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.

Immunogenicity and safety of a 14-valent pneumococcal polysaccharide conjugate vaccine (PNEUBEVAX 14™) administered to 6-8 weeks old healthy Indian Infants: A single blind, randomized, active-controlled, Phase-III study.

BACKGROUND: Introduction of pneumococcal conjugate vaccines (PCVs) reduced the number of cases of pneumococcal disease (PD). However, there is an increase in clinical and economic burden of PD from serotypes that are not part of the existing pneumococcal vaccines, particularly impacting pediatric and elder population. In addition, the regions where the PCV is not available, the disease burden remains high. In this study, immunogenicity and safety of the BE's 14-valent PCV (PNEUBEVAX 14™; BE-PCV-14) containing two additional epidemiologically important serotypes (22F and 33F) was evaluated in infants in comparison to licensed vaccine, Prevenar-13 (PCV-13). METHODS: This is a pivotal phase-3 single blind randomized active-controlled study conducted at 12 sites across India in 6-8 weeks old healthy infants at 6-10-14 weeks dosing schedule to assess immunogenic non-inferiority and safety of a candidate BE-PCV-14. In total, 1290 infants were equally randomized to receive either BE-PCV-14 or PCV-13. Solicited local reactions and systemic events, adverse events (AEs), serious AEs (SAEs), and medically attended AEs (MAAEs) were recorded. Immunogenicity was assessed by measuring anti-PnCPS (anti-pneumococcal capsular polysaccharide) IgG concentration and functional antibody titers through opsonophagocytic activity (OPA), one month after completing three dose schedule. Cross protection to serotype 6A offered by serotype 6B was also assessed in this study. FINDINGS: The safety profile of BE-PCV-14 was comparable to PCV-13 vaccine. Majority of reported AEs were mild in nature. No severe or serious AEs were reported in both the treatment groups. For the twelve common serotypes and for the additional serotypes (22F and 33F) in BE-PCV-14, NI criteria was demonstrated as defined by WHO TRS-977. Primary immunogenicity endpoint was met in terms of IgG immune responses for all 14 serotypesof BE-PCV-14. Moreover, a significant proportion of subjects (69%) seroconverted against serotype 6A, even though this antigen was not present in BE-PCV-14. This indicates that serotype 6B of BE-PCV-14 cross protects serotype 6A. BE-PCV-14 also elicited comparable serotype specific functional OPA immune responses to all the serotypes common to PCV-13. INTERPRETATIONS: BE-PCV-14 was found to be safe and induced robust and functional serotype specific immune responses to all 14 serotypes. It also elicited cross protective immune response against serotype 6B.These findings suggest that BE-PCV-14 can be safely administered to infants and achieve protection against pneumococcal disease caused by serotypes covered in the vaccine. The study was prospectively registered with clinical trial registry of India - CTRI/2020/02/023129.

Spin-State Modulation in FeII -Based Hofmann-Type Coordination Polymers: From Molecules to Materials.

Spin crossover complexes that reversibly interconvert between two stable states imitate a binary state of 0 and 1, delivering a promising possibility to address the data processing concept in smart materials. Thus, a comprehensive understanding of the modulation of magnetic transition between high spin and low spin and the factors responsible for stabilizing the spin states is an essential theme in modern materials design. In this context, the present review attempts to provide a concise outline of the design strategy employed at the molecular level for fine-tuning the spin-state switching in FeII -based Hofmann-type coordination polymers and their effects on the optical and magnetic response. In addition, development towards the nanoscale architectures of HCPs, i. e., in terms of nanoparticles and thin films, are emphasized to bridge the gap between the laboratory and reality.

Ethyl 3,4-dihydroxybenzoate (EDHB): a prolyl hydroxylase inhibitor attenuates acute hypobaric hypoxia mediated vascular leakage in brain.

Sudden exposure to altitude hypoxia is responsible for acute mountain sickness (AMS) in un-acclimatized persons. If not treated in time, AMS can worsen and leads to high altitude cerebral edema, which can be fatal. Present study explores the efficacy of ethyl 3,4-dihydroxybenzoate (EDHB), a prolyl hydroxylase enzyme inhibitor, in modulating adaptive responses to hypobaric hypoxia (HH) in rat brain. Male Sprague-Dawley rats treated with EDHB (75 mg/kg for 3 days), were subjected to acute HH exposure at 9144 m (30,000 ft) for 5 h. Animals were assessed for transvascular leakage and edema formation in brain and role of key inflammatory markers along with hypoxia responsive genes. HH stress increased transvascular permeability and edema formation in conjunction with upregulation of nuclear factor-κB (NF-κB) and its regulated proteins. There was surge in pro-inflammatory cytokines tumor necrosis factor-α, interleukin-6, interferon-γ, monocyte chemoattractant protein-1 and decrement in anti-inflammatory cytokine interleukin-10. Further, upregulation of vascular endothelial growth factor (VEGF), a vascular permeability marker and down-regulation of antioxidant and anti-inflammatory proteins hemoxygenase (HO-1) and metallothionein (MT-1) was also observed under hypoxia. EDHB supplementation effectively scaled down HH induced cerebral edema with concomitant downregulation of brain NF-κB expression. There was significant curtailment of pro-inflammatory cytokines and cell adhesion molecules. There was significant downregulation of permeability factor VEGF by EDHB with concomitant increment in hypoxia inducible factor (HIF1α) and anti-inflammatory proteins HO-1 and MT-1 compared to HH control thus accentuating the potential of EDHB as effective hypoxic preconditioning agent in ameliorating HH mediated injury in brain.

Intraperitoneal immunization of recombinant HSP70 (DnaK) of Salmonella Typhi induces a predominant Th2 response and protective immunity in mice against lethal Salmonella infection.

Heat shock proteins serve as important antigens in defense against infectious diseases. Members of HSP70 family, particularly microbial HSP70s have acquired special significance in immunity. In the present study, we evaluated the immunogenicity and protective efficacy of HSP70 of Salmonella enterica serovar Typhi against lethal infection by Salmonella in mice with or without adjuvants. The HSP70 gene was cloned and expressed in Escherichia coli BL21 and purified by affinity chromatography. Immunization of mice with HSP70 either alone or in combination with complete Freund's adjuvant (CFA) resulted in a significant increase in antibody titers as compared to control. Antibody isotyping revealed that HSP70 immunization induces both IgG1 and IgG2a antibodies to a significant extent but a higher IgG1/IgG2a ratio indicates a predominant Th2 response. There was a significant increase in lymphocyte proliferation, and levels of both Th2 and Th1 cytokines in cells isolated from immunized mice as compared to control. Immunization of mice with recombinant HSP70 either alone or in combination with CFA conferred 70-90% protection against lethal infections by Salmonella Typhi Ty2 or Salmonella Typhimurium. However, passive immunization with anti-HSP70 sera induced only partial protection in the immunized mice.

Leishmania major MAP kinase 10 is protective against experimental L. major infection.

Leishmania, a protozoan parasite that resides and replicates obligatorily within macrophages, inflicts a complex of severe diseases known as leishmaniasis. The diseases have significant socio-economic impact through gross disfiguration, morbidity and mortality worldwide. Despite these problems, an effective anti-leishmanial vaccine remains elusive. Herein, we have analyzed the immunogenicity and protective efficacy of L. major MAP kinase 10 (LmjMAPK10) against the challenge infection with the parasite. We observe significant protection against the infection by LmjMAPK10 priming of BALB/c mouse strain, a susceptible host. The resistance to the infection is generally associated with mixed Th1/Th2 responses to the infection following immunization with LmjMAPK10 DNA or protein or a combination of both DNA and protein. Therefore, LmjMAPK10 is a probable vaccine candidate against the infection.

Effect of adjuvants on immune response and protective immunity elicited by recombinant Hsp60 (GroEL) of Salmonella typhi against S. typhi infection.

Heat shock proteins (Hsps) have been reported to be dominant antigens for the host immune response to various pathogens and thus, have great potential for use in vaccination. In the present study, we evaluated the immunogenicity and protective efficacy of GroEL of Salmonella enterica serovar Typhi against lethal infection by S. typhi Ty2 in mice with or without adjuvants. Anti GroEL-IgG titers were significantly higher in mice immunized with either GroEL-alone or in combination with alum/Complete Freund's adjuvant (CFA) as compared to the control. Analysis of antibody isotypes suggested predominance of Th2 type immune response in GroEL + alum immunized animals as revealed by higher IgG1/IgG2a ratio. Whereas, immunization of animals with GroEL + CFA or GroEL-alone shifted the immune response toward Th1 phenotype. Mice immunized with GroEL with or without adjuvants, showed a significant increase in lymphocyte proliferation and cytokine levels. The animals immunized with GroEL + CFA or GroEL-alone showed higher IFN-gamma and IL-2 levels than alum group, indicating Th1 response whereas IL-4 levels (Th2 response) were found to be highest in alum group as compared to other two immunized groups. Immunization of mice with GroEL-alone, GroEL + alum, and GroEL + CFA provided 70, 50 and 80% protection, respectively, against lethal challenge by S. typhi in mice. The differences in the percentage protection among various groups were attributed to the differences in the immune responses generated by respective immunizations. The present study shows that GroEL forms an ideal candidate molecule to develop a recombinant protein based vaccine against human typhoid.

Cloning, expression and characterization of heat shock protein 60 (groEL) of Salmonella enterica serovar Typhi and its role in protective immunity against lethal Salmonella infection in mice.

Heat shock proteins (Hsps) represent dominant antigens in numerous microbial infections, suggesting a potential use of pathogen-derived Hsps for vaccination. The present study evaluates the immunogenicity and protective efficacy of groEL (Hsp60) of Salmonella enterica serovar Typhi against lethal challenge by S. Typhi Ty2 and Salmonella enterica serovar Typhimurium in mice. The groEL gene was cloned and expressed in Escherichia coli BL21 and purified by affinity chromatography. Immunization of mice with groEL resulted in a significant increase in antibody titers. Antibody isotyping revealed that groEL immunization induces both IgG1 and IgG2a antibodies. There was a significant increase in lymphocyte proliferation, interleukin-4 and interferon-gamma levels in cells isolated from immunized mice as compared to control. Immunization of mice with recombinant groEL protein with or without adjuvant conferred 70-90% protection against lethal infections either by S. Typhi Ty2 or S. Typhimurium. Passive immunization with anti-groEL sera also protected 50% mice against lethal infection.

Studies on immunogenicity and protective efficacy of DnaJ of Salmonella Typhi against lethal infection by Salmonella Typhimurium in mice.

In the present study DnaJ (HSP40) of Salmonella enterica serovar Typhi has been evaluated for its immunogenicity and efficacy in protecting mice against lethal challenge by S. enterica serovar Typhimurium infection. DnaJ was amplified by PCR of the genomic DNA of S. Typhi and subsequently cloned in pQE-30 expression vector. The protein was induced by IPTG and purified using Ni-NTA chromatography under denaturing conditions. After refolding in vitro the immune response was evaluated by injecting 40 microg DnaJ protein/mouse i.p. on 0th, 7th and 28th day. The results showed a significant increase in antibody titre and lymphocyte proliferation in animals immunised with DnaJ as compared to control. Further there was an appreciable increase in IL-2, IL-4, IFN-gamma production in lymphocytes isolated from immunised mice as compared to control. In this limited study, immunisation of mice with DnaJ was found to provide 70% protection against lethal challenge by S. Typhimurium indicating the possible use of DnaJ as vaccine candidate against typhoid.

Immunogenicity and protective efficacy of DnaJ (hsp40) of Streptococcus pneumoniae against lethal infection in mice.

The present study was carried out to evaluate the immunogenicity and protective efficacy of DnaJ (hsp40) of Streptococcus pneumoniae, by cloning the full-length DnaJ of S. pneumoniae and expressing in heterologous host E. coli BL-21 (DE3). PCR amplified DnaJ was ligated in pQE-30 expression vector and subsequently transformed in E. coli DH5alpha strain. Cloning of DnaJ was confirmed by double digestion and PCR, followed by DNA sequencing. The His-tag containing recombinant protein was purified by Ni-NTA affinity chromatography. To determine the immunogenicity of DnaJ, the mice (10 mice/group) were immunized by injecting 40 microg DnaJ protein/mouse i.p. There was a significant increase in IgG titres (2 x 10(5)) in mice immunized with DnaJ protein. Isotyping studies revealed that antibodies produced are predominantly IgG2a type indicating the predominance of Th1 response. A significant increase in lymphocyte proliferation was observed in mice immunized with DnaJ protein as compared to the control mice. Further, there was a significant increase in IL-2 and gamma-IFN levels in culture supernatants of splenocytes isolated from immunized mice. To determine the efficacy of DnaJ vaccination in eliciting protection, the mice were challenged with 1 x 10(5)cells of S. pneumoniae A66 type 3 capsular strain intra-nasally after 7 days of last immunization. All the control mice died within 2 days of post-infection, while 70% of animals immunized with DnaJ survived the lethal challenge by S. pneumoniae. The study reveals that immunization of mice with DnaJ elicits protective immunity against S. pneumoniae infection.