Quantitative determination of C-polysaccharide in Streptococcus pneumoniae capsular polysaccharides by enzyme-linked immunosorbent assay.

Capsular polysaccharides of Streptococcus pneumoniae are used in pneumococcal polysaccharide and protein-conjugate vaccines. Cell-wall polysaccharide (C-Ps) is a critical impurity that must be kept at low levels in purified polysaccharide preparations. Hence, accurate and precise methods for determining C-Ps are needed. Currently available methods include nuclear magnetic resonance (NMR) spectroscopy and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both these methods suffer from their own limitations; therefore, we developed a simple and efficient enzyme-linked immunosorbent assay (ELISA) for accurate and precise quantification of C-Ps in samples of any serotype of pneumococcal capsular polysaccharide without interference. We quantified C-Ps in preparations of 14 serotype polysaccharides using newly developed ELISA method and compared the results with C-Ps values obtained using two previously reported methods, 1H NMR and HPAEC-PAD. The C-Ps value determined using 1H NMR for serotype 5 was 21.08%, whereas the values obtained using HPAEC-PAD and ELISA were 2.38% and 2.89% respectively, indicating some interference in 1H NMR method. The sensitivity of the ELISA method is higher because the sample is used directly unlike HPAEC-PAD method where sample is subjected to harsh treatment, such as acid digestion and quantify C-Ps based on peak area of ribitol or AAT. Furthermore, 1H NMR and HPAEC-PAD are expensive and laborious methods. Our work, underscores the simple and efficient ELISA that can be used for quantification of C-Ps in pneumococcal polysaccharide preparations.

O-phthalaldehyde based quantification of polysaccharide modification in conjugate vaccines.

Polysaccharide-based vaccines cannot stimulate long-lasting immune response in infants due to their inability to elicit a T-cell-dependent immune response. This has been addressed using conjugation technology, where conjugates were produced by coupling a carrier protein to polysaccharides using different conjugation chemistries, such as cyanylation, reductive amination, ethylene diamine reaction, and others. Many glycoconjugate vaccines that are manufactured using different conjugation technologies are already in the market for neonates, infants and young children (e.g., Haemophilus influenzae type-b, Streptococcus pneumoniae and Neisseria meningitidis vaccines), and all of them elicit a T-cell dependent immune response. To manufacture glycoconjugate vaccines, the capsular polysaccharide is first activated by converting its hydroxyl groups to aldehyde-, cyanyl-, or cyanate ester groups, depending on the conjugation chemistry selected. The oxidized and reduced aldehyde functional groups of the polysaccharides are subsequently reacted with the amino groups of carrier protein by reductive amination to form a stable amide bond. In CDAP-based conjugation, the polysaccharide -OH groups are activated to form cyanyl-, or cyanate ester groups to react with the amino groups of carrier protein and forms an isourea bond. Understanding the extent of polysaccharide activation/modification is essential since it directly influences the molar mass of the conjugate, its stability, and the immunogenicity of the product. Reported methods are available to estimate the aldehyde groups of polysaccharides generated by reductive amination. However, no method is available to quantify the cyanyl or cyanate ester (-OCN) groups generated by cyanylation with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP). We report a novel strategy using an O-phthalaldehyde (OPA) derivatization process followed by size-exclusion chromatography (SEC) high-performance liquid chromatography (HPLC) separation and UV detection. The cyanate ester groups on the activated polysaccharide directly reveal the extent of polysaccharide activation/modification and the residual activated groups in the purified conjugates. This method would be useful for conjugate vaccine manufacturing using CDAP chemistry.

Vitamin C and its therapeutic potential in the management of COVID19.

COVID19 has emerged as one of the worst pandemics in the history of mankind. Several vaccines have been approved by different government agencies worldwide, but data on their efficacy and safety are limited, and distribution remains a massive challenge. As per WHO, personal immunity is vital for protection against COVID19. Earlier, Vitamin C-mediated pathways have been shown to play critical role in boosting immunity attributed to its antioxidant properties. Recently, the involvement of such pathways in protection against COVID19 has been suggested. The controlled doses of Vitamin C administered through intravenous (IV) injections are being studied for determining its role in the prognosis of COVID19. In this article, we have discussed the potential role of Vitamin C in the management in COVID19 patients and presented recent clinical trials data. Additionally, we have elaborated the possibility of administering Vitamin C through inhalers in order to achieve local high concentration and the challenges of such approach.

The extended (p)ppGpp family: New dimensions in Stress response.

Second messenger (p)ppGpp mediated stress response plays a crucial role in bacterial persistence and multiple drug resistance. In E. coli, (p)ppGpp binds to RNA polymerase and upregulates the transcription of genes essential for stress response while concurrently downregulating the expression of genes critical for growth and metabolism. Recently, the family of alarmone molecules has expanded to pppGpp, ppGpp, pGpp & (pp)pApp as distinct members. These molecules may help in fine-tuning stress responses in different hostile conditions. Do all of these molecules bind to RNA polymerase? Do they compete with each other or complement each other's functions is still not clear. Earlier, others and we have synthesized artificial analogs of (p)ppGpp that inhibited (p)ppGpp synthesis and long-term survival in M. smegmatis and in B. subtilis suggesting that analogs could compete with each other. Understanding the interplay of these molecules will allow deciphering novel pathways that can be potentially subjected to the therapeutic intervention. In this article, we have reviewed newly characterized second messengers and discussed their mode of action. We have also documented the progress made to-date in understanding the molecular basis of regulation of transcription by second messenger ppGpp, pppGpp, and pGpp.

2-Phenoxyethanol: A novel reagent for improved sensitivity of carbohydrate quantification.

Anthrone is a routinely used reagent for estimating carbohydrates (Polysaccharides) in research, development and pharmaceutical applications. In presence of sulphuric acid, the polysaccharide gets hydrolyzed to monosaccharides in the form of hydroxymethyl furfural or furfural. The furfural then reacts with anthrone to form a green color complex with a maximum absorbance at 625 nm. Though anthrone reacts well with polysaccharides containing hexoses (such as glucose and galactose) and rhamnose, it is less reactive with uronic acids (such as glucuronic acid and galacturonic acid) and hexosamines (such as fucosamine, glucosamine, galactosamine, mannosamine, pneumosamine). Here, we report a novel reagent, 2-Phenoxyethanol, which reacts with furfural or hydroxymethyl furfural resulting in higher absorptivity. This method is rapid, sensitive, simple and direct, and can be used for quantitative determination of any type of carbohydrate that contains neutral sugars and uronic acids. For these saccharides, the sensitivity of the assay using 2-Phenoxyethanol (2-PE) is twice over anthrone method. Uronic acids show improved sensitivity using 2-PE over Phenol and it is more than twice with glucuronic acid. 2-PE reagent method has greater application for quantification of carbohydrates when present in low concentration in vaccines/biologicals.