Quality control for new vaccine candidates.
It has been almost a year and a half since our chemists, together with Cuban partners, produced novel glycoconjugates that can be used as new vaccines against the bacterial pathogens of pneumonia and meningitis. Now the scientists have developed an analytical method to subject the potential vaccine candidates to a sophisticated quality control. With that, the next step towards application has been taken.
Pneumococci and meningococci cause serious diseases and can trigger life-threatening inflammatory reactions, especially in young children and immunocompromised individuals. However, vaccines for this group of patients are not effective against certain manifestations or against all variants of the pathogens. They are therefore constantly being improved. But the road to vaccine optimization is full of pitfalls and stumbling blocks. Hurdle number 1: The pathogens encapsulate themselves with a thick protective shell of branched chains of sugar molecules. These polysaccharides tend to act as weak antigens in the organism. In contrast to protein-based antigens, capsular polysaccharides can only trigger the mechanisms of innate immune response. They are not able to activate T-helper cells and thus the pathway of acquired immunity. Consequently, T cell-based stimulation of B lymphocytes and the production of pathogen-specific antibodies fail to occur. Long-term immunity will not arise. This scenario plays out both in case of infection with the pathogens and in case of vaccination with capsular polysaccharides as the only immunogenic agent.
Only a binding of capsular polysaccharides to suitable carrier proteins enables a pneumococcal vaccine to activate the T cell response, which leads to the production of different antibodies against both the carrier protein and the pathogen's capsular molecules. Such conjugated vaccines have been available on the market for about 20 years. Currently, tetanus and diphtheria toxoids are used as carrier proteins in various vaccinations - not only against pneumococci. However, this carries the risk of epitope suppression - a weakening of the specific anti-polysaccharide immune response caused by already existing anti-carrier antibodies produced by previous vaccinations against pneumococci or other pathogens.
The second major challenge is the variability of the pathogen. Pneumococci form more than 90 distinct serotypes worldwide, which differ slightly in the makeup of their capsular polysaccharides. Since the acquired immunity's memory function is not naturally activated upon infection with these pathogens, each individual serotype is treated as a new pathogen that, upon re-infection, is repeatedly attacked by innate immunity responses. For vaccines to effectively protect against multiple serotypes, they need to consist of a mixture of multiple capsular polysaccharide antigens. The latest generation of pneumococcal vaccines contains up to 20 different capsular polysaccharide antigens of the most common serotypes. Each individual antigen is coupled to its own carrier protein, which enhances epitope suppression.
One could mitigate epitope suppression by binding different capsular polysaccharides to the same carrier protein - and that is exactly what the Halle-Havana team achieved in fall 2020. In their article published in Bioconjugate Chemistry of the American Chemical Society, they report on the successful coupling of two different pneumococcal polysaccharide antigens to a single tetanus toxoid carrier protein by multicomponent reaction. The resulting bivalent glycoconjugate reliably elicited a T-cell immune response in rabbits with corresponding production of functional specific antibodies against both polysaccharide antigens. Thus, a single carrier protein was sufficient to initiate the production of antibodies against both capsular polysaccharides in the course of anti-carrier antibody production.
In their current publication in Journal of Pharmaceutical and Biomedical Analysis, the authors have now combined capsular polysaccharides from five different serotypes of Streptococcus pneumoniae and from four different Neisseria meningitidis serotypes. As a result they obtained eight different bivalent glycoconjugates containing either two different pneumococcal polysaccharides or a combination of one pneumococcal and one meningococcal antigen each. A good vaccine preparation against both pathogens would include all of the eight glycoconjugates (and probably more) - so the question arose: how to check for a balanced ratio of the individual capsular polysaccharide antigens? How can further parameters, such as the ratio of polysaccharide to carrier protein, the proportion of conjugated and non-conjugated capsular polysaccharides, or even the existence of undesired impurities be analyzed?
Conventional methods like mass spectrometry proved to be unsuitable for the analysis of these complex bivalent glycoconjugates. Quantitative NMR, on the other hand, allowed the scientists to detect characteristic signals for the individual polysaccharide types. Thus, it was possible to identify the capsular polysaccharides type and quantity in the vaccine mixture. Using HPLC and spectrophotometric methods, the chemists also calculated the amount of free carrier protein and quantified the total protein and carbohydrates. In the end, all main components of the conjugates could be qualitatively and quantitatively determined.
The ratio between the serotypic polysaccharide antigens in the vaccine mixture was balanced, so the scientists assumed an equal antibody production against all serotypes or against both pathogens after vaccination. Similarly, the ratio of capsular polysaccharides and carrier proteins was in accordance with WHO guidelines for commercial conjugate vaccines. If there was too little carrier protein, the scientists speculate, T-cell activation and general antibody production might be inadequate. With too much carrier protein, on the other hand, T cells would likely be activated well, but anti-carrier antibodies might be primarily produced, rather than the desired anti-polysaccharide antibodies.
Multicomponent reactions, the authors conclude, are proving to be a promising approach for the rapid and cost-effective development of complex multivalent vaccines with fewer conjugation and purification steps. Subsequent quality control with qNMR is ideally suited for analysis and characterization of the novel glycoconjugates.
