SBIR-STTR Award

Pneumococcal pneumonia remains the leading cause of bacterial pneumonia in both children under 5 years of age and adults over 65 years of age. The standard, preventative therapy is the conjugate vaccine, Prevnar-13, which consists of an immunogenic carrier protein covalently attached to one of thirteen pneumococcal capsular polysaccharides. Although Prevnar-13 has significantly reduced the burden of pneumococcal disease, it only protects against 13 of the 90 plus pneumococcal serotypes; furthermore, current methods employed to expand the serotype coverage are notoriously slow requiring complex synthetic chemistries to link a new pneumococcal capsular polysaccharide to the immunogenic carrier protein. Over the last decade, we have been pioneering an innovative approach to conjugate vaccine development that drastically simplifies the production of glycoconjugates. This glycoengineering strategy, consisting of the exploitation of bacterial glycosylation machineries to generate “bioconjugates”, eliminates the need of intricate chemical conjugation methods by employing conjugating enzymes to attach polysaccharides to acceptor proteins in Escherichia coli. Two conjugating enzymes, PglB and PglL, have been commercially utilized to generate bioconjugates as they are able to transfer a wide variety of polysaccharides to proteins; however, neither are able to transfer polysaccharides containing glucose at the reducing end (the first sugar of a growing polysaccharide chain). This seemingly simple observation has enormous implications as approximately 80% of pneumococcal capsules contain glucose at the reducing end. Recently, we have identified and patented the first conjugating enzyme that is able to efficiently transfer pneumococcal capsular polysaccharides containing glucose at the reducing end to an acceptor protein. Based on this observation, we will couple our novel conjugating enzyme technology with carrier proteins previously utilized in conjugate vaccine formulations, streamlining the generation of a superior pneumococcal vaccine with broader serotype coverage. Importantly, our glycoengineering strategy does not require pathogenic organisms as a source of polysaccharide nor chemical reactions to link polysaccharides to proteins. The proposed research in this phase I application will focus on (Aim 1) glycoengineering three commercial carrier proteins (exotoxin A, tetanus toxin fragment C, and CRM197) to contain a modular glycotag with pneumococcal capsular polysaccharides generating a new bioconjugate vaccine for pneumococcal serotypes 8, 9V, 14, and 15b. Subsequently (Aim 2) we will demonstrate the immunogenicity and efficacy of our pneumococcal specific bioconjugate vaccine compared to the standard preventative therapy Prevnar-13. Our next step for phase II funding is to expand the serotype coverage included in our bioconjugate vaccine, develop a large-scale purification scheme for obtaining our bioconjugate vaccine, as well as pre-clinical studies to further demonstrate the safety, potency, and efficacy of our next generation glycoengineered pneumococcal bioconjugate vaccine.

Public Health Relevance Statement:
PROJECT NARRATIVE The proposal seeks to further develop an innovative technology that can be used to make conjugate vaccines against pneumococcus with broader coverage without the need of chemical procedures. For the first time ever, our group has identified a conjugating enzyme that is able to attach pneumococcal polysaccharides to an acceptor protein, dramatically simplifying the synthesis of protein-polysaccharide conjugates. In this application, we propose to apply our novel conjugating enzyme technology to carrier proteins previously utilized in conjugate vaccine formulations, streamlining the generation of a superior pneumococcal vaccine with broader serotype coverage.

Project Terms:
5 year old; Adult; Age-Years; Amino Acids; Bacteremia; Bacterial Pneumonia; Biological Products; Biopharmaceutics; capsule; Carrier Proteins; chemical reaction; Chemicals; Child; Chronic; Clinical; commercialization; Communities; Complex; Conjugate Vaccines; cost; cross reacting material 197; Development; Enzymes; Epitopes; Escherichia coli; Exotoxins; Funding; Generations; Glucose; Glycoconjugates; glycosylation; Heterogeneity; Immune response; immunogenic; immunogenicity; Immunoglobulin G; Immunologic Memory; Infant; innovation; innovative technologies; Joints; Killings; Klebsiella; Legal patent; Link; Longevity; Meningitis; Methods; Natural immunosuppression; next generation; novel; Otitis Media; pathogen; Pathogenicity; Patients; Phase; Phase I Clinical Trials; Pneumococcal conjugate vaccine; Pneumococcal Infections; Pneumococcal Pneumonia; Pneumococcal vaccine; Pneumonia; Polysaccharides; preclinical study; Preventive therapy; Prevnar; Procedures; Production; Protein Biosynthesis; Proteins; Recombinants; Research; Safety; Scheme; Serotyping; Shigella dysenteriae bacterium; Site; Source; Streptococcus pneumoniae; success; sugar; Synthesis Chemistry; Technology; tetanus toxin fragment C; Time; Translations; vaccine development; Vaccines; World Health Organization

Pneumococcal conjugate vaccines (PCVs), composed of a pneumococcal polysaccharide covalently linked to a carrier protein, are life-saving prophylactics used to prevent pneumococcal disease. Importantly, PCVs provide immunity for all age groups, including, infants and children under the age of two, which is not the case for purely polysaccharide vaccines. Like all conjugate vaccines, PCVs are manufactured using chemical conjugation, which is notoriously complex, labor intensive, and ultimately hinders the development of better versions that provide immunity to more disease-causing serotypes. As an example, the PCV, Prevnar 13, was licensed in 2010 and only protects against 13 pneumococcal serotypes; whereas, the purely polysaccharide vaccine, Pneumovax 23, was licensed in 1983 and protects against 23 serotypes. Pneumovax 23 is approved for use in the elderly; however, it does not provide protection to infants and children. Thus, for more than three decades, infants and children have not had a vaccine option that protects against 20+ disease causing pneumococcal serotypes. In order to provide a 20+ valent PCV for use in all age groups, VaxNewMo has been developing a method for manufacturing pneumococcal and other conjugate vaccines that bypasses the dependency of chemical conjugation and instead exploits prokaryotic glycosylation systems in process termed bioconjugation. VaxNewMo’s proprietary bioconjugation platform relies on a conjugating enzyme to transfer a bacterial polysaccharide, like a pneumococcal capsular polysaccharide, to a carrier protein all within the lab safe bacterium E. coli. Since bioconjugation is an enzyme driven technology, the conjugates produced are homogenous and readily purified. Importantly, bioconjugation can be used to rapidly produce a plethora of conjugates against many serotypes simply by introducing the genetic information encoding for a different pneumococcal serotype into a bioconjugation competent strain of E. coli. Thus, bioconjugation can be used for the streamlined development of a PCV covering more than 20 serotypes. In Phase I of this project, we successfully established proof of principle that VaxNewMo’s bioconjugation platform could generate PCVs containing conventional vaccine carriers. Moreover, VaxNewMo’s PCVs were both immunogenic and protective against pneumococcal disease. The proposed research in this Phase II SBIR application will focus on (Aim 1) establishing bioprocessing capabilities for large volumetric production of VNM8, a serotype 8 pneumococcal bioconjugate. Establishing bioprocessing procedures for a single serotype bioconjugate is an important first step towards commercial scale production and will help streamline future upstream processing for other pneumococcal bioconjugates. Subsequently, (Aim 2) we will confirm that VNM8 produced in a large volumetric bioprocess is protective using in vitro and in vivo models. In addition, (Aim 3) we will generate a library of E. coli strains capable of producing a 24 valent PCV. For Phase IIB, will seek to formulate a 24 valent PCV (24vPneumo) for pharmacokinetic and toxicity studies and prepare for pre-IND meetings with the FDA.

Public Health Relevance Statement:
PROJECT NARRATIVE Pneumococcal conjugate vaccines (PCVs) prevent colonization and disease for some serotypes of Streptococcus pneumoniae. The leading PCV targets only 13 of the more than 90 pneumococcal serotypes. This application seeks to continue advanced development of PCV covering 24 serotypes, manufactured in a streamlined bioconjugation platform using a lab safe bacterium, Escherichia coli, as a host.

Project Terms:
Adjuvant; Advanced Development; Age; age group; Air; Antibody Response; Applications Grants; Bacteria; Bacterial Polysaccharides; bioprocess; Bypass; Carrier Proteins; Chemicals; Chemistry; Child; Childhood; clinically relevant; Complex; Conjugate Vaccines; cost; Dependence; Development; Disease; Dose; Drug Addiction; Drug Kinetics; Elderly; Engineering; Enzymes; Escherichia coli; Formulation; Foundations; Future; Generations; genetic information; Glucose; Glycoconjugates; glycosylation; Immunity; immunogenic; immunogenicity; Immunologic Memory; In Vitro; in vivo; in vivo Model; Infant; Infection; Libraries; Life; Link; meetings; Methodology; Methods; mortality; mouse model; Mus; Pharmaceutical Preparations; Phase; Pneumococcal conjugate vaccine; Pneumococcal Infections; Polysaccharides; Polyvalent pneumococcal vaccine; Positioning Attribute; prevent; Prevnar; Procedures; Process; Production; prophylactic; Proteins; Recombinants; Research; research clinical testing; Sales; Savings; Serotyping; Serum; Small Business Innovation Research Grant; Streptococcus pneumoniae; sugar; System; Technology; Toxic effect; vaccine development; Vaccine Production; Vaccines