The safety and immunogenicity of a bivalent conjugate vaccine against Salmonella enterica Typhi and Paratyphi A in healthy Indian adults: a phase 1, randomised, active-controlled, double-blind trial.

BACKGROUND: Enteric fever caused by Salmonella enterica Typhi and Salmonella Paratyphi A is an important public health problem, especially in low-income and middle-income countries with limited access to safe water and sanitation. We present results from, to our knowledge, the first ever human study of a bivalent paratyphoid A-typhoid conjugate vaccine (Sii-PTCV). METHODS: In this double-blind phase 1 study, 60 healthy Indian adults were randomly assigned (1:1) to receive a single intramuscular dose of either Sii-PTCV or typhoid conjugate vaccine (Typbar-TCV). Safety was assessed by observing solicited adverse events for 1 week, unsolicited events for 1 month, and serious adverse events (SAEs) over 6 months. Immunogenicity at 1 month and 6 months was assessed by measuring anti-capsular polysaccharide antigen Vi (anti-Vi) IgG and IgA against Salmonella Typhi and anti-lipopolysaccharide (LPS) IgG against Salmonella Paratyphi A by ELISA, and functional antibodies using serum bactericidal assay (SBA) against Salmonella Paratyphi A. This study is registered with Clinical Trial Registry-India (CTRI/2022/06/043608) and is completed. FINDINGS: 60 participants were enrolled. Of these 60 participants, 57 (95%) participants were male and three (5%) participants were female. Solicited adverse events were observed in 27 (90%) of 30 participants who received Sii-PTCV and 26 (87%) of 30 participants who received Typbar-TCV. The most common local solicited event was pain in 27 (90%) participants who received Sii-PTCV and in 23 (77%) participants who received Typbar-TCV. The most common solicited systemic event was myalgia in five (17%) participants who received Sii-PTCV, whereas four (13%) participants who received Typbar-TCV had myalgia and four (13%) had headache. No vaccine-related unsolicited adverse events or SAEs were reported. The seroconversion rates on day 29 were 96·7% (95% CI 82·8-99·9) with Sii-PTCV and 100·0% (88·4-100·0) with Typbar-TCV for anti-Vi IgG; 93·3% (77·9-99·2) with Sii-PTCV and 100·0% (88·4-100·0) with Typbar-TCV for anti-Vi IgA; 100·0% (88·4-100·0) with Sii-PTCV and 3·3% (0·1-17·2) with Typbar-TCV for anti-LPS (paratyphoid); and 93·3% (77·9-99·2) with Sii-PTCV and 0% (0·0-11·6) with Typbar-TCV for SBA titres (paratyphoid). Paratyphoid anti-LPS immune responses were sustained at day 181. INTERPRETATION: Sii-PTCV was safe and immunogenic for both typhoid and paratyphoid antigens indicating its potential for providing comprehensive protection against enteric fever. FUNDING: Serum Institute of India.

Acid hydrolysis conditions for quantification of meningococcal X polysaccharide in a pentavalent vaccine using HPAEC-PAD/ESI-MS.

A selective and sensitive method was evaluated for quantitation of meningococcal X (Men X) polysaccharide in pentavalent meningococcal A, C, W, Y and X conjugate vaccine using different acid hydrolysis conditions like HCl, TFA, HF, HF-TFA, and HF-HCl. High-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using CarboPac PA10 column was used to identify the hydrolyzed products based on retention time and its comparison with monosaccharide standards. Complete release of glucosamine (GlcN) from Men X in monovalent bulk and pentavalent vaccine samples was achieved using HF hydrolysis at 80 °C for 2 h. The Men X HF-hydrolyzed polysaccharide to glucosamine along with the reference standard was identified using collision-induced dissociation (CID) electrospray mass spectroscopy and the MS/MS fragments of m/z 162, m/z 144 and m/z 84. Meningococcal polysaccharide concentration was determined with a correlation coefficient r2 >0.99 using polysaccharide reference standard. The serogroups A, W, and Y were converted to their monosaccharides units and quantified using this method however, milder acid hydrolysis 0.1 M HCl 80 °C 2 h for release of sialic acid for Men C polysaccharide was found to be more suitable. These methods will provide necessary tools and prove to be beneficial to laboratories developing new saccharide-based vaccine combinations.

Effect of trifluoroacetic acid on the antigenicity of capsular polysaccharides obtained from various Streptococcus pneumoniae serotypes.

Determining the safety, antigenicity, and immunogenicity by in vitro and in vivo studies is a prerequisite for the development of new vaccines. And this study investigated it for a vaccine made from Streptococcus pneumoniae serotypes 2, 5, 12F, 18C, and 22F. The crude CPS was purified and partially depolymerized by conventional and trifluoroacetic acid methods. 1H NMR analysis confirmed the identity of the depolymerized CPS which gave similar profiles to reference polysaccharides, except for serotype 18C which was de-O-acetylated during TFA treatment. The antigenicity of the depolymerized CPS prepared by either method was comparable to that of the native CPS for serotypes 2, 5, 18C, and 22F based on multiplex bead based competitive inhibition assay. This study demonstrated a relationship between antigenicity and immunogenicity, which offers more suitable candidates for conjugation. It was found that after partial depolymerization process, the CPS with optimal molecular size resulted in higher antigenicity. The immunogenicity of S. pneumoniae serotype 2 conjugates in mice was evaluated by opsonophagocytic assay and a multiplex bead-based assay, wherein on day 42 after immunization, the total and functional IgG titer was found to be increased by 32-fold.

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.

Meningococcal ACWYX Conjugate Vaccine in 2-to-29-Year-Olds in Mali and Gambia.

BACKGROUND: An effective, affordable, multivalent meningococcal conjugate vaccine is needed to prevent epidemic meningitis in the African meningitis belt. Data on the safety and immunogenicity of NmCV-5, a pentavalent vaccine targeting the A, C, W, Y, and X serogroups, have been limited. METHODS: We conducted a phase 3, noninferiority trial involving healthy 2-to-29-year-olds in Mali and Gambia. Participants were randomly assigned in a 2:1 ratio to receive a single intramuscular dose of NmCV-5 or the quadrivalent vaccine MenACWY-D. Immunogenicity was assessed at day 28. The noninferiority of NmCV-5 to MenACWY-D was assessed on the basis of the difference in the percentage of participants with a seroresponse (defined as prespecified changes in titer; margin, lower limit of the 96% confidence interval [CI] above -10 percentage points) or geometric mean titer (GMT) ratios (margin, lower limit of the 98.98% CI >0.5). Serogroup X responses in the NmCV-5 group were compared with the lowest response among the MenACWY-D serogroups. Safety was also assessed. RESULTS: A total of 1800 participants received NmCV-5 or MenACWY-D. In the NmCV-5 group, the percentage of participants with a seroresponse ranged from 70.5% (95% CI, 67.8 to 73.2) for serogroup A to 98.5% (95% CI, 97.6 to 99.2) for serogroup W; the percentage with a serogroup X response was 97.2% (95% CI, 96.0 to 98.1). The overall difference between the two vaccines in seroresponse for the four shared serogroups ranged from 1.2 percentage points (96% CI, -0.3 to 3.1) for serogroup W to 20.5 percentage points (96% CI, 15.4 to 25.6) for serogroup A. The overall GMT ratios for the four shared serogroups ranged from 1.7 (98.98% CI, 1.5 to 1.9) for serogroup A to 2.8 (98.98% CI, 2.3 to 3.5) for serogroup C. The serogroup X component of the NmCV-5 vaccine generated seroresponses and GMTs that met the prespecified noninferiority criteria. The incidence of systemic adverse events was similar in the two groups (11.1% in the NmCV-5 group and 9.2% in the MenACWY-D group). CONCLUSIONS: For all four serotypes in common with the MenACWY-D vaccine, the NmCV-5 vaccine elicited immune responses that were noninferior to those elicited by the MenACWY-D vaccine. NmCV-5 also elicited immune responses to serogroup X. No safety concerns were evident. (Funded by the U.K. Foreign, Commonwealth, and Development Office and others; ClinicalTrials.gov number, NCT03964012.).

Quantitation of free cyanide using ion exchange chromatography in Neisseria meningitidis serogroups A, C, W, Y and X conjugates used in vaccine manufacture.

Polysaccharide vaccines essentially used in the prevention of bacterial infections are known to be good immunogens when conjugated to an immunogenic protein using various cyanylating agents. Analysis of residual cyanide in polysaccharide conjugate vaccines is an ardent task due to the complexity of the sample matrices and the lack of suitable methods. We report a selective ion chromatography method with electrochemical detection using IonPac AS7 column for estimation of residual cyanide in meningococcal serogroups A, C, W, Y and X bulk conjugates in presence of other interfering ions. Gold electrode and Ag/AgCl reference electrode ensures sensitivity and reproducibility of cyanide quantitation. The calibration curve of the method is linear having r2 ≥0.990 over the concentration range 1.45 ng/mL to 93.10 ng/mL. The recovery of cyanide in bulk conjugates ranged between 96.0% and 108.9%. The limits of detection and quantitation were 0.50 ng/mL and 1.45 ng/mL which corresponds to 0.31 ng/µg and 0.91 ng/µg of polysaccharide respectively. The method validation and feasibility study were performed using Men W and Men X bulk conjugates respectively with in house residual cyanide specification due to unavailability of pharmacopeia guidelines. The method is reproducible and can accurately quantify residual cyanide in purified meningococcal bulk conjugates.

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.

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.

Evaluation of Critical Quality Attributes of a Pentavalent (A, C, Y, W, X) Meningococcal Conjugate Vaccine for Global Use.

Towards achieving the goal of eliminating epidemic outbreaks of meningococcal disease in the African meningitis belt, a pentavalent glycoconjugate vaccine (NmCV-5) has been developed to protect against Neisseria meningitidis serogroups A, C, Y, W and X. MenA and X polysaccharides are conjugated to tetanus toxoid (TT) while MenC, Y and W polysaccharides are conjugated to recombinant cross reactive material 197 (rCRM197), a non-toxic genetic variant of diphtheria toxin. This study describes quality control testing performed by the manufacturer, Serum Institute of India Private Limited (SIIPL), and the independent control laboratory of the U.K. (NIBSC) on seven clinical lots of the vaccine to ensure its potency, purity, safety and consistency of its manufacturing. In addition to monitoring upstream-manufactured components, samples of drug substance, final drug product and stability samples were evaluated. This paper focuses on the comparison of the vaccine's critical quality attributes and reviews key indicators of its stability and immunogenicity. Comparable results were obtained by the two laboratories demonstrating sufficient levels of polysaccharide O-acetylation, consistency in size of the bulk conjugate molecules, integrity of the conjugated saccharides in the drug substance and drug product, and acceptable endotoxin content in the final drug product. The freeze-dried vaccine in 5-dose vials was stable based on molecular sizing and free saccharide assays. Lot-to-lot manufacturing consistency was also demonstrated in preclinical studies for polysaccharide-specific IgG and complement-dependent serum bactericidal activity for each serogroup. This study demonstrates the high quality and stability of NmCV-5, which is now undergoing Phase 3 clinical trials in Africa and India.

Simultaneous purification and depolymerization of Streptococcus pneumoniae serotype 2 capsular polysaccharides by trifluoroacetic acid.

Development of an effective purification process in order to provide low cost and high-quality vaccine is the necessity of glycoconjugate vaccine manufacturing industries. In the present study, we have attempted to develop a method for simultaneous purification and depolymerization process for capsular polysaccharides (CPS) derived from Streptococcus pneumoniae serotype 2. Trifluoroacetic acid (TFA) was used to precipitate impurities which were then removed by centrifugation. It was observed that the TFA treatment could simultaneously depolymerize the CPS and purify it. The purified and depolymerized CPS was analyzed for its purity, structural identity and conformity, molecular size, antigenicity to meet desired quality specifications. The obtained results showed that the purification and depolymerization of S. pneumoniae serotype 2 CPS did not affect the antigenicity of CPS.

Development of competitive inhibition ELISA as an effective potency test to analyze human rabies vaccines and assessment of the antigenic epitope of rabies glycoprotein.

The potency of all modern tissue culture human rabies vaccines is measured based on the National Institute of Health (NIH) potency test that is laborious, time-consuming, involves large test variations and requires sacrifice of large number of animals. To circumvent these limitations, several researchers and WHO expert working groups have discussed development of alternative in vitro methods to replace the NIH potency test. Although several immunochemical methods have been proposed to quantify rabies glycoprotein (G-protein) using multiple murine monoclonal antibodies, we report an In vitro competitive inhibition ELISA (CIA) method based on the use of a neutralizing rabies glycoprotein site III directed novel therapeutic human rabies monoclonal antibody (RAB1) that shows equivalence to the mice NIH potency test in recognition of neutralization site of the glycoprotein. In vitro potency testing of WHO 7th International Standard for rabies vaccine (IS) by CIA using RAB1 and In-house reference standard (IHRS) as a standard to assess its suitability for the assessment of validation parameters showed accurate and precise values with <15% coefficient variance. The method was validated using 5PL standard curve with linearity r2 > 0.98 and LLOQ of 0.125 IU/mL indicating sensitivity of the method. The method was found to be precise, robust and accurate to quantitate intact rabies glycoprotein in final vaccine and showed a strong correlation (Pearson's r = 0.81) with the NIH potency values of licensed Vero cell rabies vaccine. The CIA test using RAB1 was able to accurately quantitate degradation of rabies vaccine and assess loss in antigenicity of lyophilized and reconstituted liquid rabies vaccine under thermal stress conditions. The method was able to differentiate between potent and reduced potency vaccine samples. The new in vitro competitive inhibition ELISA method using RAB1 thus can be a valid alternative to the NIH test.

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.

Process development and immunogenicity studies on a serogroup 'X' Meningococcal polysaccharide conjugate vaccine.

Meningococcal group X (MenX) is responsible for recent outbreaks of meningitis reported in sub-Saharan region of Africa. Although protective vaccines are available for meningitis, they are not effective against MenX. An efficacious, monovalent conjugate vaccine was designed against MenX and a fed-batch fermentation process was developed. The MenX polysaccharide (PS) was purified and yield estimated to be 15-fold higher than the reported elsewhere. Structure of MenX polysaccharide was confirmed by (1)H, (13)C NMR spectroscopy analysis. Molecular weight of PS was found to be 310 kDa using HPLC-SEC coupled to refractive index (RI) detector. The MenX-Tetanus toxoid (TT) monovalent conjugate proved to be highly immunogenic in mice, and the bactericidal titers of MenX-TT conjugate were 10-fold higher than native PS. Increasing the dose of MenX-TT conjugate from 0.5 µg to 1.0 µg induced an 8-fold higher antibody titer as well as serum bactericidal titer. The current work suggests that the MenX-TT conjugate is a candidate vaccine against meningitis caused by Meningococcal group X strains.

Mapping antigenic diversity and strain specificity of mumps virus: a bioinformatics approach.

Mumps is an acute infectious disease caused by mumps virus, a member of the family Paramyxoviridae. With the implementation of vaccination programs, mumps infection is under control. However, due to resurgence of mumps epidemics, there is a renewed interest in understanding the antigenic diversity of mumps virus. Hemagglutinin-neuraminidase (HN) is the major surface antigen and is known to elicit neutralizing antibodies. Mutational analysis of HN of wild-type and vaccine strains revealed that the hypervariable positions are distributed over the entire length with no detectable pattern. In the absence of experimentally derived 3D structure data, the structure of HN protein of mumps virus was predicted using homology modeling. Mutations mapped on the predicted structures were found to cluster on one of the surfaces. A predicted conformational epitope encompasses experimentally characterized epitopes suggesting that it is a major site for neutralization. These analyses provide rationale for strain specificity, antigenic diversity and varying efficacy of mumps vaccines.