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.

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.

A phase II/III randomised, comparative study evaluating the safety and immunogenicity of Biological E's live, attenuated Measles-Rubella vaccine in 9-12 month old healthy infants.

Measles is a major cause of childhood mortality and one-third of the world's Measles deaths occur in India. Rubella causes lifelong birth defects (Congenital Rubella Syndrome). Although neither condition has a cure, the MR vaccination can successfully prevent both diseases. The safety of Biological E's live attenuated MR vaccine (BE-MR) was established in 4-5-year-old healthy children. This phase-2/3 study was conducted to assess the safety and immunogenicity of BE-MR in 9-12 month old healthy infants. Overall, 600 subjects were enrolled and equally randomized to receive either BE-MR (n = 300) or the comparator vaccine, SII MR-Vac™ (n = 300). Safety profile of BE-MR vaccine was comparable to SII MR-Vac™ with no severe or serious adverse events (AEs) reported across the study groups. The primary objective of demonstrating non inferiority by BE-MR vaccine compared to SIIL's-MR Vac™ was met. The proportion of subjects with ≥ 2-fold and ≥ 4-fold increase in antibody titre against Measles and Rubella in both the study groups was comparable. Overall, BE-MR vaccine elicited robust and protective immune response as demonstrated by high proportion of sero-protected subjects and a large increase in anti-Measles and anti-Rubella antibodies at day 42 and can be administered safely to infants below one-year of age. This study was prospectively registered with the clinical trial registry of India- CTRI/2016/07/007109.

Individual polysaccharide quantification in polyvalent pneumococcal conjugate vaccine using rate nephelometry.

The multivalent pneumococcal conjugate vaccine (PCV) contains purified polysaccharides of different serotypes conjugated to a carrier protein. Testing the final formulated product for individual serotype polysaccharide content is critical in vaccine quality control which requires an assay specific to each serotype polysaccharide present in the formulated product. Antibodies specific to the serotypes specific polysaccharide were used in rate nephelometry assay for quantifying individual serotype polysaccharides in the formulated vaccine. Generally, native polysaccharide (NP) have been used as reference standard. However, the polysaccharide antigen in the vaccine product is in the conjugate form (CRM197 linked) and hence using NP as a reference standard may not be suitable. Activated quenched polysaccharide (AQP) as a reference standard in rate nephelometry would be more appropriate. The epitope structure of AQP closely represents the polysaccharide-protein conjugate drug product (DP) after trypsin digestion. Hence, AQP was evaluated as a novel reference standard for the accurate and precise determination of individual polysaccharides in the multivalent DP. Rate nephelometry assay using AQP could be used for DP release and stability for monitoring time-dependent changes in the product and establishing the shelf life. A similar strategy could be applied to test and release monovalent or multivalent polysaccharide-protein conjugate vaccines (Meningococcal, Haemophilus influenza Type B, Typhoidal, and non-typhoidal salmonella).

High-Performance Anion-Exchange chromatography with conductivity detection method for simultaneous determination of nitrogen and phosphorus in polysaccharides.

Capsular polysaccharides of Streptococcus pneumoniae contain a characteristic mix of monosaccharides in their structure resulting in immunologically distinct serotypes. Pneumococcal capsular polysaccharides include sugars such as hexoses, uronic acids, hexosamines, methyl pentoses, other functional groups are attached to the sugars are N and O-acetyl groups, nitrogen and phosphorus. Most of these components can be quantified using different colorimetric methods. However, available methods for quantifying nitrogen and phosphorus are not sensitive enough and laborious. We report a highly sensitive high-performance anion-exchange chromatography-conductivity detector (HPAEC-CD) method for quantifying nitrogen and phosphorus present in pneumococcal capsular polysaccharides. The method is reliable, robust and reproducible with no interference. The LOQ for nitrogen and phosphorus of 3.125 and 62.5 ng/mL, respectively, is highly critical for estimating low levels of total nitrogen and total phosphorus. We have implemented this method to quantify total nitrogen in Typhoid Vi polysaccharide and phosphorus in Haemophilus influenzae type-b polysaccharide. This method has greater application for quantification of nitrogen and phosphorus present in low concentrations in polysaccharide vaccines/biologicals.

A reversed phase HPLC-UV method for the simultaneous determination of residual formaldehyde and Triton X-100 in vaccine products.

Many of the inactivated viral vaccines for human and animal use are manufactured using formaldehyde as an inactivating agent. Apart from formaldehyde, Triton X-100 is also one of the chemicals commonly used in viral vaccine manufacturing. Triton X-100 is typically used to extract the cell-associated viruses and / or components during manufacturing process. The concentration of formaldehyde and Triton X-100 in the final bulks are also reduced during vaccine purification process. Here we report a simple RP-HPLC-UV based method for the quantification of residual Triton X-100 and formaldehyde as process impurities in viral vaccines. This method is also adopted for the residual impurity determination of either formaldehyde or Triton X-100 in other non-viral vaccines, multivalent as well as sub-unit vaccines, such as liquid pentavalent, includes TT, DT, Hepatitis B (rDNA) and Haemophilus type b conjugate vaccine (adsorbed). This method is rapid and can quantify both Triton X-100 and formaldehyde in a single preparation with improved peak asymmetry. This new assay has a linearity range starting from 0.0625 to 1 µg/mL for formaldehyde and 0.625-10 µg/mL for Triton X-100. This method would be very useful for viral vaccine manufacturing and release.

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.

E6 protein of human papillomavirus 16 (HPV16) expressed in Escherichia coli sans a stretch of hydrophobic amino acids, enables purification of GST-ΔE6 in the soluble form and retains the binding ability to p53.

Recombinant E6 expressed in Escherichia coli is known to form recalcitrant inclusion bodies even when fused to the soluble GST protein. This study describes the modification of the HPV genotype-16 oncogenic protein E6 in order to obtain it in the soluble form. The modified protein (ΔE6) was expressed in E. coli BL21 as an N-terminal fusion with GST (GST-ΔE6). ΔE6 was constructed by deleting the nucleotide sequences coding for IHDIIL (31-36 a.a), one of the highly hydrophobic peptide stretches, using splicing by overextension polymerase chain reaction (SOE-PCR). The removal of IHDIIL residues rendered the GST-ΔE6 soluble and amenable for purification involving a two step process a preliminary glutathione-GST affinity chromatography followed by gel-filtration chromatography. Evaluation of purified protein fractions by HPLC suggests that GST-ΔE6 exists as a monomer. Further, the ΔE6 in GST-ΔE6 seemed to retain the binding ability to p53 as determined by the glutathione-GST capture ELISA. Purified GST-ΔE6 we reckon, might find use as an essential reagent in immunological assays, in sero-epidemiological studies, and also in studies to delineate the structure and function of HPV16 E6.

A simple and rapid method to monitor the disassembly and reassembly of virus-like particles.

Protein fluorescence spectra (~300-440 nm) could be used as a simple and sensitive method to monitor the disassembly and reassembly of virus-like particles (VLPs). Insect cell expressed and purified HPV-16 L1 VLPs show significantly high fluorescence intensity, whereas the fluorescence is almost quenched after disassembly by adding the reducing agent. By removing the reducing agent, the fluorescence was restored to its original intensity, indicating the reassembly of VLPs. The data are consistent with enzyme-linked immunosorbent assay (ELISA) reactivity using conformation-specific mouse monoclonal antibody. The same method could be extended to VLPs of other viruses.