Novel adjuvant Alum-TLR7 significantly potentiates immune response to glycoconjugate vaccines.

Although glycoconjugate vaccines are generally very efficacious, there is still a need to improve their efficacy, especially in eliciting a strong primary antibody response. We have recently described a new type of vaccine adjuvant based on a TLR7 agonist adsorbed to alum (Alum-TLR7), which is highly efficacious at enhancing immunogenicity of protein based vaccines. Since no adjuvant has been shown to potentiate the immune response to glycoconjugate vaccines in humans, we investigated if Alum-TLR7 is able to improve immunogenicity of this class of vaccines. We found that in a mouse model Alum-TLR7 greatly improved potency of a CRM197-MenC vaccine increasing anti-MenC antibody titers and serum bactericidal activity (SBA) against MenC compared to alum adjuvanted vaccine, especially with a low dose of antigen and already after a single immunization. Alum-TLR7 also drives antibody response towards Th1 isotypes. This adjuvant was also able to increase immunogenicity of all polysaccharides of a multicomponent glycoconjugate vaccine CRM197-MenACWY. Furthermore, we found that Alum-TLR7 increases anti-polysaccharide immune response even in the presence of a prior immune response against the carrier protein. Finally, we demonstrate that Alum-TLR7 adjuvant effect requires a functional TLR7. Taken together, our data support the use of Alum-TLR7 as adjuvant for glycoconjugate vaccines.

Investigating the immunodominance of carbohydrate antigens in a bivalent unimolecular glycoconjugate vaccine against serogroup A and C meningococcal disease.

Multicomponent constructs, obtained by coupling different glycans to the carrier protein, have been proposed as a way to co-deliver multiple surface carbohydrates targeting different strains of one pathogen and reduce the number of biomolecules in the formulation of multivalent vaccines. To assess the feasibility of this approach for anti-microbial vaccines and investigate the potential immunodominance of one carbohydrate antigen over the others in these constructs, we designed a bivalent unimolecular vaccine against serogroup A (MenA) and C (MenC) meningococci, with the two different oligomers conjugated to same molecule of carrier protein (CRM197). The immune response elicited in mice by the bivalent MenAC construct was compared with the ones induced by the monovalent MenA and MenC vaccines and their combinations. After the second dose, the bivalent construct induced good levels of anti-MenA and anti-MenC antibodies with respect to the controls. However, the murine sera from the MenAC construct exhibited good anti-MenC bactericidal activity, and very low anti-MenA functionality when compared to the monovalent controls. This result was explained with the diverse relative avidities against MenA and MenC polysaccharides, which were measured in the generated sera. The immunodominant effect of the MenC antigen was fully overcome following the third immunization, when sera endowed with higher avidity and excellent bactericidal activity against both MenA and MenC expressing strains were elicited. Construction of multicomponent glycoconjugate vaccines against microbial pathogens is a feasible approach, but particular attention should be devoted to study and overcome possible occurrence of immune interference among the carbohydrates.

Immunoactivity of protein conjugates of carba analogues from Neisseria meningitidis a capsular polysaccharide.

Neisseria meningitidis type A (MenA) is a Gram-negative encapsulated bacterium that is a major cause of epidemic meningitis, especially in the sub-Saharan region of Africa. The development and manufacture of a liquid glycoconjugate vaccine against MenA are hampered by the poor hydrolytic stability of its capsular polysaccharide (CPS), consisting of (1→6)-linked 2-acetamido-2-deoxy-α-d-mannopyranosyl phosphate repeating units. The replacement of the ring oxygen with a methylene group to generate a carbocyclic analogue leads to enhancement of its chemical stability. Herein, we report conjugation of carbocyclic analogue monomer, dimer, and trimer to the protein carrier CRM197. After immunization in mice, only the conjugated trimer was able to induce specific anti-MenA polysaccharide IgG antibodies with in vitro bactericidal activity, although to a lesser extent than pentadecamer and hexamer oligomers obtained from mild acid hydrolysis of the native polysaccharide conjugated to the same protein carrier. This study represents the first proof-of-concept that hydrolytically stable structural analogues of saccharide antigens can be used for the development of efficacious antimicrobial preventative therapies. Conjugates with longer carbocyclic oligomers and/or precise acetylation patterns could further increase the induced immune response to a level comparable with those of commercially available anti-meningococcal glycoconjugate vaccines.

Immunization with the RrgB321 fusion protein protects mice against both high and low pilus-expressing Streptococcus pneumoniae populations.

RrgB321, a fusion protein of the three Streptococcus pneumoniae pilus-1 backbone RrgB variants, is protective in vivo against pilus islet 1 (PI-1) positive pneumococci. In addition, antibodies to RrgB321 mediate a complement-dependent opsonophagocytosis of PI-1 positive strains at levels comparable to those obtained with antisera against glycoconjugate vaccines. In the pneumococcus, pilus-1 displays a biphasic expression pattern, with different proportions of two bacterial phenotypes, one expressing and one not expressing the pilus-1. These two populations can be stably separated in vitro giving rise to the enriched high (H) and low (L) pilus expressing populations. In this work we demonstrate that: (i) the opsonophagocytic killing mediated in vitro by RrgB321 antisera is strictly dependent on the pilus expression ratio of the strain used; (ii) during the opsonophagocytosis assay pilus-expressing pneumococci are selectively killed, and (iii) no switch towards the pilus non-expressing phenotype can be observed. Furthermore, in sepsis and pneumonia models, mice immunized with RrgB321 are significantly protected against challenge with either the H or the L pilus-expressing population of strains representative of the three RrgB variants. This suggests that the pilus-1 expression is not down-regulated, and also that the expression of the pilus-1 could be up-regulated in vivo. In conclusion, these data provide evidence that RrgB321 is protective against PI-1 positive strains regardless of their pilus expression level, and support the rationale for the inclusion of this fusion protein into a multi-component protein-based pneumococcal vaccine.

RrgB321, a fusion protein of the three variants of the pneumococcal pilus backbone RrgB, is protective in vivo and elicits opsonic antibodies.

Streptococcus pneumoniae pilus 1 is present in 30 to 50% of invasive disease-causing strains and is composed of three subunits: the adhesin RrgA, the major backbone subunit RrgB, and the minor ancillary protein RrgC. RrgB exists in three distinct genetic variants and, when used to immunize mice, induces an immune response specific for each variant. To generate an antigen able to protect against the infection caused by all pilus-positive S. pneumoniae strains, we engineered a fusion protein containing the three RrgB variants (RrgB321). RrgB321 elicited antibodies against proteins from organisms in the three clades and protected mice against challenge with piliated pneumococcal strains. RrgB321 antisera mediated complement-dependent opsonophagocytosis of piliated strains at levels comparable to those achieved with the PCV7 glycoconjugate vaccine. These results suggest that a vaccine composed of RrgB321 has the potential to cover 30% or more of all pneumococcal strains and support the inclusion of this fusion protein in a multicomponent vaccine against S. pneumoniae.