Expanding the Scope of Ugi Multicomponent Bioconjugation to Produce Pneumococcal Multivalent Glycoconjugates as Vaccine Candidates.

Conjugate vaccines against encapsulated pathogens like Streptococcus pneumoniae face many challenges, including the existence of multiple serotypes with a diverse global distribution that constantly requires new formulations and higher coverage. Multivalency is usually achieved by combining capsular polysaccharide-protein conjugates from invasive serotypes, and for S. pneumoniae, this has evolved from 7- up to 20-valent vaccines. These glycoconjugate formulations often contain high concentrations of carrier proteins, which may negatively affect glycoconjugate immune response. This work broadens the scope of an efficient multicomponent strategy, leading to multivalent pneumococcal glycoconjugates assembled in a single synthetic operation. The bioconjugation method, based on the Ugi four-component reaction, enables the one-pot incorporation of two different polysaccharide antigens to a tetanus toxoid carrier, thus representing the fastest approach to achieve multivalency. The reported glycoconjugates incorporate three combinations of capsular polysaccharides 1, 6B, 14, and 18C from S. pneumoniae. The glycoconjugates were able to elicit functional specific antibodies against pneumococcal strains comparable to those shown by mixtures of the two monovalent glycoconjugates.

Synthesis of α-Glucosyl Diacylglycerides as potential adjuvants for Streptococcus pneumoniae vaccines.

α-Glucosyl diacylglycerols (αGlc-DAGs) play an important role in providing protective immunity against Streptococcus pneumoniae infection through the engagement of the Macrophage inducible C-type lectin (Mincle). Herein, we efficiently synthesised αGlc-DAGs containing C12, C14, C16 and C18 acyl chains in 7 steps and 44-47% overall yields, and demonstrated that Mincle signaling was dependent on lipid length using mMincle and hMincle NFAT-GFP reporter cells. The greatest production of GFP in both cell types was elicited by C14 αGlc-DAG. Accordingly, C14 αGlc-DAG has potential to act as an adjuvant to augment the immune response against S. pneumoniae antigens.

Immunogenicity differences of a 15-valent pneumococcal polysaccharide conjugate vaccine (PCV15) based on vaccine dose, route of immunization and mouse strain.

Pneumococcal disease continues to be a medical need even with very effective vaccines on the market. Globally, there are extensive research efforts to improve serotype coverage with novel vaccines; therefore, conducting preclinical studies in different animal models becomes essential. The work presented herein focuses on evaluating a 15-valent pneumococcal conjugate vaccine (PCV15) in mice. Initially we evaluated several doses of PCV15 in Balb/c mice. The optimal vaccine dose was determined to be 0.4µg per pneumococcal polysaccharide (PS) (0.8µg of 6B) for subsequent studies. This PS dose was chosen for PCV evaluation in mice based on antibody levels determined by multiplexed electrochemiluminescent (ECL) assays, T-cell responses following in vitro stimulation with CRM197 peptides and protection from pneumococcal challenge. We then selected four mouse strains for evaluation: Balb/c, C3H/HeN, CD1 and Swiss Webster (SW), immunized with PCV15 by either intraperitoneal (IP) or intramuscular (IM) routes. We assessed IgG responses by ECL assays and functional antibody activity by multiplexed opsonophagocytic assays (MOPA). Every mouse strain evaluated responded to all 15 serotypes contained in the vaccine. Mice tended to have lower responses to serotypes 6B, 23F and 33F. The IP route of immunization resulted in higher antibody titers for most serotypes in Balb/c, C3H and SW. CD1 mice tended to respond similarly for most serotypes, regardless of route of immunization. Similar trends were observed with the four mouse strains when evaluating functional antibody activity. Given the differences in antibody responses based on mouse strain and route of immunization, it is critical to evaluate pneumococcal vaccines in multiple animal models to determine the optimal formulation before moving to clinical trials.

A bivalent conjugate vaccine containing PspA families 1 and 2 has the potential to protect against a wide range of Streptococcus pneumoniae strains and Salmonella Typhi.

Previously we showed that conjugation of pneumococcal surface protein A (PspA) to Vi capsular polysaccharide from Salmonella Typhi enhanced the anti-PspA response without the need to add adjuvant. In the current study conjugates consisting of the α helical regions of PspA families 1 or 2 bound to Vi were used to vaccinate mice to test their ability to protect against a lethal intravenous challenge of a range of various strains of Streptococcus pneumoniae. Conjugate vaccine containing PspA family 1 provided good protection from PspA family 1 challenge strains but offered very little protection against PspA family 2 challenge strains. Similarly, PspA family 2 conjugates provided good protection from PspA family 2 challenge strains and poor protection against PspA family 1 challenge strains. This observation was supported by the low levels of cross-reactivity of PspA antibodies seen in ELISA plates coated with the heterologous PspA family. Cytokine profiles showed a mixed Th1/Th2 response to Vi and the Vi-PspA conjugates. IgG subclass analysis of the anti-Vi response showed a shift from predominantly IgG2a/3 to IgG1 after conjugation to PspA was consistent with other polysaccharide conjugate vaccines. The results demonstrate that conjugation of the α helical region of PspA to Vi enhances its capacity to induce a protective immune response and that a vaccine based on the α helical region of PspA should contain PspA from both families 1 and 2 to achieve broad cross-protection.

A semisynthetic carbohydrate-lipid vaccine that protects against S. pneumoniae in mice.

Severe forms of pneumococcal meningitis, bacteraemia and pneumonia result in more than 1 million deaths each year despite the widespread introduction of carbohydrate-protein conjugate vaccines against Streptococcus pneumoniae. Here we describe a new and highly efficient antipneumococcal vaccine design based on synthetic conjugation of S. pneumoniae capsule polysaccharides to the potent lipid antigen α-galactosylceramide, which stimulates invariant natural killer T (iNKT) cells when presented by the nonpolymorphic antigen-presenting molecule CD1d. Mice injected with the new lipid-carbohydrate conjugate vaccine produced high-affinity IgG antibodies specific for pneumococcal polysaccharides. Vaccination stimulated germinal center formation; accumulation of iNKT cells with a T follicular helper cell phenotype; and increased frequency of carbohydrate-specific, long-lived memory B cells and plasmablasts. This new lipid-carbohydrate vaccination strategy induced potent antipolysaccharide immunity that protected against pneumococcal disease in mice and may also prove effective for the design of carbohydrate-based vaccines against other major bacterial pathogens.

Identification of serotype in culture negative pneumococcal meningitis using sequential multiplex PCR: implication for surveillance and vaccine design.

BACKGROUND: PCR-based serotyping of Streptococcus pneumoniae has been proposed as a simpler approach than conventional methods, but has not been applied to strains in Asia where serotypes are diverse and different from other part of the world. Furthermore, PCR has not been used to determine serotype distribution in culture-negative meningitis cases. METHODOLOGY: Thirty six serotype-specific primers, 7 newly designed and 29 previously published, were arranged in 7 multiplex PCR sets, each in new hierarchies designed for overall serotype distribution in Bangladesh, and specifically for meningitis and non-meningitis isolates. Culture-negative CSF specimens were then tested directly for serotype-specific sequences using the meningitis-specific set of primers. PCR-based serotyping of 367 strains of 56 known serotypes showed 100% concordance with quellung reaction test. The first 7 multiplex reactions revealed the serotype of 40% of all, and 31% and 48% non-meningitis and meningitis isolates, respectively. By redesigning the multiplex scheme specifically for non-meningitis or meningitis, the quellung reaction of 43% and 48% of respective isolates could be identified. Direct examination of 127 culture-negative CSF specimens, using the meningitis-specific set of primers, yielded serotype for 51 additional cases. CONCLUSIONS: This PCR approach, could improve ascertainment of pneumococcal serotype distributions, especially for meningitis in settings with high prior use of antibiotics.

Synthesis of Streptococcus pneumoniae type 3 neoglycoproteins varying in oligosaccharide chain length, loading and carrier protein.

The preparation is described of a range of neoglycoproteins containing synthesised fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3, that is beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->O)-(CH2)3NH2 (1), beta-D-Glcp-(1-->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->O)-(CH2)3NH2 (2), and beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->O)-(CH4)NH2 (3). A blockwise approach was developed for the synthesis of the protected carbohydrate chains, in which the carboxylic groups were introduced prior to deprotection by selective oxidation of HO-6 in the presence of HO-4 by using TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy radical). After deprotection, the 3-aminopropyl spacer of the fragments was elongated with diethyl squarate (3,4-diethoxy-3-cyclobutene-1,2-dione) and the elongated oligosaccharides were conjugated to CRM197 (cross-reacting material of diphtheria toxin), KLH (keyhole limpet hemocyanin) or TT (tetanus toxoid). The resulting neoglycoconjugates varied in oligosaccharide chain length, oligosaccharide loading and protein carrier. These well-defined conjugates are ideal probes for evaluating the influence of the different structural parameters in immunological tests.