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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - NEWCARBOVAX (New generation of carbohydrate-based vaccines via rational understanding of their immunological mechanism)

Teaser

Summary

Vaccination has had an unparalleled impact on global health and vaccines have the greatest potential for further improvement in health in the poorest countries of the world. Glycoconjugate vaccines are among the safest and most efficacious vaccines developed during the last 30 years against infectious diseases. Following the use of Haemophilus influenzae type b (Hib), Meningococcus group C (MenC) and pneumococcal glycoconjugate vaccines in national immunization programmes, drastic reductions in serious infections in target risk groups, especially infants, children and adolescents, have been observed. Glycoconjugates 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. Glycoconjugate vaccines have provided enormous health benefits globally, but they have been less successful in some populations at high risk for developing disease. As the name suggest, 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 (molecules which function is to is to bind peptide fragments derived from pathogens and display them on the APC surface for recognition by the appropriate T cells). Application of this principle resulted in a GBSIII vaccine strongly protective in a mouse model and 50â€“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 will extend 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. Innovative strategies of conjugation chemistry will be also evaluated to generate new therapeutics with chemical properties designed in-light of specific information on antigen presentation.
The goal of the proposal is therefore to develop a vaccine platform applicable to many microbial glycans based on the most advanced discovery on the immunologic mechanisms responsible for glycoconjugate processing, presentation, and T-helper cell reactivity.

Work performed

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 foundâ€”using GBSIII capsular polysaccharide (CPS) as a modelâ€”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. Recently, we have extended our observations that T cell specifically recognizing carbohydrates (Tcarbs) were critical in the response to other conjugate vaccines. Glycoconjugates made with CPSs from Salmonella Typhi, Hib, type Ib group B Streptococcus induced T carbs, while those made with group C N. meningitidis did not. 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 presentationâ€”based on the structure of the carbohydrateâ€”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.

Final results

\"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 cellâ€“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. An increasing literature is suggesting that high-affinity antibodies may be the future in the treatment of emerging infectious diseases. We think that we may have identified an entry point for creating these highly efficacious antibodies. Our working hypothesis is that these peptide glycoconjugate vaccines 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.

In the upcoming year we will investigate deeper into our observation challenging the paradigm of a direct correlation between the amount of IgG induced by a glycoconjugate and protection. A comprehensive evaluation of antibodies and immune cells generated by protein vs peptide glycoconjugates will clarify the features that make peptide vaccines extremely potent. We will also continue working on the structural characterization of the complex glycopeptide-MHCII. Different binding conditions to be tested may include pH, salt variation and presence of H2-M. Once the binding between MHCII and the glycopeptide will be confirmed, crystallization experiments and structure determination by X-ray diffraction will be performed.
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