Explore the vast range of titles available.

Background: The immunogenicity of polysaccharide conjugate vaccines is critically influenced by the choice of carrier protein, which promotes a T-cell-dependent immune response mechanism leading to strong antibody production. In this study, the cholera toxin B subunit (CTB), a non-toxic pentameric protein, was evaluated as a novel carrier protein for pneumococcal polysaccharide antigens. Methods: Recombinant CTB was produced in Escherichia coli and purified using scalable chromatographic methods. Pneumococcal polysaccharides from serotypes 7F, 22F, and 33F were chemically activated with CDAP and conjugated to CTB. Results: The resulting glycoconjugates were characterized by SEC-MALS, confirming successful conjugation, high molecular weights, consistent polysaccharide-to-protein ratios, and acceptable endotoxin levels. Immunogenicity was assessed in rabbits following immunization with alum-adjuvanted formulations. Results: Robust IgG responses were elicited by all CTB-based conjugates, with antibody levels found to be comparable to those induced by CRM197 conjugates, demonstrating the potential of CTB as a promising alternative for the next generation of conjugate vaccines.

Background/Objectives: Infection from Streptococcus pneumoniae can lead to serious complications, such as meningitis and pneumonia, in children under 2 years of age, older adults, and immunocompromised populations. Conjugate vaccines against the pathogen have been licensed for the prevention of invasive pneumococcal disease. Conjugate vaccine development is an involved process demanding extensive characterization of both the polysaccharide (PS) and protein (Pr) moieties in complex structures. One powerful tool in our analytical tool kit that can shed light on various analytical attributes of conjugate vaccines, such as molecular weight and composition and conjugation efficiency, is the size-exclusion chromatography-multi-angle light scattering detector (SEC-MALS) technique. Herein, we demonstrate the applicability of the SEC-MALS approach for pneumococcal conjugate vaccine product characterization. Methods: Capsular polysaccharides for serotypes (STs) 1, 3, 5, 10 A, 18 C, 24 F, and 33 F conjugated to rCRM197 carrier protein were chosen for this study. Results: The technique was very straightforward, with a high degree of accuracy (>90% based on standards) and repeatability (<2% RSD) for conjugate molar mass measurements. In addition, leveraging the capability of SEC-MALS for compositional analysis, we were able to get detailed information on the molecular assembly and conformation of the conjugates and further tweak the conjugation process to yield conjugates of a desired molar mass. Conclusions: Thus, this study highlights the usefulness of the SEC-MALS technique for in-depth conjugate vaccine biophysical characterization, which is critical for achieving optimal product attributes, driving manufacturing consistency and vaccine potency.

Different methods are used today for polysaccharide quantitation, including HPLC and various colorimetric assays. Among these, the anthrone-sulfuric acid assay (anthrone assay) is popular when the sample matrix is suitable, such as in purified polysaccharides and monovalent bulk conjugate components of glycoconjugate vaccines. While relatively safe, quick, and affordable, the anthrone assay requires significant operator time to complete and is not suited to high-throughput processing. Furthermore, the anthrone-sulfuric acid reagent presents a unique challenge to automation efforts due to its corrosive properties. Reported here is an automated anthrone assay via a liquid handling system (LHS). Twenty-three serotypes of pneumococcal (PNU) polysaccharide were quantified with the traditional anthrone assay and subsequently analyzed using the anthrone LHS method. The anthrone LHS method was evaluated for accuracy compared to the manual method and later validated according to ICH Q2 (R2) guidelines. To our knowledge, this is the first fully unattended and corrosion-mitigated anthrone assay validated under ICH Q2 (R2), capable of overnight batch operation. The developed assay can quantify polysaccharides with an accuracy of 81–115%, is precise to a coefficient of variation of <7.0%, and is linear between 30 and 650 µg/mL range (R2 ≥ 0.993). The assay can process eight samples per hour, can be utilized in overnight operation, and completes all pipetting, incubation, and data export steps automatically.

This study evaluated the formulation and stability of a quadrivalent glycoconjugate Shigella vaccine candidate based on four predominant strains (S. flexneri; 2a, 3a, and 6, and S. sonnei) covering ~64% of global Shigella infections. Each glycoconjugate antigen consists of a strain-specific O-polysaccharide (O-PS) covalently linked to the carrier protein IpaB, a component of the Shigella type III secretion system. First, selective competitive ELISAs were developed to measure antigenicity of the four O-PS-IpaB conjugates formulated with different adjuvants (i.e., Alhydrogel®, AH; Adju-phos®, AP; and CpG-1018®, CpG). Next, the monovalent S. sonnei O-PS-IpaB conjugate was studied to elucidate interactions with aluminum salt adjuvants (AH, AP) under different solution conditions. Third, the stability profiles of AH- or AP-adjuvanted S. sonnei O-PS-IpaB conjugate in various formulations (±CpG) were determined at different temperatures. Interestingly, incubation at 25 °C for 2 weeks resulted in increased antigenicity values when the antigen was bound to AP or AH, suggesting increased epitope exposure upon adjuvant binding. When bound to AP adjuvant at pH 5.8, the best glycoconjugate antigen stability was observed at elevated temperatures. The CpG adjuvant under these conditions, however, displayed incompatibility (i.e., material loss), presumably from precipitation due to lack of interaction with AP and presence of the detergent LDAO from the bulk antigen buffer. In contrast, the glycoconjugate antigen and CpG adjuvant were both bound to the AH adjuvant and stable at 2–8 °C, pH 7.0. This AH-CpG formulation of the O-PS-IpaB conjugate antigens was identified as a promising candidate for future animal immunogenicity testing.

K. pneumoniae is a pathogen that causes serious infections such as pneumonia and sepsis globally. The increasing prevalence of antibiotic resistance in this pathogen has complicated treatment efforts, highlighting the need for preventive therapeutic strategies such as vaccination. However, no licensed vaccines are currently available. Standardized assays to assess the immunogenicity of new vaccines are crucial for vaccine development and evaluation of other therapeutics. Therefore, we have developed assays that can assess the functionality of antibodies, which can be used to evaluate the potential of novel K. pneumoniae conjugate vaccines, and inform which antibodies are most effective for preventing disease.

Background/Objectives: Group B Streptococcus (GBS) is a significant cause of perinatal infection in neonates and infants. Complications could include neonatal sepsis and meningitis, preterm birth, stillbirth, or death. Though no GBS vaccine is currently licensed, maternal immunization is expected to be a highly effective strategy to address invasive GBS disease—particularly in low- and middle-income countries (LMICs), where the disease burden is the greatest and access to existing interventions is limited. In this study, we present a novel hexavalent GBS vaccine candidate with a unique combination of serotypes (ST)—Ia, Ib, II, III, V, and VII—that could be an efficacious and cost-effective intervention, with the broadest coverage of 99% against circulating serotypes globally. Methods: The 6-valent conjugate vaccine candidate, GBS-06, is developed using a novel approach by linking the six polysaccharides (PS) to recombinant cross-reactive material 197 (rCRM197) carrier protein derivatized with a hydrazide-polyethylene glycol-hydrazide (HZ-PEG-HZ) linker. A repeat-dose GLP toxicology study with GBS-06 was conducted at the highest clinical dose of 20 µg in rabbits with saline as the placebo control. Results: The results reveal induction of robust anti-capsular polysaccharide-specific IgG responses against each of the six serotypes after each dose with the highest antibody GMCs at Day 49 following the third dose. Conclusions: Hence, this work is the first demonstration of strong immunogenicity achieved using a linker (HZ-PEG-HZ) for GBS glycoconjugate vaccine development. The positive data from the study have strong implications in the advancement of the candidate for evaluation in clinical trials and provide a licensure pathway for maternal immunization.

Background/Objectives: Shigella is the leading bacterial cause of diarrheal disease worldwide. Although multiple vaccine candidates are under development and in clinical trials, no Shigella vaccine is currently available on the market. Shigella comprises four species: S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. S. flexneri has been recognized as the most prevalent species, particularly in low- and middle-income countries (LMICs), and the top serotypes are S. flexneri 2a, 3a and 6. Conversely, S. sonnei has a single serotype and predominates in high-income countries (HICs). Invasion plasmid antigen B (IpaB) is a critical virulence factor of Shigella type III secretion system (T3SS) that is highly conserved across Shigella serotypes. Here, we report the development of a Shigella quadrivalent O-polysaccharide-IpaB conjugate vaccine candidate (IVT Shigella-04). Methods: IVT Shigella-04 contains O-polysaccharides (O-PS) from S. flexneri 2a, 3a, 6, and S. sonnei, each individually conjugated to recombinantly expressed IpaB as the carrier protein using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) chemistry. The immunogenicity of IVT Shigella-04 was evaluated in a rabbit immunization model. Results: Baseline (day 0) IgG concentrations were low for all four Shigella serotypes (<0.5 µg/mL). Following two doses on day 0 and day 28 (2.5 µg of each conjugate per dose; total 10 µg), IgG geometric mean concentrations increased significantly (p < 0.001) by day 42, reaching 67.96 µg/mL (2a), 91.56 µg/mL (3a), 371.31 µg/mL (6), and 11.00 µg/mL (sonnei). Consistently, serum bactericidal activity (SBA) at day 42 increased 13-fold (2a), 34-fold (3a), 63-fold (6), and 224-fold (sonnei) relative to baseline (day 0). Conclusions: IVT Shigella-04 elicited robust serotype-specific humoral and functional immune responses in preclinical models, supporting its further development toward clinical evaluation.

Sinorhizobium meliloti, a legume symbiont, and Brucella abortus, a phylogenetically related mammalian pathogen, both require the bacterial-encoded BacA protein to establish chronic intracellular infections in their respective hosts. We found that the bacterial BacA proteins share sequence similarity with a family of eukaryotic peroxisomal-membrane proteins, including the human adrenoleukodystrophy protein, required for the efficient transport of very-long-chain fatty acids out of the cytoplasm. This insight, along with the increased sensitivity of BacA-deficient mutants to detergents and cell envelope-disrupting agents, led us to discover that BacA affects the very-long-chain fatty acid (27-OHC28:0 and 29-OHC30:0) content of both Sinorhizobium and Brucella lipid A. We discuss models for how BacA function affects the lipid-A fatty-acid content and why this activity could be important for the establishment of chronic intracellular infections.

Haemophilus influenzae type a (Hia) disease is associated with fatality and morbidity, predominantly in children from Northern indigenous communities in Canada and Alaska. Symptoms include but are not limited to meningitis, sepsis, pneumonia, otitis media and bacteremia. A key virulence factor associated with Hia pathogenesis is the bacterial capsular polysaccharides. Prophylactic vaccines targeting the capsular polysaccharide (CPS) antigens is considered a promising intervention strategy given the effectiveness of polysaccharide-protein conjugate vaccines against diverse bacterial pathogens. In the same vein, we in this study have presented the development of Hia conjugate vaccine involving hydrazide-polyethylene glycol-hydrazide linker with rCRM197 as a carrier protein. Preclinical immunogenicity data indicated induction of robust serum bactericidal antibody responses at a 10-μg CPS antigen dose in rabbits like the PedvaxHIB® comparator, leading to evaluation of the Hia vaccine candidate in Phase I clinical trials.

The SARS-CoV-2 pandemic necessitated effective vaccines that can endure antigenic mutations. Here we demonstrate highly immunogenic conjugate vaccines that elicit broad cross-neutralization to variants of concern (VOC) in animal studies. By utilizing protein-protein conjugation and Toll-Like Receptor (TLR) agonist adjuvants we achieve enhanced immunogenicity compared to unconjugated equivalents. These vaccine candidates induced broad cross-protection against several VOC, a characteristic lacking in early COVID-19 vaccines. Murine neutralizing antibody (nAb) titers from animals vaccinated with Beta-only conjugates were equivalent between Beta, Delta, Omicron BA.1, BA.2, and BA.4/BA.5 variants, which were circulating up to three years after the antigenic Beta strain. Additionally, Beta-Delta bivalent conjugate vaccines readily prevented disease in hamster challenge. Together this demonstrates a vaccine with remarkably broad cross-protection and potential to protect for extended periods despite mutations, without requiring modified boosters or antigen adaption. These techniques can be applied to more recent SARS-CoV-2 strains, and other viruses, highlighting the benefits of protein-protein conjugation coupled with TLR agonist secondary adjuvants. •SARS-CoV-2 spike protein conjugated to CRM-197 with TLR agonist adjuvants leads to high IgG and nAb.•Beta-Only conjugates with TLR agonists induced broadly cross-reactive nAb in mice.•Beta-only conjugate led to neutralization of variants that emerged 3 years after antigenic strain.•Bivalent conjugate vaccines effectively protect hamsters from viral challenge.•Protein conjugate platform applicable to any disease requiring enhanced immunogenicity.