and galacturonic acid, alongside an unidentified peak with a retention time of 22 minutes (Figs. 2 A and 2 B). With an aim to elucidate the nature of the unidentified peak (RT of 22 min), individual injections of various monosaccharides, including fucose, glucose, galactose, mannose, glucosamine, fucosamine, pneumosamine, and galacturonic acid, were performed (Fig. 2 A). Based on the retention time of the unidentified peak, which eluted at approximately 50 mM sodium acetate in 100 mM NaOH, it was inferred that this peak was not attributable to any monosaccharide or disaccharide, as all such compounds elute from the CarboPac PA10 column with 100 mM sodium hydroxide alone, without the necessity of sodium acetate 40 . The unidentified peak's elution in the presence of sodium acetate suggested it might be a sugar acid or a non-sugar compound. It is hypothesized that this peak represents a sugar acid or a compound containing an amino acid, potentially forming a peptide bond with galacturonic acid or another amine present in the CPS repeating unit. As we were in the process of characterizing this unidentified, Li et al. reported 21 structural studies of the serotype 38 capsular polysaccharide and they identified the unknown component as the amino acid serine conjugated to the galacturonic acid. We also confirmed the presence of serine. The discovery represents the first instance of an amino acid being identified within the repeating unit of a Streptococcus pneumoniae capsular polysaccharide. The relative peak areas for the identified monosaccharides were as follows: galactose, 72.45%; N-acetylglucosamine, 9.78%; and galacturonic acid, 8.05%, while the amino acid (Serine) peak accounted for approximately 7.83% and 1.89% of unidentified peaks of the total peak area of the chromatogram (Table 1 ). There was another small percent of peaks eluted between 5 to 8 min (Fig. 2 C) overlaid with the peaks (other than the main known sugars) obtained for PnPS serotype 5 hydrolysed with HF followed by TFA as for serotype 38 (Fig. 2 C zoomed chromatogram). This overlaying confirmed and supported the recently reported work 21 that these peaks belong to the Sug p (2-acetamido-2,6-dideoxy-D-xylo-hexos-4-ulose) moiety present in the PnPS serotype 38. This novel structural feature may influence host-pathogen interactions, immunogenicity, or present challenges in conjugation chemistry used for coupling with carrier proteins. Further investigation is warranted to explore these implications comprehensively. Table 1 Relative percent peak areas of acid hydrolyzed CPS of Spn type 38 analyzed on HPAEC-PAD Unknown peaks Glucosamine peak Galactose peak Amino acid (Serine) peak Galacturonic Acid peak 1.89 9.78 72.45 7.83 8.05 O-acetyl content by HPAEC-CD The O-acetyl content is one of the important immunogenic functional groups for capsular polysaccharide-based vaccines 27 , 28 . Hence, it is mandated to quantify it as the quality attributes of the CPS. In the ATCC-purified polysaccharide of serotype 38, under mild alkaline conditions, as mentioned previously, the O-acetyl groups were selectively hydrolysed and released as acetate ions. The hydrolysate acetate was separated from other ions by the Thermo ICS-5000 system equipped with a strong anion exchange column and guard column of IonPac AS11-HC guard column i.d. (50 × 4 mm) and IonPac AS11-HC analytical column i.d. (250 × 4 mm) were used and estimated the content. The equimolar ratio of sodium acetate as acetate was used as an assay standard from the range of 0.625 to 40µg/mL (Fig. 3 A). The polysaccharide was kept under mild alkaline conditions, 10 mM NaOH at 37°C, for two and four hours to confirm the complete release of O-acetyl groups from the polysaccharide. The standard acetate peak has a retention time of 7.2 min which overlayed with the acetate peak from the sample. The peak area obtained for two and four hours of incubation was observed to overlay (Fig. 3 B) and the O-acetyl content was calculated to be same for both two hours and four hours of incubation with a value of 5.8%. The two hours of mild alkaline condition was enough to release the total O-acetyls from the CPS of Spn type 38. Polysaccharide Net charge by DLS As described in the method section, the sample was diluted to 200 µg/mL in ASTM Type-II water to meet instrument specifications, including acceptable optical density and mean intensity (> 20 kcounts/s). With default settings, Zeta potential measurements were carried out with the Litesizer-500 (Anton Paar, India) using Omega Cuvettes and Kalliope software. The zeta potential of the polysaccharide of serotype 38 was found as -0.7 mV (Fig. 4 ). The PnPS type 1 is categorized and claimed as a zwitterionic polysaccharide elsewhere 41 , 42 . Hence for the comparison, we also measured the zeta potential for the PnPS type 1 at 200 µg/mL in ASTM Type-II water and it showed a value of -0.9 mV. This value falls within the − 10 to + 10 mV range indicating a zwitterionic nature. The nanoparticles with zeta potentials in this range are generally considered to have neutral surfaces, whereas those with absolute values exceeding ± 30 mV are classified as strongly cationic or anionic 43 . Since PnPS serotype 1 was classified as a zwitterionic polysaccharide, the similar charge of PnPS serotype 38 and the structural features (glucosasamine and galacturonic acid) implied it to be zwitterionic as well. The relative frequency distribution reveals that most particles in both samples had comparable surface charges reflecting their similar zeta potential profiles suggesting a zwitterionic nature and relatively neutral surface charge. This information is crucial for understanding the polysaccharide's behaviour in various applications and stability in different formulations. The neutral surface charge (Zwitterion) may influence its interactions with the antigen-presenting cells (APCs), ZPS-mediated T cell activation, and may trigger the T cell-dependent B cell immune response 41 . Orthogonal verification of sugar derivatives by conventional methods The sugar composition obtained by HPAEC-PAD for PnPS serotype 38 was orthogonally verified further with the conventional biochemical methods for methyl sugars (Rhamnose) and sugar acids (Uronic acid) by following the procedures reported and described previously. A good linear curve was observed with a regression value of > 0.99 for both methods (Fig. 5 A and 5 C). The OD values observed in the PnPS serotype 38 sample tested at three different concentration levels of 100, 200 and 400µg/ml were comparable to blank thereby confirming the absence of Methyl pentoses in serotype 38 capsular polysaccharide (Fig. 5 B). It was supported by the monosaccharide profiles obtained by HPAEC-PAD as there was no Rhamnose peak in the chromatogram. For the uronic acid content evaluation, the sample was tested at three different concentrations, and the obtained average % uronic acid content was determined to be 7.7 (Fig. 5 D) and the value was in line with the percent peak area obtained by HPAEC-PAD (Table 1 ). Also the estimated Hexosamines values were orthogonally verified by the Elson-Morgan method 44 using N-acetyl glucosamine as a standard, the result was in line with the percent peak area obtained by the HPAEC-PAD (Table 1 ). Thus, the percent peak areas obtained by HPAEC-PAD was used to assess the polysaccharide composition from batch to batch instead of performing multiple conventional biochemical methods which could be exploited for other polysaccharides as well. Nitrogen and Phosphorous Content by HPAEC-CD Amines (hexosamines) and phosphorus-containing sugars (ribose-5-phosphate and ribitol-5-phosphate) are important nitrogen and phosphorus sources in polysaccharides, respectively 5 . In addition to amines, protein impurities in the polysaccharides also contribute to the overall nitrogen content. Similarly, nucleic acid impurities add to the total phosphorus content. Adhering to composition specifications as outlined in recommended guidelines, it was crucial to determine the nitrogen and phosphorus content in the capsular polysaccharide sample. PnPS serotype 38 was subjected to persulfate hydrolysis to determine its nitrate (nitrogen) and phosphate (phosphorus) content using HPAEC-CD. The quantification was performed against standard linear calibration curves established with urea for nitrogen content and sodium phosphate monobasic for phosphorus content (Fig. 6 A and 6 B). In the chromatogram, a nitrate peak was detected, whereas no phosphate peak was observed in the sample (Fig. 6 C). The nitrogen content was estimated to be 3.67% with no detectable phosphorus content. Based on these findings and using the ATCC polysaccharide as a reference standard (since it is used as a standard in the respective immunological assays) for monitoring the critical quality attributes, we inferred that during the purification of PnPS serotype 38, the nitrogen content should be in between 3 to 4%. The phosphorus content should be limited to a range of 0 to 1%, taking into account the minimal nucleic acid contamination present in the capsular polysaccharide (CPS). Immunochemical properties With an aim to assess the validity of our hypothesis that the unknown sugar (Sug p ) moiety of serotype 38 polysaccharide may constitute a portion of an epitope recognized by the host immune system, we compared the HPAEC-PAD profiles and sugar composition of PnPS serotype 38 with that of serotype 5. Overlapping unidentified peaks were observed, potentially generated from the unknown sugars by acid hydrolysis using hydrofluoric acid (HF) followed by trifluoroacetic acid (TFA) (Fig. 2 C). As described earlier, the CPS of Spn type 38 has a net charge of -0.7mV indicating that it is in zwitterionic form like a CPS of Spn type 1 41,42,45 has a net charge of -0.9mV (Fig. 4 ). Thus, we have performed the SLOTBLOT assay using serotype 1 and serotype 5 monoclonal antibodies from AbMax and polyclonal antibodies from SSI, both of which possess unknown, rare sugars and a zwitterionic form of their polysaccharide repeating units. Our results revealed a clear blue blot upon the addition of TMB substrate in the slot where the serotype 5 and serotype 1 polyclonal were loaded against the serotype 38 lane onto the membrane (Fig. 7 ). In each set, lanes 1, 2, and 3 correspond to PnPS 1, 5, and 38, respectively, with the left spots (2nd vertical line) representing the 2 µg/mL concentration and the right spots (1st vertical line) representing the 1 µg/mL concentration. The intensity and pattern of the spots in each lane and concentration provide insights into the antibody specificity and binding affinity for the respective PnPS. These findings provide evidence supporting the hypothesis that serotype 38 possesses a similar epitope characteristic of serotype 5, and partially of serotype 1, as the unknown sugar in its capsular polysaccharide repeating unit. Consequently, we anticipate that serotype 38 will offer cross-protection against invasive pneumococcal disease caused by serotype 5 and vice versa. The findings of this study revealed crucial insights into the immunological characteristics of Streptococcus pneumoniae serotype 38, particularly its interaction with antibodies from other serotypes. Notably, serotype 38 exhibits cross-reactivity with polyclonal antibodies of serotypes 5 and serotype 1, yet does not react with the corresponding monoclonal antibodies. This discrepancy highlights the complexity of the immune response and the potential structural similarities and differences in capsular polysaccharides among these serotypes. The cross-reactivity observed with polyclonal antibodies indicates that there are shared epitopes between serotype 38 and serotypes 5 and 1. The polyclonal antibodies, being a heterogeneous mix, could recognize multiple antigenic sites on a pathogen. This broad recognition suggested that certain polysaccharide structures within serotype 38’s capsule were similar enough to those in serotypes 5 and 1 to elicit an immune response. Such shared epitopes might include common sugar components or structural motifs that were conserved across these serotypes. In contrast, the lack of reaction with monoclonal antibodies, which were specific to a single epitope, suggested that the unique epitopes targeted by these antibodies in serotypes 5 and 1 were absent or significantly different in serotype 38. This implies that while there are similarities in the capsular polysaccharide structures, serotype 38 possesses distinct antigenic features that set it apart from serotypes 5 and 1. These unique structural elements are not recognized by the monoclonal antibodies, which could be due to differences in the oligosaccharide units or specific side groups attached to the polysaccharides. The zwitterionic state of serotype 38’s capsular polysaccharide, as revealed by zeta potential measurements, suggests a potential for eliciting a T cell-dependent B cell-mediated immune response. ZPS presentation is known to influence T cell activation and abscess formation where antigen-presenting cells produce TNFα activating TH1 cells which secrete cytokines like IL-17 in response to ZPS presented on MHC class II molecules. The interaction between ZPS-MHC class II and αßTCRs has been shown to drive the development of abscesses, demonstrating the integration of innate and adaptive immune responses 41 , 45 , 46 . This characteristic might contribute to the observed cross-reactivity with polyclonal antibodies as the immune system can generate a broader array of antibodies in response to a more complex antigenic structure. These findings could enable formulation of therapeutic strategies and are highly relevant for vaccine design. The cross-reactivity with polyclonal antibodies suggested that current vaccines targeting serotypes 5 and 1 might offer some degree of protection against serotype 38. However, the lack of reaction with monoclonal antibodies underscored the necessity for further research to identify the unique epitopes of serotype 38. The serotype 38 interaction with polyclonal and monoclonal antibodies from serotypes 5 and 1 enhanced our understanding of the immunological landscape of S. pneumoniae . Critical quality attributes of serotype 38 capsular polysaccharide Establishing specific percentage limits for each functional group within polysaccharides is crucial to assess the integrity of immunogenic functional groups and ensure the preservation of a vaccine's immunogenic properties. In this study, we have evaluated the biochemical characteristics of PnPS Type 38 by estimating the CQAs and proposing specific limits (Table 2 ). These defined CQA limits provide a preliminary reference for assessing the purified Capsular Polysaccharide (CPS) quality of Type 38 produced by vaccine manufacturers. Defining Critical Quality Attributes (CQAs) could be challenging with a single source of CPS. However, using ATCC Capsular Polysaccharide as a reference material is commonly employed as an internal standard for immunological assays by vaccine manufacturers, hence proposed tentative CQA limits could be validated further by comparing multiple sources of CPS of Spn type 38. Table 2 Critical quality attribute results of CPS of Spn type 38 Quality attribute Results Proposing limits (concerning the ATCC CPS as a reference) Molar mass (kg/mol) 768 ≤ 800 Net charge (mV) 0.7 Neutral (+ 10 to -10) O-Acetyl Groups (%) 5.8 ≥ 6 Total Nitrogen (%) 3.67 4 to 5 Phosphorus (%) 0 (No peak) 0 to 1 Amino acid (Serine) (relative % peak area by HPAEC-PAD) 7.83 ≥ 8 Uronic acid (%) 8.05 ≥ 8 Hexosamines (relative % peak area by HPAEC-PAD) 9.78 ≥ 10 Methyl Pentose (%) Nil Nil Conclusion This study provides a comprehensive characterization of the newly prevalent non-vaccine serotype 38 of Streptococcus pneumoniae . Through detailed analysis of its biochemical properties, such as size, molar mass, sugar composition, O-acetyl content, and net charge, we have characterized the structural attributes of this serotype. The immunological assessment, particularly the observed cross-reactivity with polyclonal antibodies from serotype 5 and serotype 1, underscores the potential for cross-protection. This highlights the significance of inclusion of serotype 38 in vaccine development. Moreover, the existence of an amino acid in the polysaccharide repeating unit of this serotype for the first time is particularly notable, as it may play an vital role in host-pathogen interactions. The data presented in this study provide vital information for research and development of therapeutics including vaccines. Together, addressing the challenge posed by non-vaccine serotypes like serotype 38 is vital for improving global health outcomes and reducing the burden of IPD across all age groups. Further research is warranted to explore the potential inclusion of serotype 38 in future vaccine formulations and to monitor its epidemiological trends and impact on public health. Abbreviations CPS, Capsular polysaccharide; CQA, Critical Quality Attributes; HPAEC-PAD/CD, High-performance anion-exchange pulsed amperometry detection/Conductivity detector; IPD, Invasive pneumococcal disease; DLS, Dynamic Light Scattering system; SEC-MALS-RI, Size exclusion chromatography connected with multi-angle light scattering and Refractive index detectors; Spn, Streptococcus Pneumonia ; PnPS, Pneumococcal Polysaccharide; ZPS, Zwitterionic Polysaccharide. Declarations Conflicts of interest There are no conflicts to declare. Author Contribution MVNJR, KS conceptualized the study.MVNJR, YD carried out experiments.MVNJR, YD, BR, and KS analyzed data and wrote manuscript.All authors reviewed it. Acknowledgements Authors acknowledge Department of Biotechnology, Government of India for DBT-RLS Re-entry fellowship to KS. Authors acknowledge BITS Pilani Hyderabad campus for funding. YD acknowledge UGC, Delhi for fellowship. We extend our sincere gratitude to Dr. Ramesh Matur, Head of Vaccine R&D, and Dr. Rajendar Burki, Head of Analytical R&D, Biological E. Limited for their invaluable technical support in data interpretation and for granting us access to the necessary instrumentation for this study. Data Availability All data generated or analysed during this study are included in this published article. References Ryan Gierke, M. P. H., Patricia Wodi, A. & Miwako Kobayashi, M. M. M. D. in U S Centers Disease Control Prev. (2024). O'Brien, K. L. et al. Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates. Lancet 374 10.1016/S0140-6736(09)61204-6 (2009). Matanock, A. et al. Use of 13-Valent Pneumococcal Conjugate Vaccine and 23-Valent Pneumococcal Polysaccharide Vaccine Among Adults Aged ≥ 65 Years: Updated Recommendations of the Advisory Committee on Immunization Practices. MMWR Morb. Mortal. Wkly Rep. 68 10.15585/mmwr.mm6846a5 (2019). Hausdorff, W. P., Bryant, J., Paradiso, P. R. & Siber, G. R. Which pneumococcal serogroups cause the most invasive disease: Implications for conjugate vaccine formulation and use, part I. Clin. Infect. Dis. 30 10.1086/313608 (2000). Geno, K. A. et al. Pneumococcal capsules and their types: Past, present, and future. Clin. Microbiol. Rev. 28 , 871–899. 10.1128/CMR.00024-15 (2015). Soininen, A., Karpala, M., Wahlman, S. L., Lehtonen, H. & Käyhty, H. Specificities and opsonophagocytic activities of antibodies to pneumococcal capsular polysaccharides in sera of unimmunized young children. Clin. Diagn. Lab. Immunol. 9 10.1128/CDLI.9.5.1032-1038.2002 (2002). Lortan, J. E., Kaniuk, A. S. & Monteil, M. A. Relationship of in vitro phagocytosis of serotype 14 Streptococcus pneumoniae to specific class and IgG subclass antibody levels in healthy adults. Clin. Exp. Immunol. 91 10.1111/j.1365-2249.1993.tb03353.x (1993). Winkelstein, J. A. The role of complement in the host's defense against streptococcus pneumoniae. Rev. Infect. Dis. 3 10.1093/clinids/3.2.289 (1981). Briles, D. E., Crain, M. J., Gray, B. M., Forman, C. & Yother, J. Strong association between capsular type and virulence for mice among human isolates of Streptococcus pneumoniae. Infect. Immun. 60 10.1128/iai.60.1.111-116.1992 (1992). Pilishvili, T. et al. Sustained reductions in invasive pneumococcal disease in the era of conjugate vaccine. J. Infect. Dis. 201 10.1086/648593 (2010). Masomian, M., Ahmad, Z., Gew, L. T. & Poh, C. L. in Vaccines Vol. 8 (2020). Cannon, K. et al. A trial to evaluate the safety and immunogenicity of a 20-valent pneumococcal conjugate vaccine in populations of adults ≥ 65 years of age with different prior pneumococcal vaccination. Vaccine 39 10.1016/j.vaccine.2021.10.032 (2021). Brooks, L. R. K. & Mias, G. I. in Front. Immunol. 9 (2018). (Frontiers Media S.A.. Wattal, C., Goel, N. & Byotra, S. P. Prevalence of Pneumococcal Serotypes in Adults ≥ 50 Years of Age. Ind. J. Med. Microbiol. 35 , 95–100. 10.4103/IJMM.IJMM_16_132 (2017). Wijayasri, S. et al. The shifting epidemiology and serotype distribution of invasive pneumococcal disease in Ontario, Canada, 2007–2017. PLoS ONE . 14 10.1371/journal.pone.0226353 (2019). Scott, N. R., Mann, B., Tuomanen, E. I. & Orihuela, C. J. in Vaccines Vol. 9 1–17MDPI AG, (2021). Sharew, B. et al. Serotype distribution of streptococcus pneumoniae isolates causing invasive and non-invasive infections using whole-genome sequencing in Ethiopia. Infect. Drug Resist. 14 10.2147/IDR.S293578 (2021). Isturiz, R. E. et al. Pneumococcal epidemiology among us adults hospitalized for community-acquired pneumonia. Vaccine 37 10.1016/j.vaccine.2019.04.087 (2019). Løchen, A., Croucher, N. J. & Anderson, R. M. Divergent serotype replacement trends and increasing diversity in pneumococcal disease in high income settings reduce the benefit of expanding vaccine valency. Sci. Rep. 10 10.1038/s41598-020-75691-5 (2020). Manoharan, A. et al. Invasive pneumococcal disease in children aged younger than 5 years in India: a surveillance study. Lancet. Infect. Dis . 17 10.1016/S1473-3099(16)30466-2 (2017). Li, X., Puvanesarajah, V. & Berti, F. Structure of the type 38 Streptococcus pneumoniae capsular polysaccharide. Carbohydr. Res. 541 10.1016/j.carres.2024.109165 (2024). Bednar, B. & Hennessey, J. P. Molecular size analysis of capsular polysaccharide preparations from Streptococcus pneumoniae. 243–243 (1993). Sangareddy, V. et al. Identifying and optimization of critical process parameters for the modulation of polysaccharide molecular size in Streptococcus pneumoniae serotype-1. Discover Appl. Sci. 6 10.1007/s42452-024-06096-6 (2024). Theisen, A., Johann, C., Deacon, M. P. & Harding, S. E. Refractive Increment Data-Book for Polymer and Biomolecular Scientists (Nottingham University, 2000). Striegel, A. M. Specific Refractive Index Increment (∂n/∂c) of Polymers at 660 nm and 690 nm. Chromatographia 80 , 989–996. 10.1007/s10337-017-3294-2 (2017). Talaga, P., Vialle, S. & Moreau, M. Development of a high-performance anion-exchange chromatography with pulsed-amperometric detection based quantification assay for pneumococcal polysaccharides and conjugates. 2474–2484 (2002). McNeely, T. B., Staub, J. M., Rusk, C. M., Blum, M. J. & Donnelly, J. J. Antibody Responses to Capsular Polysaccharide Backbone and O-Acetate Side Groups of Streptococcus pneumoniae Type 9V in Humans and Rhesus Macaques. 3705–3710 (1998). Spencer, B. L., Saad, J. S., Shenoy, A. T., Orihuela, C. J. & Nahm, M. H. Position of O-acetylation within the capsular repeat unit impacts the biological properties of pneumococcal serotypes 33A and 33F. Infect. Immun. 85 10.1128/IAI.00132-17 (2017). Kao, G. & Tsai, C. M. Quantification of O-acetyl, N-acetyl and phosphate groups and determination of the extent of O-acetylation in bacterial vaccine polysaccharides by high-performance anion-exchange chromatography with conductivity detection (HPAEC-CD). Vaccine 22 , 335–344. 10.1016/j.vaccine.2003.08.008 (2004). Rajendar, B. et al. High-Performance Anion-Exchange chromatography with conductivity detection method for simultaneous determination of nitrogen and phosphorus in polysaccharides. J. Chromatogr. B: Anal. Technol. Biomedical Life Sci. 1207 10.1016/j.jchromb.2022.123367 (2022). Dische, Z., Shettles, L. B., A NEW SPECTROPHOTOMETRIC TEST FOR & THE DETECTION OF METHYLPENTOSE. J. Biol. Chem. 192 , 579–582, doi: 10.1016/S0021-9258(19)77781-3 (1951). Bitter, T. & Muir, H. M. A modified uronic acid carbazole reaction. Anal. Biochem. 4 10.1016/0003-2697(62)90095-7 (1962). Filisetti-Cozzi, T. M. C. C. & Carpita, N. C. Measurement of uronic acids without interference from neutral sugars. Anal. Biochem. 197 10.1016/0003-2697(91)90372-Z (1991). GmbH, A. P. Instruction Manual Litesizer™ 500 (2016). Jansson, P. E., Lindberg, B. & Lindquist, U. Structural studies of the capsular polysaccharide from Streptococcus pneumoniae type 5. Carbohydr. Res. 140 , 101–110. 10.1016/0008-6215(85)85053-9 (1985). Stroop, C. J. M., Xu, Q., Retzlaff, M., Abeygunawardana, C. & Bush, C. A. Structural analysis and chemical depolymerization of the capsular polysaccharide of Streptococcus pneumoniae type 1. Carbohydr. Res. 337 , 335–344. 10.1016/S0008-6215(01)00318-4 (2002). Schumann, B. et al. Development of an Efficacious, Semisynthetic Glycoconjugate Vaccine Candidate against Streptococcus pneumoniae Serotype 1. ACS Cent. Sci. 4 , 357–361. 10.1021/acscentsci.7b00504 (2018). Abcam Dot blot protocol. doi: (2023). https://www.abcam.com/en-us/technical-resources/protocols/dot-blot WHO. Recommendations to assure the quality, safety and efficacy of pneumococcal conjugate vaccines, World Health Organization,. WHO-Technical Report Series, No. 977, 91–151. (2009). Hotchkiss, A. T. & Hicks, K. B. Analysis of oligogalacturonic acids with 50 or fewer residues by high-performance anion-exchange chromatography and pulsed amperometric detection. Anal. Biochem. 184 , 200–206. 10.1016/0003-2697(90)90669-Z (1990). Cobb, B. A. & Kasper, D. L. Cell. Microbiol. 7 1398–1403 (2005). Mertens, J. et al. Streptococcus pneumoniae serotype 1 capsular polysaccharide induces CD8 + CD28- regulatory T lymphocytes by TCR crosslinking. PLoS Pathog. 5 10.1371/journal.ppat.1000596 (2009). Patravale, V., Dandekar, P. & Jain, R. Characterization techniques for nanoparticulate carriers. Nanoparticulate Drug Delivery . 87–121. 10.1533/9781908818195.87 (2012). Blumenkrantz, N. & Asboe-Hansen, G. An assay for total hexosamine and a differential assay for glucosamine and galactosamine. Clin. Biochem. 9 , 269–274. 10.1016/S0009-9120(76)80075-6 (1976). Gallorini, S. et al. Introduction of Zwitterionic Motifs into Bacterial Polysaccharides Generates TLR2 Agonists Able to Activate APCs. J. Immunol. 179 , 8208–8215. 10.4049/jimmunol.179.12.8208 (2007). Mazmanian, S. K., Cui, H. L., Tzianabos, A. O. & Kasper, D. L. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 122 , 107–118. 10.1016/j.cell.2005.05.007 (2005). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5880261","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":414553067,"identity":"10f30566-dbe5-4806-9399-8addde8360ac","order_by":0,"name":"M V N Janardhan Reddy","email":"","orcid":"","institution":"Birla Institute of Technology and Sciences-Pilani","correspondingAuthor":false,"prefix":"","firstName":"M","middleName":"V N Janardhan","lastName":"Reddy","suffix":""},{"id":414553068,"identity":"d5023558-8cf2-44fb-b70b-af82f0fa175a","order_by":1,"name":"Yogeshwar Devarakonda","email":"","orcid":"","institution":"Birla Institute of Technology and Sciences-Pilani","correspondingAuthor":false,"prefix":"","firstName":"Yogeshwar","middleName":"","lastName":"Devarakonda","suffix":""},{"id":414553069,"identity":"f9493f01-bc8a-42f7-b86e-00287b50a338","order_by":2,"name":"Burki Rajendar","email":"","orcid":"","institution":"Research \u0026 Development","correspondingAuthor":false,"prefix":"","firstName":"Burki","middleName":"","lastName":"Rajendar","suffix":""},{"id":414553071,"identity":"a25adc4f-5f62-44b3-b7df-507c2e4c21be","order_by":3,"name":"Kirtimaan Syal","email":"data:image/png;base64,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","orcid":"","institution":"Birla Institute of Technology and Sciences-Pilani","correspondingAuthor":true,"prefix":"","firstName":"Kirtimaan","middleName":"","lastName":"Syal","suffix":""}],"badges":[],"createdAt":"2025-01-22 11:16:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5880261/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5880261/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77615733,"identity":"5c86576e-5380-4b13-9cbe-717c4b2bf137","added_by":"auto","created_at":"2025-03-03 14:59:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":36564,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEC-MALS Profile of Molar mass Vs Time of PnPS serotype 38.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5880261/v1/eab6a22944f8f18cf17c5e03.jpg"},{"id":77619627,"identity":"b3bfa6cc-5fe6-4404-9a21-8187cea40f15","added_by":"auto","created_at":"2025-03-03 15:23:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1040896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eOverlayed chromatogram of TFA hydrolysed PnPS type 38 and the individual monosaccharides injected onto CarboPac PA10 column connected to HPAEC-PAD. \u003cstrong\u003eB.\u003c/strong\u003e Overlayed chromatogram of TFA hydrolysed PnPS type 38 and its respective monosaccharides injected onto to CarboPac PA10 column connected to HPAEC-PAD. \u003cstrong\u003eC. \u003c/strong\u003eOverlayed chromatogram of TFA digested PnPS type 38, HF followed by TFA hydrolysed PnPS type 38 and PnPS type 5 injected onto CarboPac PA10 column connected to HPAEC-PAD along with zoomed profile from 0 to 15 min (Inset).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5880261/v1/11b7112759828a6ff1229a98.jpg"},{"id":77617078,"identity":"51a9cc0f-d348-4bbe-9d4e-aced8110ac1d","added_by":"auto","created_at":"2025-03-03 15:07:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":71247,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Overlayed profiles of Acetate standards (0.625 to 40 µg/mL) from HPAEC-CD and are separated by using AS11 HC column and 5 mM NaOH as an eluent. \u003cstrong\u003eB\u003c/strong\u003e. Overlayed profiles of PnPS type 38 from HPAEC-CD analysis of acetyl groups released for two and four hours of mild alkaline treatment and are separated using an AS11 HC column and 5 mM NaOH eluent.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5880261/v1/cf42710c3ae61edf5736fc48.jpg"},{"id":77615734,"identity":"8bc165ab-6b58-472c-961d-f0440612059f","added_by":"auto","created_at":"2025-03-03 14:59:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":71448,"visible":true,"origin":"","legend":"\u003cp\u003eThe zeta potential distribution profile for PnPS serotype 38 and serotype 1. Both serotypes exhibited similar net charge values with their zeta potentials centered near to 0 mV. The near-identical charge value of PnPS serotype 38 also suggested it to be in a zwitterionic state.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5880261/v1/f1df451ff34571959ddafb6e.jpg"},{"id":77619548,"identity":"ffc6a88c-6ba8-4948-a8b0-da6c5a4e2875","added_by":"auto","created_at":"2025-03-03 15:23:49","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":72360,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Linear standard curve for Rhamnose obtained using the Cysteine-HCl method. \u003cstrong\u003eB.\u003c/strong\u003e Sample results showing the percentage of methyl pentoses tested at three different concentration levels. \u003cstrong\u003eC.\u003c/strong\u003eLinear curve for Uronic acid determined using the Carbazole method. \u003cstrong\u003eD.\u003c/strong\u003e Results table displaying the percentage of Uronic acid content in the sample, tested at three different concentration levels.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5880261/v1/2a89acbd7682ff7806a31177.jpg"},{"id":77615741,"identity":"33cc77e8-beb8-4439-a706-8e9ecf6624c7","added_by":"auto","created_at":"2025-03-03 14:59:47","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":55533,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Overlayed chromatographs of standard concentrations and linearity curve of Nitrate (0.1 to 1.5 µg/mL). \u003cstrong\u003eB\u003c/strong\u003e. Overlayed Standards and linearity curve of Phosphate (1.0 to 10 µg/mL). \u003cstrong\u003eC\u003c/strong\u003e. Chromatogram of PnPS type 38. All the standards and samples were injected into the Ion PAC AS15 (4X250mm) column with guard (4X50mm) by pumping the 38mM Sodium Hydroxide as a mobile phase with the flow rate of 1.2mL/min and detected with suppressed conductivity detection, ADRS auto suppression recycle mode, 113mA current.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5880261/v1/52cf7763aa312bc8f4272b01.jpg"},{"id":77615744,"identity":"190461b4-46f3-416d-aba9-22b1e16cfe5f","added_by":"auto","created_at":"2025-03-03 14:59:47","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":34333,"visible":true,"origin":"","legend":"\u003cp\u003eSLOTBLOT analysis- the horizontal lanes 1, 2, and 3 represented PnPS 1, 5, and 38, loaded in, respectively, at two different concentrations: 1 µg/mL and 2 µg/mL. The analysis were performed across four sets (A, B, C, D). The Blot of A \u0026amp; B were tested with Anti-PnPS type1 monoclonal antibodies (mAb) and polyclonal antibodies (pAb), respectively. C \u0026amp; D were tested with Anti-PnPS type5 monoclonal antibodies and polyclonal antibodies respectively.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5880261/v1/4f5045f25e98bb3d3eda6ae8.jpg"},{"id":83030604,"identity":"ac27ec61-8988-416c-be4c-6ea86ac3cac3","added_by":"auto","created_at":"2025-05-19 09:02:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2363398,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5880261/v1/7fe23941-9c17-4400-8d5c-a4647e590fd1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biochemical and Immunological Insights into Capsular Polysaccharide of Streptococcus pneumoniae Serotype 38 (American type 71)- An emerging non-vaccine serotype","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eStreptococcus pneumoniae-\u003c/em\u003e a Gram-positive bacterium with a polysaccharide capsule, can potentially cause both non-invasive and invasive pneumococcal disease (IPD) involving respiratory system predominantly in children under five and the elderly\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Though more than 100 serotypes of \u003cem\u003eS. pneumoniae\u003c/em\u003e are known\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, a relatively small subset (less than 30) is responsible for the majority of pneumococcal infections in humans\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The diversity of pneumococcal serotypes is primarily determined by variations in the chemical structure of the bacterial capsule's polysaccharides such as differences in the oligosaccharide units or attached side groups\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The capsular polysaccharide acts as a virulence factor for \u003cem\u003eS. pneumoniae\u003c/em\u003e aiding its survival inside host. Vaccines mediated immune response against capsular polysaccharides involves protective antibodies with opsonophagocytic activity (OPA). Such antibodies facilitate the complement-mediated uptake and killing of pneumococci by human phagocytic cells\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Evidently, the introduction of the pneumococcal capsular polysaccharide conjugate vaccine (PCV7) has reduced IPD-related deaths among children under five in the United States from 2000 to 2007\u003csup\u003e10\u003c/sup\u003e. The PPSV 23 (PNEUMOVAX\u0026reg; 23) is a widely used pneumococcal polysaccharide-based vaccine for adults (fifty years old and above) whereas the PCV13 conjugate vaccine is preferred for children. The PCV13 was further improvised to PCV20 (PREVNAR 20\u0026reg;), which could protect against seven additional serotypes including 8, 10A, 11A, 12F, 15B, 22F, and 33F\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The new serotypes that have not been characterized continues to pose challenges due to their unknown capsular polysaccharide structures\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The characterization of such new serotypes of \u003cem\u003eS. pneumoniae\u003c/em\u003e involving examination the biological and serological characteristics of capsular polysaccharides may facilitate their inclusion in the vaccine designs. The coverage of existing PCV 10, 13, 20, and PPSV 23 vaccines has been observed at 16%, 24%, 48%, and 66%, respectively\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Moreover, approximately 30% of the total \u003cem\u003eS. pneumoniae\u003c/em\u003e isolates belong to non-vaccine serotypes (NVTs), highlighting the need to include these serotypes in vaccine coverage to improve population protection\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The prevalence of pneumonia caused by non-vaccine serotypes varies globally\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In 2019 alone, an estimated one million deaths among children under five were attributed to pneumonia\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In India, around 21% of the IPD cases in 2016 were attributed to non-vaccine serotypes\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe non-vaccine \u003cem\u003eS. pneumoniae\u003c/em\u003e serotypes are continuously emerging as evident by their increased incidence rates and spread\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The most common serotypes that infect children include 10A/F, 7C, 35A/B, 16F, 19A, 3, and \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e38\u003c/span\u003e\u003csup\u003e15,17\u003c/sup\u003e. Surveillance data from countries such as the USA, Australia, Finland, France, Norway, Canada, and Ethiopia indicate that serotype 38 has become more prevalent causing IPD across all age groups\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In India, Manoharan et al. identified serotype 38 in more than 1% of the \u003cem\u003eS. pneumoniae\u003c/em\u003e clinical isolates\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Together, the understanding of its CPS biochemical characteristics, immunological properties, and critical quality attributes (CQAs) has become vital considering its widespread distribution, high incident rates, and identification in clinical isolates.\u003c/p\u003e \u003cp\u003eEarlier, the structure of the type 38 polysaccharide was studied by NMR analysis, however, it did not analyze the zwitterionic state of the polysaccharide, phosphorus content, and the cross-reactivity with other serotypes\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In his study, in addition to biochemical characterization, zwitterionic state was determined, phosphorus content was estimated, and immunological cross reactivity with selected serotypes was assessed thereby providing essential insights for designing effective therapeutic strategies against IPD caused by serotype 38.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003ePurified pneumococcal polysaccharide serotype 38 was procured from the American Type Culture Collection (ATCC 543-X). Glucose (Glc), Galactose (Gal), Mannose (Man), Fucose (Fuc), Rhamnose (Rha), Glucuronic acid (GlcA), Galacturonic acid (GalA), Glucosamine (GlcN), Galactosamine (GalN), N-Acetylglucosamine (GlcNAc), and N-Acetyl galactosamine (GalNAc) were purchased from Sigma-Aldrich Co. (St. Louis, USA). N-Acetyl Fucosamine (FucNAc), N-acetyl-Pneumosamine (PneNAc) were purchased from Omicron Biochemicals Inc. USA. Trifluoroacetic acid (TFA) and hydrofluoric acid (HF) obtained from Merck, India. Sodium acetate and sodium hydroxide 50% solution were purchased from Sigma-Aldrich Co. (St. Louis, USA). Mouse Monoclonal Antibodies of serotype 1 and serotype 5 were procured from AbMax-China, and Rabbit polyclonal antisera and capsular polysaccharide of serotype 1 and serotype 5 were procured from Statens Serum Institute Diagnostica, Denmark. Secondary antibodies of Goat Anti-mouse IgG peroxidase and Anti Rabbit IgG peroxidase purchased from Bio-Rad laboratories-India. All the chemicals were ACS reagent-grade and IC-grade with a purity specification of \u0026ge;\u0026thinsp;90\u0026ndash;99% (Fluka, Sigma\u0026ndash;Aldrich).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of CPS of serotype 38 and the sugar standards\u003c/h2\u003e \u003cp\u003eThe stock solution of purified polysaccharide of serotype 38 was prepared by dissolving it in ASTM Type-II water at a concentration of 2 mg/mL (w/v). All other sugar master stocks were prepared as 1mg/mL (w/v) in ASTM Type-II water which were subsequently diluted to the final concentration of 10\u0026micro;g/mL (for each sugar). The buffered serotype stock, originally had sodium chloride in its lyophilized form, has been subjected to desalting procedure wherever needed.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSEC-UV-MALS-RI\u003c/h3\u003e\n\u003cp\u003eThe pneumococcal polysaccharide (PnPS) physical parameters such as molar mass was determined by SEC-UV-MALS-RI. It is crucial to maintain the molecular size (MS) of PnPS within the specified range for the design of conjugate vaccines either through physical or acid hydrolysis before use in the conjugation process\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The purified polysaccharide obtained from ATCC was initially dissolved in ASTM Type-II water to achieve a concentration of 2 mg/mL (w/v). Subsequently, a 100 \u0026micro;L aliquot of this solution was injected into a size exclusion chromatography (SEC) column that was connected to UV-MALS-RI detectors and are calibrated using a BSA monomer standard of 2 mg/mL. The HPLC system utilized for the analysis consisted of SHODEX SB 806HQ and SHODEX SB 803HQ connected in series with 100mM sodium phosphate and 0.05% sodium azide pH 7.2 serving as the mobile phase. The sample separation was performed by pumping the mobile phase at a flow rate of 0.5mL/min through the connected analytical columns. The Agilent 1260 series HPLC, equipped with miniDAWN\u0026reg; TREOS\u0026reg; and Optilab\u0026reg; T-rEX RI detectors from Wyatt Technology-USA, was used to carry out the analysis. The molar mass and size distribution pattern was determined using the refractive index as a concentration source-detector by inputting the dn/dc of 0.133 mL/g\u003csup\u003e24,25\u003c/sup\u003e to the ASTRA software.\u003c/p\u003e\n\u003ch3\u003eHPAEC-PAD\u003c/h3\u003e\n\u003cp\u003eThe 10 \u0026micro;g/mL of polysaccharide treated with 10M TFA with the ratio of 4:1 (v/v) into a glass vial, sealed with caps equipped with Teflon faced rubber insert, and was subjected to incubation at 121\u0026deg;C for 2 hours in the digital dry bath with safety lid. Post incubation, the sample was subjected to nitrogen evaporation set at 45\u0026deg;C for 30min to evaporate the TFA. The polysaccharide smear in the vial was then dissolved in 1mL of ASTM type-II water and then filtered through 0.22\u0026micro;m syringe filter into the HPLC vial. In parallel set, 10 \u0026micro;g/mL of polysaccharide was first treated with HF followed by the TFA as per the referred protocol\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The 20 \u0026micro;L of depolymerized polysaccharide and sugar standards were injected onto Carbopac PA10 (4.6X250mm) column and guard (4.6X50mm) connected to the ICS-5000 (Thermo Fisher Scientific, Sunnyvale, CA, USA) system equipped with Electrochemical detector that includes an Ag/AgCl reference electrode and a disposable gold working electrode. The mobile phase of A; 18mM NaOH, B; 100mM NaOH and C;1M Sodium acetate in 100mm NaOH were pumped into the column as a gradient program with the flow rate of 1mL/min. The temperature of column and autosampler (ASAP) were set at 30\u0026deg;C and 15\u0026deg;C, respectively. The carbohydrate (Standard quad) waveform was applied to the ECD. All the data was processed using Chromeleon\u0026trade; software.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHPAEC-CD\u003c/h2\u003e \u003cp\u003eThe O-acetyl content determines the immunogenic response of the polysaccharides as reported\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The O- acetyl content of the capsular polysaccharides was examined. The polysaccharide at a concentration of 200 \u0026micro;g/mL was treated with 0.1 N NaOH and incubated at 37\u0026deg;C for 2 and 4 hours. Following the incubation, the mixture was passed through 10kDa nanosep centrifugal filters that had been pre-rinsed three times with ASTM Type-II water at 10000 rpm for 15 minutes. To avoid any dilution of the sample filtrate with droplets from the filter holder used for the pre-rinse process, the filtrate was collected into a fresh 10kDa filter holder. A working standard was devised using Sodium acetate (as acetate source) in a concentration range of 0.625 to 40 \u0026micro;g/mL. The standard was diluted with ASTM Type-II water and the sample filtrate was analyzed as described elsewhere\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe total nitrogen (contribution from hexosamines and residual protein impurity) content was estimated with in the polysaccharide as per the referred protocol\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The nitrogen content is not solely attributed to hexosamines but is also influenced by any protein content present in the capsular polysaccharides therefore may help in estimating protein impurities. In addition to amines, phosphates may also exist as sugar derivative components, such as ribitol phosphate and ribose phosphate, within these polysaccharides. Moreover, nucleic acid impurities in the purified capsular polysaccharides may contribute significantly to the overall nitrogen and phosphorus levels. Therefore, it is essential to quantify these components within the polysaccharides, in order to provide indirect insights into the impurity levels of residual proteins and nucleic acids, serving as an orthogonal verification method alongside conventional OD 260/280 measurements. In this study, 200 \u0026micro;g/mL of serotype 38 polysaccharide was subjected to digestion using 0.5 M potassium persulfate in 0.5 M NaOH at 100\u0026deg;C for 16 hours. Following digestion, the sample was diluted before the analysis by HPAEC-CD. The analysis was performed using a Thermo ICS-5000 system, equipped with an IonPac AS15 guard column (50 \u0026times; 4 mm) and an IonPac AS15 analytical column (250 \u0026times; 4 mm), to separate and estimate the nitrogen and phosphorus content as previously reported\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOrthogonal verification of sugar composition and its derivatives\u003c/h3\u003e\n\u003cp\u003eThe bacterial capsular polysaccharide structure comprises common sugar derivatives like methyl pentoses and uronic acids, along with the common sugar forms of hexoses and pentoses. The sugar composition obtained by HPAEC-PAD was verified by the conventional colorimetric methods such as Cysteine-HCl method for methyl pentoses\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e and Carbazole method for uronic acids\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. As reported, rhamnose was used as a standard for methyl pentose assay, and glucuronic acid was used as a standard for uronic acid assay. Briefly, for the methyl pentose assay, 100 \u0026micro;L of rhamnose standard ranging from 2 to 40\u0026micro;g/mL was prepared alongside the diluted sample. Then, 500 \u0026micro;L of reagent A (1 in 6 v/v Water/ H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) was added to the standard solutions and the diluted sample followed by incubation at 90˚C for 7 min in water bath. Then 40 \u0026micro;L of reagent B (3% w/v Cysteine HCl) was added to all the tubes and incubated at 37˚C for 30 min. After incubation, 200 \u0026micro;L of each standard and sample were transferred into 96 well plate and the absorbance was measured at 396nm and 430 nm. As previously reported, the Carbazole method was used for estimating the uronic acid content. Briefly, the 100 \u0026micro;L of glucuronic acid standard range from 5 to 50\u0026micro;g/mL was prepared, and the 100 \u0026micro;L of diluted sample was added to 500 \u0026micro;L of reagent A (0.030M Borate buffer / H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) and incubated at 90˚C for 15 min in water bath. After the addition of 20 \u0026micro;L of reagent B (0.125% Carbazole in ethanol), all tubes were incubated at 90˚C for 15 min. After incubation, 200 \u0026micro;L of each standard and sample were transferred to 96 well plate and the absorbance was measured at 530nm and 660 nm\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eDynamic Light Scattering (DLS)\u003c/h3\u003e\n\u003cp\u003eThe zeta potential was measured in series mode using Omega Cuvettes with the light scattering instrument of Litesizer-500 (Anton Paar, India) by Kalliope software, and the default measurement parameters were set\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The lyophilized polysaccharide powder of serotype 38 was received from ATCC and it had high sodium chloride content (6M) which interfered with the charge measurement. Therefore, the polysaccharide powder was dissolved in ASTM Type-II water and desalted by passing it through a 10kDa cutoff centrifugal filter to remove the sodium chloride content. Sodium chloride hinders the zeta potential measurement due to its high conductivity and may lead to charring of the electrode in cuvette. The buffer exchanged sample is further diluted to 200\u0026micro;g/mL with ASTM Type-II water to meet the instrument measurement specifications of the sample such as accepted filter optical density and sufficient mean intensity (\u0026gt;\u0026thinsp;20 kcounts/s) values. Before analyzing the sample, the instrument was calibrated with the known zeta potential standards.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSLOTBLOT\u003c/h2\u003e \u003cp\u003eThe capsular polysaccharide of serotype 38 consisted of an unknown sugar in its repeating unit identified as \"Sug\u003csub\u003e\u003cem\u003ep\u003c/em\u003e\u003c/sub\u003e\" as described in the reference and we also could not decipher this unknown sugar\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, similar sugar (2-acetamido-2,6-dideoxy-D-xylo-hexos-4-ulose) was also present in another pneumococcal serotype i.e. PnPS serotype 5\u003csup\u003e35\u003c/sup\u003e. Additionally, PnPS serotype 1 also featured a unique and rare amino sugar moiety, AAT sugar (2-acetamido-4-amino-2,4,6-trideoxy-D-galactose), which was previously noted for its immunogenicity\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Therefore, we tested if the serotype 38 polysaccharide may elicit antibodies functionally similar to those elicited by the polysaccharides derived from serotypes 1 and 5. Such antibody response is expected only if the unknown sugar acted as an antigenic epitope in the serotype 38 polysaccharide. We performed a SLOTBLOT assay to test our hypothesis \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e with an aim to evaluate the cross-reactivity of serotype 38 against antibodies specific to serotypes 1 and 5. Briefly, PnPS serotypes 1, 5, and 38 were adhered to a nitrocellulose membrane at two different concentrations in four sets. The 1% skim milk powder solution in PBS was used as a blocking reagent over the membrane. Following blocking, the membrane was washed three times with PBST (PBS with 0.05% Tween 20). With the optimized dilutions of Monoclonal and polyclonal antibodies specific to serotypes 1 and 5 were added to designated blots and incubated for one hour. After incubation, the membrane was washed again with PBST and incubated with respective secondary antibodies labeled with horseradish peroxidase. The reaction was developed using a TMB substrate to visualize the antibody binding on the membrane concerning the lanes of the loaded antigens.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCritical quality attributes of capsular polysaccharide\u003c/h2\u003e \u003cp\u003eCapsular polysaccharides content should meet stringent standards for identity, purity, potency, and safety set by regulatory authorities like the FDA, EMA, and WHO for inclusion in vaccine design\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The vaccine's reproducibility depends on various factors including surface charge that affects stability and particle distribution. Based on the regulatory guidelines, monitoring and adhering to critical quality attribute (CQA) is essential for characterization of antigenic content and demonstration of their immunogenic properties in order to ensure optimal efficacy and protection against the corresponding pneumococcal serotype. The chemical composition, including sugars and functional groups, must be precise to maintain the immunogenic properties and mimic the native bacterial surface. Establishing specific percentage limits for each functional group within the polysaccharides is necessary to evaluate the integrity of the immunogenic functional groups and confirm the preservation of the vaccine's immunogenic characteristics. The CQAs for these polysaccharides include several factors that can significantly impact the performance and reliability of the final vaccine product. The most important attributes to assess the PnPS quality are; molecular size, purity, sugar composition, and surface charge. The polysaccharides that are too small may be ineffective, while those too large can complicate manufacturing due to excessive viscosity. Consistent molar mass and size distribution are also necessary for uniform vaccine quality\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Impurities such as proteins, nucleic acids, and endotoxins may reduce vaccine effectiveness and safety. Rigorous purification is essential to minimize these contaminants. Ensuring these CQAs are met is crucial for producing safe and effective vaccines. Hence we attempted to propose the limits of CQAs with the ATCC-purified polysaccharide as it is used as a reference standard by the manufacturers to develop immunological-related methods.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSize and Molar mass by SEC-MALS\u003c/h2\u003e \u003cp\u003eThe molar mass and size distribution pattern of purified CPS are essential quality attributes for glycoconjugate vaccine manufacturing, as they indicate the consistency of upstream and downstream processes. In this study, a sample of purified Spn type 38 CPS (2 mg/mL) was injected into size exclusion columns connected to an Agilent HPLC system equipped with a UV-MALS-RI detector. The normalization was carried out with the BSA monomer prior to analysis. The refractive index increment, dn/dc of the polysaccharide (0.133 mL/g), was used to determine the molar mass, rms radius moments (rz), and size distribution pattern using ASTRA software. The polysaccharide peak was observed between 22 and 30 minutes, with the size distribution graph showing a range of 1.0x107 to 1.0x105 g/mol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The average molar mass of the purified CPS of Spn type 38 was determined to be 7.684x105 g/mol (\u0026plusmn;\u0026thinsp;3.72%) with a polydispersity (Mw/Mn) value of 1.451 (\u0026plusmn;\u0026thinsp;4.460%) and z-average radius of gyration (Rz) value of 90 nm. These results can be used as a reference value for assessing the consistency of the purification process, both in terms of fermentation and downstream purification, for purified CPS of Spn type 38.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMonosaccharide composition by HPAEC-PAD\u003c/h2\u003e \u003cp\u003eAs described in the method section, the depolymerized samples were analyzed in ion- chromatography system using a CarboPac PA10 column coupled with electrochemical detection (ECD). The monosaccharide components identified in the CPS included N-acetylglucosamine, galactose, and galacturonic acid, alongside an unidentified peak with a retention time of 22 minutes (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). With an aim to elucidate the nature of the unidentified peak (RT of 22 min), individual injections of various monosaccharides, including fucose, glucose, galactose, mannose, glucosamine, fucosamine, pneumosamine, and galacturonic acid, were performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Based on the retention time of the unidentified peak, which eluted at approximately 50 mM sodium acetate in 100 mM NaOH, it was inferred that this peak was not attributable to any monosaccharide or disaccharide, as all such compounds elute from the CarboPac PA10 column with 100 mM sodium hydroxide alone, without the necessity of sodium acetate\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The unidentified peak's elution in the presence of sodium acetate suggested it might be a sugar acid or a non-sugar compound. It is hypothesized that this peak represents a sugar acid or a compound containing an amino acid, potentially forming a peptide bond with galacturonic acid or another amine present in the CPS repeating unit. As we were in the process of characterizing this unidentified, Li et al. reported\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e structural studies of the serotype 38 capsular polysaccharide and they identified the unknown component as the amino acid serine conjugated to the galacturonic acid. We also confirmed the presence of serine. The discovery represents the first instance of an amino acid being identified within the repeating unit of a \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e capsular polysaccharide. The relative peak areas for the identified monosaccharides were as follows: galactose, 72.45%; N-acetylglucosamine, 9.78%; and galacturonic acid, 8.05%, while the amino acid (Serine) peak accounted for approximately 7.83% and 1.89% of unidentified peaks of the total peak area of the chromatogram (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). There was another small percent of peaks eluted between 5 to 8 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) overlaid with the peaks (other than the main known sugars) obtained for PnPS serotype 5 hydrolysed with HF followed by TFA as for serotype 38 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC zoomed chromatogram). This overlaying confirmed and supported the recently reported work\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e that these peaks belong to the Sug\u003csub\u003e\u003cem\u003ep\u003c/em\u003e\u003c/sub\u003e (2-acetamido-2,6-dideoxy-D-xylo-hexos-4-ulose) moiety present in the PnPS serotype 38. This novel structural feature may influence host-pathogen interactions, immunogenicity, or present challenges in conjugation chemistry used for coupling with carrier proteins. Further investigation is warranted to explore these implications comprehensively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRelative percent peak areas of acid hydrolyzed CPS of Spn type 38 analyzed on HPAEC-PAD\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnknown peaks\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlucosamine peak\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGalactose peak\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAmino acid (Serine) peak\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGalacturonic Acid peak\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eO-acetyl content by HPAEC-CD\u003c/h2\u003e \u003cp\u003eThe O-acetyl content is one of the important immunogenic functional groups for capsular polysaccharide-based vaccines\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Hence, it is mandated to quantify it as the quality attributes of the CPS. In the ATCC-purified polysaccharide of serotype 38, under mild alkaline conditions, as mentioned previously, the O-acetyl groups were selectively hydrolysed and released as acetate ions. The hydrolysate acetate was separated from other ions by the Thermo ICS-5000 system equipped with a strong anion exchange column and guard column of IonPac AS11-HC guard column i.d. (50 \u0026times; 4 mm) and IonPac AS11-HC analytical column i.d. (250 \u0026times; 4 mm) were used and estimated the content. The equimolar ratio of sodium acetate as acetate was used as an assay standard from the range of 0.625 to 40\u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The polysaccharide was kept under mild alkaline conditions, 10 mM NaOH at 37\u0026deg;C, for two and four hours to confirm the complete release of O-acetyl groups from the polysaccharide. The standard acetate peak has a retention time of 7.2 min which overlayed with the acetate peak from the sample. The peak area obtained for two and four hours of incubation was observed to overlay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and the O-acetyl content was calculated to be same for both two hours and four hours of incubation with a value of 5.8%. The two hours of mild alkaline condition was enough to release the total O-acetyls from the CPS of Spn type 38.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePolysaccharide Net charge by DLS\u003c/h2\u003e \u003cp\u003eAs described in the method section, the sample was diluted to 200 \u0026micro;g/mL in ASTM Type-II water to meet instrument specifications, including acceptable optical density and mean intensity (\u0026gt;\u0026thinsp;20 kcounts/s). With default settings, Zeta potential measurements were carried out with the Litesizer-500 (Anton Paar, India) using Omega Cuvettes and Kalliope software. The zeta potential of the polysaccharide of serotype 38 was found as -0.7 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The PnPS type 1 is categorized and claimed as a zwitterionic polysaccharide elsewhere\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Hence for the comparison, we also measured the zeta potential for the PnPS type 1 at 200 \u0026micro;g/mL in ASTM Type-II water and it showed a value of -0.9 mV. This value falls within the \u0026minus;\u0026thinsp;10 to +\u0026thinsp;10 mV range indicating a zwitterionic nature. The nanoparticles with zeta potentials in this range are generally considered to have neutral surfaces, whereas those with absolute values exceeding\u0026thinsp;\u0026plusmn;\u0026thinsp;30 mV are classified as strongly cationic or anionic\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Since PnPS serotype 1 was classified as a zwitterionic polysaccharide, the similar charge of PnPS serotype 38 and the structural features (glucosasamine and galacturonic acid) implied it to be zwitterionic as well. The relative frequency distribution reveals that most particles in both samples had comparable surface charges reflecting their similar zeta potential profiles suggesting a zwitterionic nature and relatively neutral surface charge. This information is crucial for understanding the polysaccharide's behaviour in various applications and stability in different formulations. The neutral surface charge (Zwitterion) may influence its interactions with the antigen-presenting cells (APCs), ZPS-mediated T cell activation, and may trigger the T cell-dependent B cell immune response\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eOrthogonal verification of sugar derivatives by conventional methods\u003c/h2\u003e \u003cp\u003eThe sugar composition obtained by HPAEC-PAD for PnPS serotype 38 was orthogonally verified further with the conventional biochemical methods for methyl sugars (Rhamnose) and sugar acids (Uronic acid) by following the procedures reported and described previously. A good linear curve was observed with a regression value of \u0026gt;\u0026thinsp;0.99 for both methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The OD values observed in the PnPS serotype 38 sample tested at three different concentration levels of 100, 200 and 400\u0026micro;g/ml were comparable to blank thereby confirming the absence of Methyl pentoses in serotype 38 capsular polysaccharide (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). It was supported by the monosaccharide profiles obtained by HPAEC-PAD as there was no Rhamnose peak in the chromatogram. For the uronic acid content evaluation, the sample was tested at three different concentrations, and the obtained average % uronic acid content was determined to be 7.7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) and the value was in line with the percent peak area obtained by HPAEC-PAD (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Also the estimated Hexosamines values were orthogonally verified by the Elson-Morgan method\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e using N-acetyl glucosamine as a standard, the result was in line with the percent peak area obtained by the HPAEC-PAD (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Thus, the percent peak areas obtained by HPAEC-PAD was used to assess the polysaccharide composition from batch to batch instead of performing multiple conventional biochemical methods which could be exploited for other polysaccharides as well.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eNitrogen and Phosphorous Content by HPAEC-CD\u003c/h2\u003e \u003cp\u003eAmines (hexosamines) and phosphorus-containing sugars (ribose-5-phosphate and ribitol-5-phosphate) are important nitrogen and phosphorus sources in polysaccharides, respectively\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In addition to amines, protein impurities in the polysaccharides also contribute to the overall nitrogen content. Similarly, nucleic acid impurities add to the total phosphorus content. Adhering to composition specifications as outlined in recommended guidelines, it was crucial to determine the nitrogen and phosphorus content in the capsular polysaccharide sample. PnPS serotype 38 was subjected to persulfate hydrolysis to determine its nitrate (nitrogen) and phosphate (phosphorus) content using HPAEC-CD. The quantification was performed against standard linear calibration curves established with urea for nitrogen content and sodium phosphate monobasic for phosphorus content (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In the chromatogram, a nitrate peak was detected, whereas no phosphate peak was observed in the sample (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The nitrogen content was estimated to be 3.67% with no detectable phosphorus content. Based on these findings and using the ATCC polysaccharide as a reference standard (since it is used as a standard in the respective immunological assays) for monitoring the critical quality attributes, we inferred that during the purification of PnPS serotype 38, the nitrogen content should be in between 3 to 4%. The phosphorus content should be limited to a range of 0 to 1%, taking into account the minimal nucleic acid contamination present in the capsular polysaccharide (CPS).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eImmunochemical properties\u003c/h2\u003e \u003cp\u003eWith an aim to assess the validity of our hypothesis that the unknown sugar (Sug\u003csub\u003e\u003cem\u003ep\u003c/em\u003e\u003c/sub\u003e) moiety of serotype 38 polysaccharide may constitute a portion of an epitope recognized by the host immune system, we compared the HPAEC-PAD profiles and sugar composition of PnPS serotype 38 with that of serotype 5. Overlapping unidentified peaks were observed, potentially generated from the unknown sugars by acid hydrolysis using hydrofluoric acid (HF) followed by trifluoroacetic acid (TFA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). As described earlier, the CPS of Spn type 38 has a net charge of -0.7mV indicating that it is in zwitterionic form like a CPS of Spn type 1\u003csup\u003e41,42,45\u003c/sup\u003e has a net charge of -0.9mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Thus, we have performed the SLOTBLOT assay using serotype 1 and serotype 5 monoclonal antibodies from AbMax and polyclonal antibodies from SSI, both of which possess unknown, rare sugars and a zwitterionic form of their polysaccharide repeating units. Our results revealed a clear blue blot upon the addition of TMB substrate in the slot where the serotype 5 and serotype 1 polyclonal were loaded against the serotype 38 lane onto the membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In each set, lanes 1, 2, and 3 correspond to PnPS 1, 5, and 38, respectively, with the left spots (2nd vertical line) representing the 2 \u0026micro;g/mL concentration and the right spots (1st vertical line) representing the 1 \u0026micro;g/mL concentration. The intensity and pattern of the spots in each lane and concentration provide insights into the antibody specificity and binding affinity for the respective PnPS. These findings provide evidence supporting the hypothesis that serotype 38 possesses a similar epitope characteristic of serotype 5, and partially of serotype 1, as the unknown sugar in its capsular polysaccharide repeating unit. Consequently, we anticipate that serotype 38 will offer cross-protection against invasive pneumococcal disease caused by serotype 5 and vice versa.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe findings of this study revealed crucial insights into the immunological characteristics of \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e serotype 38, particularly its interaction with antibodies from other serotypes. Notably, serotype 38 exhibits cross-reactivity with polyclonal antibodies of serotypes 5 and serotype 1, yet does not react with the corresponding monoclonal antibodies. This discrepancy highlights the complexity of the immune response and the potential structural similarities and differences in capsular polysaccharides among these serotypes. The cross-reactivity observed with polyclonal antibodies indicates that there are shared epitopes between serotype 38 and serotypes 5 and 1. The polyclonal antibodies, being a heterogeneous mix, could recognize multiple antigenic sites on a pathogen. This broad recognition suggested that certain polysaccharide structures within serotype 38\u0026rsquo;s capsule were similar enough to those in serotypes 5 and 1 to elicit an immune response. Such shared epitopes might include common sugar components or structural motifs that were conserved across these serotypes.\u003c/p\u003e \u003cp\u003eIn contrast, the lack of reaction with monoclonal antibodies, which were specific to a single epitope, suggested that the unique epitopes targeted by these antibodies in serotypes 5 and 1 were absent or significantly different in serotype 38. This implies that while there are similarities in the capsular polysaccharide structures, serotype 38 possesses distinct antigenic features that set it apart from serotypes 5 and 1. These unique structural elements are not recognized by the monoclonal antibodies, which could be due to differences in the oligosaccharide units or specific side groups attached to the polysaccharides.\u003c/p\u003e \u003cp\u003eThe zwitterionic state of serotype 38\u0026rsquo;s capsular polysaccharide, as revealed by zeta potential measurements, suggests a potential for eliciting a T cell-dependent B cell-mediated immune response. ZPS presentation is known to influence T cell activation and abscess formation where antigen-presenting cells produce TNFα activating TH1 cells which secrete cytokines like IL-17 in response to ZPS presented on MHC class II molecules. The interaction between ZPS-MHC class II and α\u0026szlig;TCRs has been shown to drive the development of abscesses, demonstrating the integration of innate and adaptive immune responses\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. This characteristic might contribute to the observed cross-reactivity with polyclonal antibodies as the immune system can generate a broader array of antibodies in response to a more complex antigenic structure.\u003c/p\u003e \u003cp\u003eThese findings could enable formulation of therapeutic strategies and are highly relevant for vaccine design. The cross-reactivity with polyclonal antibodies suggested that current vaccines targeting serotypes 5 and 1 might offer some degree of protection against serotype 38. However, the lack of reaction with monoclonal antibodies underscored the necessity for further research to identify the unique epitopes of serotype 38. The serotype 38 interaction with polyclonal and monoclonal antibodies from serotypes 5 and 1 enhanced our understanding of the immunological landscape of \u003cem\u003eS. pneumoniae\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCritical quality attributes of serotype 38 capsular polysaccharide\u003c/h2\u003e \u003cp\u003eEstablishing specific percentage limits for each functional group within polysaccharides is crucial to assess the integrity of immunogenic functional groups and ensure the preservation of a vaccine's immunogenic properties. In this study, we have evaluated the biochemical characteristics of PnPS Type 38 by estimating the CQAs and proposing specific limits (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These defined CQA limits provide a preliminary reference for assessing the purified Capsular Polysaccharide (CPS) quality of Type 38 produced by vaccine manufacturers. Defining Critical Quality Attributes (CQAs) could be challenging with a single source of CPS. However, using ATCC Capsular Polysaccharide as a reference material is commonly employed as an internal standard for immunological assays by vaccine manufacturers, hence proposed tentative CQA limits could be validated further by comparing multiple sources of CPS of Spn type 38.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCritical quality attribute results of CPS of Spn type 38\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuality attribute\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResults\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProposing limits (concerning the ATCC CPS as a reference)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMolar mass (kg/mol)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e768\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;800\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNet charge (mV)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNeutral (+\u0026thinsp;10 to -10)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eO-Acetyl Groups (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Nitrogen (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 to 5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhosphorus (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (No peak)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 to 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmino acid (Serine) (relative % peak area by HPAEC-PAD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUronic acid (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHexosamines (relative % peak area by HPAEC-PAD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethyl Pentose (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides a comprehensive characterization of the newly prevalent non-vaccine serotype 38 of \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e. Through detailed analysis of its biochemical properties, such as size, molar mass, sugar composition, O-acetyl content, and net charge, we have characterized the structural attributes of this serotype. The immunological assessment, particularly the observed cross-reactivity with polyclonal antibodies from serotype 5 and serotype 1, underscores the potential for cross-protection. This highlights the significance of inclusion of serotype 38 in vaccine development. Moreover, the existence of an amino acid in the polysaccharide repeating unit of this serotype for the first time is particularly notable, as it may play an vital role in host-pathogen interactions. The data presented in this study provide vital information for research and development of therapeutics including vaccines. Together, addressing the challenge posed by non-vaccine serotypes like serotype 38 is vital for improving global health outcomes and reducing the burden of IPD across all age groups. Further research is warranted to explore the potential inclusion of serotype 38 in future vaccine formulations and to monitor its epidemiological trends and impact on public health.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCPS, Capsular polysaccharide; CQA, Critical Quality Attributes; HPAEC-PAD/CD, High-performance anion-exchange pulsed amperometry detection/Conductivity detector; IPD, Invasive pneumococcal disease; DLS, Dynamic Light Scattering system; SEC-MALS-RI, Size exclusion chromatography connected with multi-angle light scattering and Refractive index detectors; Spn, \u003cem\u003eStreptococcus Pneumonia\u003c/em\u003e; PnPS, Pneumococcal Polysaccharide; ZPS, Zwitterionic Polysaccharide.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of interest\u003c/h2\u003e \u003cp\u003eThere are no conflicts to declare.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMVNJR, KS conceptualized the study.MVNJR, YD carried out experiments.MVNJR, YD, BR, and KS analyzed data and wrote manuscript.All authors reviewed it.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eAuthors acknowledge Department of Biotechnology, Government of India for DBT-RLS Re-entry fellowship to KS. Authors acknowledge BITS Pilani Hyderabad campus for funding. YD acknowledge UGC, Delhi for fellowship. We extend our sincere gratitude to Dr. Ramesh Matur, Head of Vaccine R\u0026amp;D, and Dr. Rajendar Burki, Head of Analytical R\u0026amp;D, Biological E. Limited for their invaluable technical support in data interpretation and for granting us access to the necessary instrumentation for this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRyan Gierke, M. P. H., Patricia Wodi, A. \u0026amp; Miwako Kobayashi, M. M. M. D. in \u003cem\u003eU S Centers Disease Control Prev.\u003c/em\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO'Brien, K. L. et al. Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates. \u003cem\u003eLancet\u003c/em\u003e \u003cb\u003e374\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0140-6736(09)61204-6\u003c/span\u003e\u003cspan address=\"10.1016/S0140-6736(09)61204-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatanock, A. et al. Use of 13-Valent Pneumococcal Conjugate Vaccine and 23-Valent Pneumococcal Polysaccharide Vaccine Among Adults Aged\u0026thinsp;\u0026ge;\u0026thinsp;65 Years: Updated Recommendations of the Advisory Committee on Immunization Practices. \u003cem\u003eMMWR Morb. Mortal. Wkly Rep.\u003c/em\u003e \u003cb\u003e68\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.15585/mmwr.mm6846a5\u003c/span\u003e\u003cspan address=\"10.15585/mmwr.mm6846a5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHausdorff, W. P., Bryant, J., Paradiso, P. R. \u0026amp; Siber, G. R. Which pneumococcal serogroups cause the most invasive disease: Implications for conjugate vaccine formulation and use, part I. \u003cem\u003eClin. Infect. Dis.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1086/313608\u003c/span\u003e\u003cspan address=\"10.1086/313608\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeno, K. A. et al. Pneumococcal capsules and their types: Past, present, and future. \u003cem\u003eClin. Microbiol. Rev.\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e, 871\u0026ndash;899. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/CMR.00024-15\u003c/span\u003e\u003cspan address=\"10.1128/CMR.00024-15\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoininen, A., Karpala, M., Wahlman, S. L., Lehtonen, H. \u0026amp; K\u0026auml;yhty, H. Specificities and opsonophagocytic activities of antibodies to pneumococcal capsular polysaccharides in sera of unimmunized young children. \u003cem\u003eClin. Diagn. Lab. Immunol.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/CDLI.9.5.1032-1038.2002\u003c/span\u003e\u003cspan address=\"10.1128/CDLI.9.5.1032-1038.2002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLortan, J. E., Kaniuk, A. S. \u0026amp; Monteil, M. A. Relationship of in vitro phagocytosis of serotype 14 Streptococcus pneumoniae to specific class and IgG subclass antibody levels in healthy adults. \u003cem\u003eClin. Exp. Immunol.\u003c/em\u003e \u003cb\u003e91\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1365-2249.1993.tb03353.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2249.1993.tb03353.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWinkelstein, J. A. The role of complement in the host's defense against streptococcus pneumoniae. \u003cem\u003eRev. Infect. Dis.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/clinids/3.2.289\u003c/span\u003e\u003cspan address=\"10.1093/clinids/3.2.289\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1981).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBriles, D. E., Crain, M. J., Gray, B. M., Forman, C. \u0026amp; Yother, J. Strong association between capsular type and virulence for mice among human isolates of Streptococcus pneumoniae. \u003cem\u003eInfect. Immun.\u003c/em\u003e \u003cb\u003e60\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/iai.60.1.111-116.1992\u003c/span\u003e\u003cspan address=\"10.1128/iai.60.1.111-116.1992\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1992).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePilishvili, T. et al. Sustained reductions in invasive pneumococcal disease in the era of conjugate vaccine. \u003cem\u003eJ. Infect. Dis.\u003c/em\u003e \u003cb\u003e201\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1086/648593\u003c/span\u003e\u003cspan address=\"10.1086/648593\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMasomian, M., Ahmad, Z., Gew, L. T. \u0026amp; Poh, C. L. in \u003cem\u003eVaccines\u003c/em\u003e Vol. 8 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCannon, K. et al. A trial to evaluate the safety and immunogenicity of a 20-valent pneumococcal conjugate vaccine in populations of adults\u0026thinsp;\u0026ge;\u0026thinsp;65 years of age with different prior pneumococcal vaccination. \u003cem\u003eVaccine\u003c/em\u003e \u003cb\u003e39\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2021.10.032\u003c/span\u003e\u003cspan address=\"10.1016/j.vaccine.2021.10.032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrooks, L. R. K. \u0026amp; Mias, G. I. in \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e (2018). (Frontiers Media S.A..\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWattal, C., Goel, N. \u0026amp; Byotra, S. P. Prevalence of Pneumococcal Serotypes in Adults\u0026thinsp;\u0026ge;\u0026thinsp;50 Years of Age. \u003cem\u003eInd. J. Med. Microbiol.\u003c/em\u003e \u003cb\u003e35\u003c/b\u003e, 95\u0026ndash;100. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4103/IJMM.IJMM_16_132\u003c/span\u003e\u003cspan address=\"10.4103/IJMM.IJMM_16_132\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWijayasri, S. et al. The shifting epidemiology and serotype distribution of invasive pneumococcal disease in Ontario, Canada, 2007\u0026ndash;2017. \u003cem\u003ePLoS ONE\u003c/em\u003e. \u003cb\u003e14\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0226353\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0226353\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScott, N. R., Mann, B., Tuomanen, E. I. \u0026amp; Orihuela, C. J. in \u003cem\u003eVaccines\u003c/em\u003e Vol. 9 1\u0026ndash;17MDPI AG, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharew, B. et al. Serotype distribution of streptococcus pneumoniae isolates causing invasive and non-invasive infections using whole-genome sequencing in Ethiopia. \u003cem\u003eInfect. Drug Resist.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2147/IDR.S293578\u003c/span\u003e\u003cspan address=\"10.2147/IDR.S293578\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIsturiz, R. E. et al. Pneumococcal epidemiology among us adults hospitalized for community-acquired pneumonia. \u003cem\u003eVaccine\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2019.04.087\u003c/span\u003e\u003cspan address=\"10.1016/j.vaccine.2019.04.087\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL\u0026oslash;chen, A., Croucher, N. J. \u0026amp; Anderson, R. M. Divergent serotype replacement trends and increasing diversity in pneumococcal disease in high income settings reduce the benefit of expanding vaccine valency. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-020-75691-5\u003c/span\u003e\u003cspan address=\"10.1038/s41598-020-75691-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManoharan, A. et al. Invasive pneumococcal disease in children aged younger than 5 years in India: a surveillance study. \u003cem\u003eLancet. Infect. Dis\u003c/em\u003e. \u003cb\u003e17\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S1473-3099(16)30466-2\u003c/span\u003e\u003cspan address=\"10.1016/S1473-3099(16)30466-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, X., Puvanesarajah, V. \u0026amp; Berti, F. Structure of the type 38 Streptococcus pneumoniae capsular polysaccharide. \u003cem\u003eCarbohydr. Res.\u003c/em\u003e \u003cb\u003e541\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.carres.2024.109165\u003c/span\u003e\u003cspan address=\"10.1016/j.carres.2024.109165\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBednar, B. \u0026amp; Hennessey, J. P. Molecular size analysis of capsular polysaccharide preparations from Streptococcus pneumoniae. 243\u0026ndash;243 (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSangareddy, V. et al. Identifying and optimization of critical process parameters for the modulation of polysaccharide molecular size in Streptococcus pneumoniae serotype-1. \u003cem\u003eDiscover Appl. Sci.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s42452-024-06096-6\u003c/span\u003e\u003cspan address=\"10.1007/s42452-024-06096-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTheisen, A., Johann, C., Deacon, M. P. \u0026amp; Harding, S. E. \u003cem\u003eRefractive Increment Data-Book for Polymer and Biomolecular Scientists\u003c/em\u003e (Nottingham University, 2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStriegel, A. M. Specific Refractive Index Increment (\u0026part;n/\u0026part;c) of Polymers at 660 nm and 690 nm. \u003cem\u003eChromatographia\u003c/em\u003e \u003cb\u003e80\u003c/b\u003e, 989\u0026ndash;996. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10337-017-3294-2\u003c/span\u003e\u003cspan address=\"10.1007/s10337-017-3294-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalaga, P., Vialle, S. \u0026amp; Moreau, M. Development of a high-performance anion-exchange chromatography with pulsed-amperometric detection based quantification assay for pneumococcal polysaccharides and conjugates. 2474\u0026ndash;2484 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcNeely, T. B., Staub, J. M., Rusk, C. M., Blum, M. J. \u0026amp; Donnelly, J. J. Antibody Responses to Capsular Polysaccharide Backbone and O-Acetate Side Groups of Streptococcus pneumoniae Type 9V in Humans and Rhesus Macaques. 3705\u0026ndash;3710 (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpencer, B. L., Saad, J. S., Shenoy, A. T., Orihuela, C. J. \u0026amp; Nahm, M. H. Position of O-acetylation within the capsular repeat unit impacts the biological properties of pneumococcal serotypes 33A and 33F. \u003cem\u003eInfect. Immun.\u003c/em\u003e \u003cb\u003e85\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/IAI.00132-17\u003c/span\u003e\u003cspan address=\"10.1128/IAI.00132-17\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKao, G. \u0026amp; Tsai, C. M. Quantification of O-acetyl, N-acetyl and phosphate groups and determination of the extent of O-acetylation in bacterial vaccine polysaccharides by high-performance anion-exchange chromatography with conductivity detection (HPAEC-CD). \u003cem\u003eVaccine\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e, 335\u0026ndash;344. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2003.08.008\u003c/span\u003e\u003cspan address=\"10.1016/j.vaccine.2003.08.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajendar, B. et al. High-Performance Anion-Exchange chromatography with conductivity detection method for simultaneous determination of nitrogen and phosphorus in polysaccharides. \u003cem\u003eJ. Chromatogr. B: Anal. Technol. Biomedical Life Sci.\u003c/em\u003e \u003cb\u003e1207\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jchromb.2022.123367\u003c/span\u003e\u003cspan address=\"10.1016/j.jchromb.2022.123367\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDische, Z., Shettles, L. B., A NEW SPECTROPHOTOMETRIC TEST FOR \u0026amp; THE DETECTION OF METHYLPENTOSE. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e192\u003c/b\u003e, 579\u0026ndash;582, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0021-9258(19)77781-3\u003c/span\u003e\u003cspan address=\"10.1016/S0021-9258(19)77781-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1951).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBitter, T. \u0026amp; Muir, H. M. A modified uronic acid carbazole reaction. \u003cem\u003eAnal. Biochem.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/0003-2697(62)90095-7\u003c/span\u003e\u003cspan address=\"10.1016/0003-2697(62)90095-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1962).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFilisetti-Cozzi, T. M. C. C. \u0026amp; Carpita, N. C. Measurement of uronic acids without interference from neutral sugars. \u003cem\u003eAnal. Biochem.\u003c/em\u003e \u003cb\u003e197\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/0003-2697(91)90372-Z\u003c/span\u003e\u003cspan address=\"10.1016/0003-2697(91)90372-Z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1991).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGmbH, A. P. \u003cem\u003eInstruction Manual Litesizer\u0026trade; 500\u003c/em\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJansson, P. E., Lindberg, B. \u0026amp; Lindquist, U. Structural studies of the capsular polysaccharide from Streptococcus pneumoniae type 5. \u003cem\u003eCarbohydr. Res.\u003c/em\u003e \u003cb\u003e140\u003c/b\u003e, 101\u0026ndash;110. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/0008-6215(85)85053-9\u003c/span\u003e\u003cspan address=\"10.1016/0008-6215(85)85053-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1985).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStroop, C. J. M., Xu, Q., Retzlaff, M., Abeygunawardana, C. \u0026amp; Bush, C. A. Structural analysis and chemical depolymerization of the capsular polysaccharide of Streptococcus pneumoniae type 1. \u003cem\u003eCarbohydr. Res.\u003c/em\u003e \u003cb\u003e337\u003c/b\u003e, 335\u0026ndash;344. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0008-6215(01)00318-4\u003c/span\u003e\u003cspan address=\"10.1016/S0008-6215(01)00318-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchumann, B. et al. Development of an Efficacious, Semisynthetic Glycoconjugate Vaccine Candidate against Streptococcus pneumoniae Serotype 1. \u003cem\u003eACS Cent. Sci.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, 357\u0026ndash;361. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acscentsci.7b00504\u003c/span\u003e\u003cspan address=\"10.1021/acscentsci.7b00504\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbcam Dot blot protocol. doi: (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.abcam.com/en-us/technical-resources/protocols/dot-blot\u003c/span\u003e\u003cspan address=\"https://www.abcam.com/en-us/technical-resources/protocols/dot-blot\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWHO. Recommendations to assure the quality, safety and efficacy of pneumococcal conjugate vaccines, World Health Organization,. WHO-Technical Report Series, No. 977, 91\u0026ndash;151. (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHotchkiss, A. T. \u0026amp; Hicks, K. B. Analysis of oligogalacturonic acids with 50 or fewer residues by high-performance anion-exchange chromatography and pulsed amperometric detection. \u003cem\u003eAnal. Biochem.\u003c/em\u003e \u003cb\u003e184\u003c/b\u003e, 200\u0026ndash;206. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/0003-2697(90)90669-Z\u003c/span\u003e\u003cspan address=\"10.1016/0003-2697(90)90669-Z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1990).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCobb, B. A. \u0026amp; Kasper, D. L. \u003cem\u003eCell. Microbiol.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e 1398\u0026ndash;1403 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMertens, J. et al. Streptococcus pneumoniae serotype 1 capsular polysaccharide induces CD8\u0026thinsp;+\u0026thinsp;CD28- regulatory T lymphocytes by TCR crosslinking. \u003cem\u003ePLoS Pathog.\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.ppat.1000596\u003c/span\u003e\u003cspan address=\"10.1371/journal.ppat.1000596\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatravale, V., Dandekar, P. \u0026amp; Jain, R. Characterization techniques for nanoparticulate carriers. \u003cem\u003eNanoparticulate Drug Delivery\u003c/em\u003e. 87\u0026ndash;121. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1533/9781908818195.87\u003c/span\u003e\u003cspan address=\"10.1533/9781908818195.87\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlumenkrantz, N. \u0026amp; Asboe-Hansen, G. An assay for total hexosamine and a differential assay for glucosamine and galactosamine. \u003cem\u003eClin. Biochem.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 269\u0026ndash;274. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0009-9120(76)80075-6\u003c/span\u003e\u003cspan address=\"10.1016/S0009-9120(76)80075-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1976).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGallorini, S. et al. Introduction of Zwitterionic Motifs into Bacterial Polysaccharides Generates TLR2 Agonists Able to Activate APCs. \u003cem\u003eJ. Immunol.\u003c/em\u003e \u003cb\u003e179\u003c/b\u003e, 8208\u0026ndash;8215. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4049/jimmunol.179.12.8208\u003c/span\u003e\u003cspan address=\"10.4049/jimmunol.179.12.8208\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMazmanian, S. K., Cui, H. L., Tzianabos, A. O. \u0026amp; Kasper, D. L. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. \u003cem\u003eCell\u003c/em\u003e \u003cb\u003e122\u003c/b\u003e, 107\u0026ndash;118. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cell.2005.05.007\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2005.05.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2005).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Streptococcus pneumoniae, non-vaccine serotype 38, Capsular Polysaccharides, Zwitterionic Polysaccharide, Pneumococcal vaccines","lastPublishedDoi":"10.21203/rs.3.rs-5880261/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5880261/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInvasive pneumococcal disease presents a threat to humankind predominantly affecting children and the elderly. Despite the availability of high-valency pneumococcal polysaccharide vaccine of PPSV23 (PNEUMOVAX\u0026reg; 23) and conjugate vaccines such as VAXNEUVANCE and PREVNAR 20\u0026reg;, non-vaccine serotypes continue to contribute to higher mortality rates. The characterization of non-vaccine serotypes is becoming increasingly crucial considering an increase in their prevalence. In this study, biochemical characteristics, immunological properties, and critical quality attributes of the capsular polysaccharide isolated from prevalent non-vaccine serotype 38 (American type 71) have been examined. Advanced analytical techniques, including multi-angle light scattering (MALS), ion chromatography, dynamic light scattering in addition to conventional biochemical methods and SLOTBLOT analysis were employed. We observed that serotype 38 capsular polysaccharide has a molar mass of 768 kDa with a distribution of 1.451 (\u0026plusmn;\u0026thinsp;4.460%) and a z-average radius of gyration (\u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003eg\u003c/em\u003e\u003c/sub\u003e) is 90 nm. The polysaccharide composition included approximately 72% galactose, 9.78% N-acetylglucosamine, and 8.05% galacturonic acid, while the unknown peak accounted for approximately 7.83% of the total peak area of the chromatogram. The O-acetyl content of polysaccharide was determined to be nearly 6% and it lacked methyl pentoses (rhamnose). Zeta potential measurements revealed its zwitterionic state which suggested its potential to trigger T cell-dependent B cell-mediated immunological response. Serotype 38 polysaccharide showed immunological cross-reactivity with serotype 5 and serotype 1 polyclonal sera, likely due to a shared common epitope region having an unknown sugar component (Sug\u003csub\u003e\u003cem\u003ep\u003c/em\u003e\u003c/sub\u003e) in their polysaccharide repeating units and zwitterionic properties. The findings highlight novel features of serotype 38 polysaccharide, including its amino acid content and zwitterionic nature, which may contribute to the development of new therapeutics and improved vaccines.\u003c/p\u003e","manuscriptTitle":"Biochemical and Immunological Insights into Capsular Polysaccharide of Streptococcus pneumoniae Serotype 38 (American type 71)- An emerging non-vaccine serotype","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-03 14:59:42","doi":"10.21203/rs.3.rs-5880261/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"36bad89e-9d6e-4a0a-90ba-2e7444d17481","owner":[],"postedDate":"March 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":44206246,"name":"Biological sciences/Biochemistry"},{"id":44206247,"name":"Biological sciences/Chemical biology"},{"id":44206248,"name":"Biological sciences/Immunology"},{"id":44206249,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-05-19T08:54:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-03 14:59:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5880261","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5880261","identity":"rs-5880261","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}