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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.

Mumps epidemics in Canada and the United States prompted us to review evidence for the effectiveness of 5 different vaccine strains. Early trials with the Jeryl Lynn vaccine strain demonstrated an efficacy of ∼95%, but in epidemic conditions, the effectiveness has been as low as 62%; this is still considerably better than the effectiveness of another safe strain, Rubini (which has an effectiveness of close to 0% in epidemic conditions). The Urabe vaccine strain has an effectiveness of 54%-87% but is prone to cause aseptic meningitis. Little epidemiological information is available for other vaccines. The Leningrad-Zagreb vaccine strain, which is widely used in developing countries and costs a fraction of what vaccines cost in the developed world, seems to have encouraging results; in 1 study, the effectiveness of this vaccine exceeded 95%. Aseptic meningitis has also been reported in association with this vaccine, but the benign nature of the associated meningitis was shown recently in Croatia. Also, the Leningrad-3 strain seems to be effective but causes less-benign meningitis. No mumps vaccine equals the best vaccines in quality, but the virtually complete safety of some strains may not offset their low effectiveness. Epidemiological data are pivotal in mumps, because serological testing is subject to many interpretation problems.

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.

► Recently, a recombinant human IgG1 monoclonal antibody (SII RMab) was developed to replace serum-derived immunoglobulin. ► A randomized dose-escalation phase 1 study along with rabies vaccine was conducted in healthy adults. ► SII RMab was well tolerated with similar reactions to the licensed human rabies immunoglobulin (HRIG). ► The antibody titers in the 10IU/kg cohort were comparable to the vaccine plus HRIG cohort. ► Future post-exposure prophylaxis studies are planned. Rabies is an essentially fatal disease that is preventable with the timely administration of post-exposure prophylaxis (PEP). The high cost of PEP, which includes vaccine and hyperimmune globulin, is an impediment to the goal of preventing rabies in the developing world. Recently a recombinant human IgG1 anti-rabies monoclonal antibody (SII RMab) has been developed in India to replace serum-derived rabies immunoglobulin. The present study was conducted to demonstrate the safety of SII RMab and to determine the dose resulting in neutralizing serum antibody titers comparable to human rabies immunoglobulin (HRIG) when administered in conjunction with rabies vaccine in a simulated PEP regimen. This randomized, open label, dose-escalation phase 1 study was conducted in healthy adults at a large tertiary care, referral, public hospital in India. Safety was assessed by active surveillance for adverse events along with standard laboratory evaluations and measurement of anti-drug antibodies (ADA). Anti-rabies antibody levels were measured by rapid fluorescent focus inhibition test (RFFIT) and ELISA. The study duration was 365 days. SII RMab was well tolerated with similar frequency of local injection site reactions to HRIG. The geometric mean concentrations of rabies neutralizing antibody in the vaccine plus SII RMab 10IU/kg cohort were comparable to the vaccine plus HRIG 20IU/kg cohort throughout the 365-day study period; day 14 geometric mean concentrations 23.4IU/ml (95% CI 14.3, 38.2) vs. 15.3IU/ml (95% CI 7.72, 30.3; p=NS), respectively. Future post-exposure prophylaxis studies of SII RMab at a dose of 10IU/kg in conjunction with vaccine are planned.

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.

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.

Background. Group A Neisseria meningitidis has been a major cause of bacterial meningitis in the sub-Saharan region of Africa in the meningitis belt. Neisseria meningitidis is an encapsulated pathogen, and antibodies against the capsular polysaccharide are protective. Polysaccharide–protein conjugate vaccines have proven to be highly effective against several different encapsulated bacterial pathogens. Purified polysaccharide vaccines have been used to control group A meningococcal (MenA) epidemics with minimal success. Methods. A monovalent MenA polysaccharide–tetanus toxoid conjugate was therefore developed. This vaccine was developed by scientists working with the Meningitis Vaccine Project, a partnership between PATH and the World Health Organization. Results. A high-efficiency conjugation method was developed in the Laboratory of Bacterial Polysaccharides in the Center for Biologics Evaluation and Research and transferred to the Serum Institute of India, Ltd, which then developed methods for purification of the group A polysaccharide and used its tetanus toxoid as the carrier protein to produce the now-licensed, highly effective MenAfriVac conjugate vaccine. Conclusions. Although many years of application of meningococcal polysaccharide vaccines have had minimal success in preventing meningococcal epidemics in the meningitis belt of Africa, our collaborative efforts to develop a MenA conjugate vaccine yielded a safe and highly effective vaccine.

Background. In 2002, the Meningitis Vaccine Project (MVP) chose the Serum Institute of India, Ltd (SIIL), as its manufacturing partner to establish a product development partnership (PDP) with the Meningitis Vaccine Project (MVP). MVP was a collaboration between PATH and the World Health Organization (WHO) to develop meningococcal conjugate vaccines for sub-Saharan Africa. Method. From the outset, SIIL recognized that a partnership with MVP carried some risk but also offered important opportunities for accessing new conjugate vaccine technology and know-how. Over 3 years, SIIL successfully accepted technology transfer for the group A meningococcal polysaccharide from SynCo Bio Partners and a conjugation method from the US Food and Drug Administration. Results. SIIL successfully scaled up production of a group A meningococcal conjugate vaccine that used SIIL tetanus toxoid as the carrier protein. Phase 1 studies began in India in 2005, followed by phase 2/3 studies in Africa and India. A regulatory dossier was submitted to the Indian authorities in April 2009 and WHO in September 2009. Export license was granted in December 2009, and WHO prequalification was obtained in June 2010. Vaccine was introduced at public scale in Burkina Faso that December. The group A meningococcal conjugate vaccine was named MenAfriVac, and is the first internationally qualified vaccine developed outside of big pharma. Conclusions. The project proved to be a sound investment for SIIL and is a concrete example of the potential for PDPs to provide needed products for resource-poor countries.