Evaluation of structural modification induced activation of pneumococcal polysaccharide by GC-MS for the conjugate vaccine.

Polysaccharide (Ps) activation evaluation is an imperative quality attribute in a conjugate vaccine. Pneumococcal polysaccharide (PnPs) serotypes 5, 6B, 14, 19A and 23F were cyanylated for 3 and 8 min. The cyanylated and non-cyanylated polysaccharides were methanolysed and derivatized to assess the activation of each sugar by GC-MS. The activation of 22 and 27% serotype 6B and 11 and 36% in serotype 23 F Ps at 3 and 8 min respectively showed controlled conjugation kinetics with CRM197 carrier protein estimated by SEC-HPLC and optimal absolute molar mass by SEC-MALS. The Glc and Gal are the most commonly activated sugars of all PnPs serotypes while N-acetyl sugars PneuNAc, GalNAc and Rha in serotypes 5, 14 and 19A respectively showed >50% activation which contributes to conjugate aggregate formation at 8 min compared to 3 min cyanylation. The GC-MS analysis of structural modifications at functional groups entails important information to characterize the activated polysaccharide for consistent conjugate vaccine manufacturing.

Transferrin binding protein-B from Neisseria meningitidis C as a novel carrier protein in glycoconjugate preparation: an in silico approach.

The linking of polysaccharide in glycoconjugate vaccine with carrier protein is an imperative step to develop a strong memory response. The excessive use of similar carrier protein known to result in bystander immunity warrants an urgent need for new carrier protein. The preparation of the glycoconjugate vaccine using cyanylation chemistry is to link the active cyanate ester site of polysaccharide with the carrier protein. In the present study, transferrin binding protein-B (Tbp-B) has been explored as a new carrier protein to develop in silico pneumococcal polysaccharide serotype-5 (PnPs-5) conjugate vaccine. The homology model of Tbp-B was constructed using the Prime module and stereochemically validated using ProSA, PDBsum and ProQ. The selected model revealed a Z-score of -5.6 within the X-ray region in ProSA analysis, LGscore: 9.776, and MaxSub: 0.8 in protein quality predictor suggesting its preferred use. Loop modeling and active site analysis followed by in silico PnPs-5 activation with cyanalyting agent CDAP was docked with Tbp-B using Glide module. The complex stability of cyanate esters with Tbp-B, analyzed by molecular dynamics (MD) simulation, revealed an average RMSD of 2.49 Å for its binding to the receptor. The RMSF values of cyanate ester-1, -2, and -3 were observed to be 1.06, 1.39 and 0.79 Å, respectively. The higher RMSF of 1.39 Å of cyanate ester-2 was further found unstable which corroborates its non-binding to the protein and also incurring conformational changes to a carrier protein. Molecular simulations revealed that cyanate ester-1 and cyanate ester-3 formed stable conjugates with carrier protein Tbp-B. Communicated by Ramaswamy H. Sarma.

Quantitation of endotoxin by gas chromatography-mass spectrometry in Neisseria meningitidis serogroups A, C, W, Y and X during polysaccharide purification used in conjugate vaccine.

Bacterial lipopolysaccharide (LPS) responsible for endotoxin effect induces inflammatory reactions. The endotoxins are difficult to separate from the gram-negative polysaccharide (PS) during polysaccharide purification. The most common method to quantify LPS is the limulus amebocyte lysate (LAL) test which interferes with the agents used during PS purification. The gas chromatography-mass spectrometry (GC-MS) provides a suitable alternative by estimating lipid-A chain anchored 3-hydroxy fatty acid methyl ester (FAME) to estimate LPS however, there are no reports of its application in natural polysaccharides used for vaccine preparation. The transesterification of LPS and meningococcal PS yielded primary target 3-O-acetylated myristic acid which was detected by GC-MS and provided quantitative estimation of endotoxin. The GC-MS method was found in agreement with the LAL values showing lower endotoxin content< 10Eu/µg in meningococcal C and Y serogroup polysaccharides in comparison to higher endotoxin 177-523 Eu/µg in meningococcal A, W and X serogroups. The high endotoxin content in purified polysaccharide was attributed to it being detected in its intermediate stage by GC-MS unlike the LAL test. Thus GC-MS serves as a valuable method for endotoxin monitoring and quantitation in gram-negative meningococcal intermediate and purified PS during vaccine preparation.

Evaluation of a sensitive GC-MS method to detect polysorbate 80 in vaccine preparation.

Polysorbates are the most versatile and common surfactants used as protein stabilizers. Analysis of residual polysorbate 80 (PS 80) in conjugate vaccine is challenging due to complexity of conjugate matrices and heterogeneity of the structure of the PS 80 analyte. The direct approach using high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) and gas chromatography-mass spectrometry (GC-MS) that is based on oleic acid methyl ester formation followed by transesterification have been evaluated to quantitate residual PS 80 in meningococcal serogroups A, C, W, Y and X bulk conjugates. HPLC-ELSD method was observed to be less sensitive in comparison to the GC-MS method. The GC-MS method showed promise for quantitation of residual polysorbate 80 with advantages of higher sensitivity, simple sample preparation and mass spectral characterization compared to methods reported to literature. The oleic acid methyl ester was solubilized in hexane and injected in GC-MS to separate on highly polar capillary CP-WAX 52 CB Column. The mass spectral analysis showed characteristic ions at m/z 180, 222 and 264. The method was validated with linearity r2 > 0.99 over the concentration range of 0.5 to 100 µg/mL with LOD and LOQ of 0.3 and 0.91 respectively using PS 80 standard. The GC-MS method provides a simple, fast and label free technique for the precise quantitation of residual PS 80 in meningococcal bulk conjugate vaccine sample, achieved with accuracy between 85-105%.

Quantitation of residual sodium dodecyl sulfate in meningococcal polysaccharide by gas chromatography-mass spectrometry.

Sodium dodecyl sulfate (SDS) is a commonly used surfactant in protein solubilization and also during the polysaccharide purification. A GC-MS method has been developed to quantitate residual SDS in meningococcal polysaccharide serogroups A,C,W,Y and X circumventing the need of spectroscopic assays and HPLC based methods which are either unstable or requires the confirmation by MS. The developed method is based on quantitative conversion of SDS to 1-dodecanol at elevated temperature. Meningococcal polysaccharides and SDS standards were treated with methanolic-HCl and extracted in n-Hexane. The conversion of SDS to 1-dodecanol was confirmed by mass spectra and separation was achieved using a DB-5ms column. The mass spectral analysis of 1-dodecanol showed characteristic ions at m/z 168, 140 and 125. The GC-MS method validation performed on intermediate and purified meningococcal polysaccharides showed linearity with r2 > 0.99 over the concentration range of 2.5-200 µg/ml with LOD and LOQ of 1.27 and 3.85 respectively. The method was found to be precise, robust and accurate with spike recovery ranging 83-117%. The GC-MS method can be used in the quantitation of residual SDS during polysaccharide purification and provides valuable information about consistency of polysaccharide manufacturing process for development of pentavalent meningococcal conjugate vaccine.

Detection of in vitro and in vivo released antigens of diagnostic interest in Mycobacterium tuberculosis by immunoblotting.

Identification of in vitro and in vivo released mycobacterial antigens are of considerable interest in diagnosis of Mycobacterium tuberculosis. Isolation of in vitro released antigen from M. tb excretory-secretory culture filtrate protein and in vivo released circulating tuberculous antigen from smear positive pulmonary tuberculosis sera by ammonium sulphate precipitation is reported. The antigens were resolved by SDS-PAGE and immunoblotting was performed using pooled serum of smear positive, smear negative pulmonary tuberculosis sera and normal sera to identify reactive tuberculous antigens. In vitro and in vivo released mycobacterial antigens showed reactivity at 100, 31, 43 and 20 kDa with smear positive and smear negative pulmonary tuberculosis patients. Further, the in vitro released antigen showed strong reactivity exclusively at 55 kDa antigen with smear positive and 24 kDa antigen with smear negative pulmonary tuberculosis sera. In vivo released antigen reacted exclusively at 170 and 16 kDa with smear positive and 19 kDa antigen with smear negative pulmonary tuberculosis patients. Antigens of 24 and 19 kDa which are reactive with sputum negative sera will be of diagnostic interest and need further study in patients with low bacillary load. The in vitro and in vivo released mycobacterial 100, 31,43 and 20 kDa antigens, reactive with patients sera are of diagnostic interest in tuberculosis.

Isolation of Mycobacterium tuberculosis protein antigens ES-3 1, ES-43 and EST-6 of diagnostic interest from tubercle bacilli by affinity chromatography.

Immunodiagnostically useful M. tuberculosis H37Ra protein antigens ES-31, ES-43 and EST-6 were isolated from detergent soluble sonicate (DSS) antigen using monospecific antibodies by affinity chromatography and compared with similar antigens isolated from M. tuberculosis culture filtrate for seroreactivity in tuberculosis sera by Indirect Enzyme Linked Immunosorbent Assay. Recovery of affinity purified ES-31, ES-43 and EST-6 antigen from DSS antigen was approximately 3, 3.5 and 4% respectively, compared to 10, 9 and 6.3% from culture filtrate. Affinity purified ES-31, ES-43 and EST-6 antigens from both culture filtrate as well as DSS antigen showed similar seroreactivity with overall sensitivity 85, 80 and 75% respectively and specificity of 85% at optimum concentration of 50 pg protein of each antigen. The results suggest that DSS antigen may be a promising antigen source for isolating antigens of diagnostic interest obviating the need for cumbersome, time-consuming culture techniques of mycobacteria.

Isolation and analysis of circulating tuberculous antigens in Mycobacterium tuberculosis.

BACKGROUND: Serological techniques like enzyme linked immunosorbent assay (ELISA) and immunoblotting are useful for detection of mycobacterial antigens of diagnostic importance in tuberculosis. AIM: To isolate and identify circulating tuberculous antigens reactive with sputum positive and sputum negative pulmonary tuberculosis (PTB) sera. METHODS: Circulating tuberculous antigen was isolated by ammonium sulphate fractionation from the sera of sputum positive and sputum negative (clinically and radiologically diagnosed) PTB cases. The circulating antigen fractions and individual patients' serum samples were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting was performed using anti M.tb sonicate IgG as a probe to detect antigens. RESULTS: Anti M.tb sonicate IgG was found to be reactive with mycobacterial proteins 170 kDa, 140 kDa, 85 kDa, 55 kDa, 43 kDa, 20 kDa and 16 kDa in the antigen fraction isolated from sputum positive tuberculosis sera by immunoblotting. However only 85 kDa, 55kDa, 43 kDa and 20 kDa antigenic proteins were found to be recognized by anti sonicate IgG in the antigen isolated from sputum negative sera. These observations were further confirmed by analysis of individual S+ and S- PTB serum by immunoblotting. CONCLUSION: Seroreactive studies of circulating tuberculous antigens showed the presence of 170 kDa, 140 kDa, 85 kDa, 55 kDa, 43 kDa, 20 kDa and 16 kDa protein antigens in sputum positive sera, while 85 kDa, 55 kDa, 43 kDa and 20 kDa antigens were found to be present in sputum negative PTB which need further evaluation for their use in serological diagnosis of tuberculosis.

Detection of antigen and antibody in childhood tuberculous meningitis.

OBJECTIVE: Mycobacterium tuberculosis excretory secretory 31 kDa, a serine protease antigen (M. tb ES-31), prepared from Mycobacterium tuberculosis H37Ra culture medium has been shown to have potential in detecting tuberculosis. Precise diagnosis and management of tuberculous meningitis, in children in particular, is essential to curtail mortality and morbidity. METHODS: In this study, M. tb ES-31 antigen, was used in Indirect ELISA to detect tuberculous IgG antibody, in sera and CSF samples while affinity purified anti ES-31 goat antibody was used in sandwich ELISA for detection of tuberculous antigen. In sixty-five samples each of CSF and sera from cases with neurotuberculosis and control with non-tuberculous diseases were collected from Kasturba Hospital, Sevagram. RESULTS: Among the 20 patients suffering from neurotuberculosis the IgG antibody was detected in 17(85%) of CSF and 16(80%) of sera samples, while antigen was detected in 18 (90%) in CSF and 16 (80%) in sera. Overall specificity of the assay for both IgG antibody and antigen detection in CSF was 96% while in sera it was 94% for IgG antibody and 96% for antigen detection. CONCLUSION: This study showed the usefulness of mycobacterial serine protease antigen and its antibody in detecting neurotuberculosis.

A cocktail of affinity-purified antibodies reactive with diagnostically useful mycobacterial antigens ES-31, ES-43, and EST-6 for detecting the presence of Mycobacterium tuberculosis.

A cocktail of affinity-purified antibodies against diagnostically useful Mycobacterium tuberculosis H37Ra excretory-secretory protein antigens ES-31, ES-43, and EST-6 was explored for detection of circulating free and immune-complexed (IC) antigen in sera of patients with confirmed tuberculosis (TB) by sandwich enzyme-linked immunosorbent assay and compared with monospecific anti-ES-31 antibody. Out of 68 smear-positive TB cases studied, using cocktail antibody, a sensitivity of 97% (66/68) for immune-complexed cocktail antigen and 91% (62/68) for free cocktail-antigen detection was observed, compared to 91% (62/68) for immune-complexed ES-31 and 79% (54/68) for free ES-31 antigen when anti-ES-31 antibody was used alone. Thus, combinatorial use of antibodies showed improved sensitivity and was thus observed to be better than single antibody. The specificity was observed to be 99% for immune-complexed antigen using cocktail antibody. Furthermore, analysis of different groups of TB sera showed that circulating immune-complexed antigen is a sensitive marker than free antigen.

IgG subclass antibody response to mycobacterial serine protease at different stages of pulmonary tuberculosis.

BACKGROUND: Tuberculosis (TB) is a chronic bacterial infection caused by M. Tuberculosis. Studies of antibody response in TB have focussed mainly on their usefulness as a diagnostic serological tool, with little attention given to analysis of antibodies at the isotype and subclass level in relation to disease pathogenesis. Hence the present study was done to analyse IgG subclass response at different stages of tuberculosis, in order to understand the immunological events associated with disease development. MATERIAL/METHODS: Sera samples were collected from 104 subjects: 79 tuberculosis patients (fresh, relapse and chronic cases) and 25 healthy normals. IgG subclass antibody response was analysed by indirect plate peroxidase ELISA against previously reported mycobacterial serine protease (ES-31) antigen. RESULTS: Fresh cases of tuberculosis showed increased IgG1 and IgG3 antibodies, while a few cases showed moderately increased IgG2. IgG1 and IgG3 were found to be elevated with increased bacillary load. Relapse and chronic cases showed increased IgG1 and IgG3, while positivity to IgG2 was decreased. Chronic cases showed a moderate increase in IgG4 antibody. Thus IgG1 and IgG3 were predominant in all forms of tuberculosis. CONCLUSIONS: The elevated levels of IgG1 and IgG3 antibodies to mycobacterial serine protease in active tuberculosis observed in this study provide an additional marker for diagnosis of tuberculosis. Furthermore, the higher level of these antibodies with high bacillary load patients and in chronic cases of tuberculosis may provide valuable insight into their possible role in disease progression.

Effectivity of crude versus purified mycobacterial secretory proteins as immunogen for optimum antibody production.

Monospecific antibodies have been successfully utilized in antigen detection, which is better indicator of active infection. Mycobacterium tuberculosis excretory secretory (M tb ES) antigens such as ES 31, ES 41 and ES 43 (31 kDa, 41 kDa and 43 kDa protein, respectively) have been shown to be present in Mycobacterium tuberculosis H37Ra culture filtrate and are of diagnostic interest. To study the immunogenic potential of crude versus purified antigen, goat was immunized with M tb detergent soluble sonicate (DSS) antigen as well as purified antigen fraction (ESAS 7) containing ES 31 antigen. Both anti-DSS IgG antibody and anti ESAS 7 IgG antibody were found to be reactive with ES 31 antigen upto 1 ng concentration of antibody by ELISA. Crude DSS antigen was found to be quite effective in producing high titre antibodies and showed further high reactivity with other ES antigens (ES 41 and ES 43) of diagnostic interest.

Levels of antibody, free antigen and immune-complexed antigen by ELISA in different grades of sputum positive patients of pulmonary tuberculosis.

The ES-31 (31 kDa protein) antigen was isolated from culture filtrate of Mycobacterium tuberculosis H37Ra and was shown to have potential in immunodiagnosis of pulmonary tuberculosis. Serum samples from 38 confirmed sputum positive pulmonary tuberculosis patients were grouped into AFB+, AFB++, AFB+++ based on sputum bacillary load. Analysis of tubercular antibody, circulating free and immune complexed antigen (CIC-Ag) was done in serum samples by Indirect and Sandwich ELISA using ES-31 antigen and affinity purified anti ES-31 antibody respectively. The analysis of Geometric mean titre (GMT) of all the three groups showed that GMT of tubercular antibody was considerably decreased compared to elevated levels of CIC-Ag (Antibody: 1360 to 816 and CIC-Ag: 534 to 1744) from low bacillary sample to high bacillary samples, whereas there is no significant change in the titre of circulating free antigen. Low levels of detectable antibody is observed possibly due to removal of antibody from circulation by immune complex formation as confirmed by its elevated levels in sputum AFB+++ positive patients.

Serodiagnosis of tuberculosis using two ELISA systems.

Serodiagnosis by ELISA has been widely explored over the years, in the diagnosis of tuberculosis. Two ELISA systems were evaluated for detection of mycobacterial antibodies in pulmonary and extra pulmonary tuberculosis. The two test assays explored were ERBA LISA (TB IgG) test (Anda Biologicals) which uses A60 antigen complex found in the cytosol of typical and atypical mycobacteria, and SEVA TB (IgG) ELISA, which uses a 31 kDa, glycoprotein antigen purified fromM. tb H(37)Ra culture filtrate. Sera from 98 proven tuberculosis [pulmonary TB (48), tuberculous lymphadenopathy (30), tuberculous meningitis (15) & genitourinary TB (5)] were studied along with 32 healthy controls. The overall positivity obtained using ERBA LISA (TB IgG) test and SEVA TB (IgG) ELISA test was 72.9% and 91.6% in pulmonary tuberculosis, 43.3% and 76.6% in tuberculous lymphadenopathy respectively. The sensitivity of ERBA LISA test in tuberculous meningitis and genito-urinary TB was significantly low (26.6% & 40% respectively) compared to sensitivity obtained using SEVA TB ELISA (86.6% & 60% respectively) with overall specificity of 60% and 87.5%. Thus SEVA TB IgG ELISA test was found to be more sensitive than ERBA LISA in detecting IgG antibodies in tuberculous sera, in particular in extra pulmonary tuberculosis cases.

Analysis of SEVA TB ES-31 antigen specific immunoglobulins IgM, IgA and IgG in sera of sputum and culture positive pulmonary tuberculosis.

Tuberculosis remains major health problem in India and developing countries Immunodiagnosis has important role in screening, diagnosis and management of tuberculosis. SEVA TB ES-31 antigen has shown potential in detecting tuberculous IgG antibody in earlier studies from our laboratory. In the present study we have analysedSEVA TB ES-31 antigen specific immunoglobulinsIgM, IgA and IgG in clinically and bacteriologically confirmed pulmonary tuberculosis cases to determine the usefulness of specific immunoglobulin class in the diagnosis of patients attending the hospital.Of the 30 cases of pulmonary tuberculosis 25 (83.3%) were positive for IgG, 19 (63.3%) for IgM and 16 (53.3%) for IgA. On combining IgG and IgM positivity, sensitivity was increased to 93.3%. While combining IgG and IgA positivity, sensitivity increased to 90%. However specificity was decreased to 66.6% and 70% for both of these combinations respectively. It could be envisaged from this study that IgG antibody detection against ES-31 antigen showed acceptable sensitivity (83.3%) and specificity (86.6%) compared to IgM or IgA alone or in combination. When immune responses were analysed according to degree of sputum positivity, IgG response was observed to be predominant in all grades, compared to IgM or IgA antibody. The addition of IgM or IgA as an adjunct test increases the sensitivity but at the cost of specificity. Hence the detection of IgG alone is more useful compared to IgM or IgA assay, in detecting tuberculosis disease cases coming to the hospital.