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New generation of carbohydrate-based vaccines via rational understanding of their immunological mechanism

Periodic Reporting for period 2 - NEWCARBOVAX (New generation of carbohydrate-based vaccines via rational understanding of their immunological mechanism)

Período documentado: 2019-01-01 hasta 2019-12-31

Glycoconjugates are among the safest and most efficacious vaccines developed against infectious diseases. They have been developed to improve the immunogenicity of polysaccharide vaccines which are T cell independent, poorly immunogenic in infants and young children < 2 years of age, do not induce immunological memory and can lead to hyporesponsiveness to repeat vaccination. Glycoconjugates have provided enormous health benefits globally, but they have been less successful in some populations at high risk for developing disease. They are composed by a sugar antigen covalently linked to a carrier protein. The traditional hypothesis of immune activation by glycoconjugate vaccines suggests that only peptides generated from glycoconjugate processing can be presented to and recognized by T cells, and this contribution is crucial for their immunogenicity. Recently, new findings offer a rational explanation for how conjugates work and may render vaccine development a more straightforward process. In contrast with the classical mechanism, this new model suggests that carbohydrate presentation to the T cell by antigen-presenting cell (APC) may strongly enhance antibody response. The key strategy is to conjugate the carbohydrate to peptides which anchor the conjugate via MHC class II (antigen-presenting molecule). Application of this principle resulted in a GBSIII vaccine strongly protective in a mouse model and 50–100 times more immunogenic than a traditional vaccine composed by random linking of the sugar on a protein carrier. Although the principle has been demonstrated much remains to be done to generally apply the concept to generate vaccines for clinical use. In the proposed study, we have extended the approach by analyzing different variables (peptide carrier, glycan chain length, conjugation chemistry and microbial antigen), with the aim of using the increased understanding of basic immunological mechanisms to develop a new translational platform for optimized and cost-effective carbohydrate-based vaccines.

Throughout NEWCARBOVAX, we have developed new-generation glycoconjugate vaccines – by a rational approach based on their immunological mechanism - and showed an improved protective capacity compared to traditional glycoconjugates. We have characterized the humoral response and identified possible correlate of the higher functional activity, and started a structural investigation of MHCII-antigen interaction. The information obtained will direct the rational design of a new generation of vaccines.
We have been developing a platform to optimize glycoconjugate vaccine design by determining optimal polysaccharide chain length and peptide conjugation sites. We learned that the use of a full-length polysaccharide and conjugation to the C-terminal end of the peptide induce optimal immunogenicity. When immunizing mice with such glycopeptide vaccines, we found—using GBSIII capsular polysaccharide (CPS) as a model—that these glycoconjugates induced full protection in the challenge model. We extended the platform to develop a prototype vaccine for Francisella tularensis, coupling the O polysaccharide (OPS) to an optimized peptide and observing very significant protection. Remarkably, protection with a glycoconjugate vaccine against F. tularensis had not been shown previously. Thus, we were greatly surprised when we measured antibodies to the GBSIII CPS and the OPS of F. tularensis and found that they were of very low titer but very highly protective. We have further characterized the humoral response induced by both glycoprotein and glycopeptide vaccines looking at subclasses, functionality, and relative affinity/avidity. Antibodie induced by glycopeptide vaccines showed higher relative affinity/avidity and functional activity. In the future, we are planning to expand the investigation on polysaccharide specific B cells at single cell transcriptional analysis level of as well as by hybridoma technology.
We have also extended our observations that T cell specifically recognizing carbohydrates (Tcarbs) were critical in the response to other conjugate vaccines (Salmonella Typhi, Hib, type Ib group B Streptococcus) but not group C N. meningitidis. In the latter case, digestion of the acid-sensitive group C N. meningitidis polysaccharide in the endosome resulted in a dominant CD4+ T-cell response to peptides in the context of MHCII presentation. Our results show that different mechanisms of presentation—based on the structure of the carbohydrate—are operative in responses to different glycoconjugate vaccines.
In addition, we are working on a structural investigation of the interaction between glycopeptides and MHCII molecules. We have produced two glycopeptides, using GBSIII CPS as carbohydrate source and generating oligosaccharides corresponding to a degree of polymerization (i.e. number of repeating units) of respectively 3 and 4, both conjugated to OVA peptide. Preliminary binding experiments were performed.
In the context of designing a new class of vaccines via rational approaches, we have developed a Francisella tularensis glycoconjugate vaccine by using a genetically enlarged O-antigen able to induce protective antibodies in mice. Also, we have contributed to the synthesis of Neisseria Meningitidis A carba analogues as hydrolytically stable antigens for anti-meningococcal glycoconjugate vaccines.

With regard to exploitation and dissemination, we are currently evaluating the possibility to protect the intellectual property of results generated by patents or similar mechanisms. The NEWCARBOVAX action has been presented in several conferences and symposiums, as well as exposed to Industrial, Academic and Start-up environments. So far, four publications in top-leading journals have been produced with reference to the action, and few more are expected to come in the next months.
"The concept of carbohydrate presentation to T cells in the context of vaccines is totally novel. In most cases, glycoconjugate vaccine construction has been a random process of linking two molecules (carbohydrate and protein) without due consideration of optimal design based on scientific principles. The innovative use of the carbohydrate as the major T helper cell–activating antigen offers an opportunity for rational chemical design of a new generation of glycoconjugate vaccines that embody optimal presentation of carbohydrate to T cells. Our recent finding that peptide-carrier glycoconjugate vaccines are highly protective even at low antibody concentrations is intriguing and potentially disruptive. We have applied this concept to two different conjugate vaccine platform (Francisellla Tularensis and GBSIII). The induction of high-efficacy antibodies has been a ""holy grail"" in the vaccine field. We think that we may have identified an entry point for creating these highly efficacious antibodies. Our hypothesis is that these peptide glycoconjugates induce antibodies with very high affinity and/or avidity and that the production of these antibodies corresponds to improved functionality and an enhanced ability to confer protection. The information obtained will direct the rational design of a new generation of vaccines. Since carbohydrates decorate the surface of nearly all bacterial cells, the outcome of NEWCARBOVAX action has the potential to create vaccines against bacterial species that are so far intractable to vaccine intervention."