Identification of a Novel Keto Sugar Component in Streptococcus pneumoniae Serotype 12F Capsular Polysaccharide and Impact on Vaccine Immunogenicity.

Implementation of conjugate vaccine technology revolutionized the ability to effectively elicit long-lasting immune responses to bacterial capsular polysaccharides. Although expansion of conjugate vaccine serotype coverage is designed to target residual disease burden to pneumococcal serotypes not contained in earlier vaccine versions, details of polysaccharide Ag structure, heterogeneity, and epitope structure components contributing to vaccine-mediated immunity are not always clear. Analysis of Streptococcus pneumoniae serotype 12F polysaccharide by two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry revealed a partial substitution of N-acetyl-galactosamine by the keto sugar 2-acetamido-2,6-dideoxy-xylo-hexos-4-ulose (Sug) in up to 25% of the repeat units. This substitution was not described in previous published structures for 12F. Screening a series of contemporary 12F strains isolated from humans (n = 17) identified Sug incorporation at varying levels in all strains examined. Thus, partial Sug substitution in S. pneumoniae serotype 12F may have always been present but is now detectable by state-of-the-art analytical techniques. During the steps of conjugation, the serotype 12F Sug epitope is modified by reduction, and both polysaccharide PPSV23 and conjugate PCV20 vaccines contain 12F Ags with little to no Sug epitope. Both PCV20 and PPSV23 vaccines were evaluated for protection against circulating 12F strains with varying amounts of Sug in their repeat unit based on an opsonophagocytic killing assay involving HL-60 cells and rabbit complement. Both vaccines elicited human-derived neutralizing Abs against serotype 12F, independent of Sug level between ∼2 and 25 mol%. These findings suggest that the newly identified serotype 12F Sug epitope is likely not an essential epitope for vaccine-elicited protection.

Preclinical Immunogenicity and Efficacy of Optimized O25b O-Antigen Glycoconjugates To Prevent MDR ST131 E. coli Infections.

Multivalent O-antigen polysaccharide glycoconjugate vaccines are under development to prevent invasive infections caused by pathogenic Enterobacteriaceae. Sequence type 131 (ST131) Escherichia coli of serotype O25b has emerged as the predominant lineage causing invasive multidrug-resistant extraintestinal pathogenic E. coli (ExPEC) infections. We observed the prevalence of E. coli O25b ST131 among a contemporary collection of isolates from U.S. bloodstream infections from 2013 to 2016 (n = 444) and global urinary tract infections from 2014 to 2017 (n = 102) to be 25% and 24%, respectively. To maximize immunogenicity of the serotype O25b O antigen, we investigated glycoconjugate properties, including CRM197 carrier protein cross-linking (single-end versus cross-linked "lattice") and conjugation chemistry (reductive amination chemistry in dimethyl sulfoxide [RAC/DMSO] versus ((2-((2-oxoethyl)thio)ethyl)carbamate [eTEC] linker). Using opsonophagocytic assays (OPAs) to measure serum functional antibody responses to vaccination, we observed that higher-molecular-mass O25b long-chain lattice conjugates showed improved immunogenicity in mice compared with long- or short-chain O antigens conjugated via single-end attachment. The lattice conjugates protected mice from lethal challenge with acapsular O25b ST131 strains as well as against hypervirulent O25b isolates expressing K5 or K100 capsular polysaccharides. A single 1-µg dose of long-chain O25b lattice conjugate constructed with both chemistries also elicited robust serum IgG and OPA responses in cynomolgus macaques. Our findings show that key properties of the O-antigen carrier protein conjugate such as saccharide epitope density and degree of intermolecular cross-linking can significantly enhance functional immunogenicity.

A Novel Hexavalent Capsular Polysaccharide Conjugate Vaccine (GBS6) for the Prevention of Neonatal Group B Streptococcal Infections by Maternal Immunization.

BACKGROUND: Group B streptococcus (GBS) causes serious diseases in newborn infants, often resulting in lifelong neurologic impairments or death. Prophylactic vaccination of pregnant women prior to delivery could provide comprehensive protection, as early onset and late-onset disease and maternal complications potentially could be addressed. METHODS: Capsular polysaccharide conjugate vaccine GBS6 was designed using surveillance data yielded by whole-genome sequencing of a global collection of recently recovered GBS isolates responsible for invasive neonatal GBS disease. Capsular polysaccharides were isolated, oxidized using sodium periodate, and conjugated to CRM197 by reductive amination in dimethyl sulfoxide. Immune responses in mice and rhesus macaques were measured in a multiplex Luminex immunoglobulin G (IgG) assay and opsonophagocytic activity assays. RESULTS: The optimized conjugates were immunogenic, alone and in combination, in mice and rhesus macaques, inducing IgG antibodies that mediated opsonophagocytic killing. Active immunization of murine dams with GBS6 prior to mating resulted in serotype-specific protection of pups from a lethal challenge with GBS. Protection following passive administration of serotype-specific IgG monoclonal antibodies to dams demonstrated conclusively that anticapsular polysaccharide IgG alone is sufficient for protection. CONCLUSIONS: The findings support the ongoing clinical evaluation of maternal GBS6 vaccination as a potential alternative method to prevent GBS disease in infants.

Production and Characterization of Chemically Inactivated Genetically Engineered Clostridium difficile Toxoids.

A recombinant Clostridium difficile expression system was used to produce genetically engineered toxoids A and B as immunogens for a prophylactic vaccine against C. difficile-associated disease. Although all known enzymatic activities responsible for cytotoxicity were genetically abrogated, the toxoids exhibited residual cytotoxic activity as measured in an in vitro cell-based cytotoxicity assay. The residual cytotoxicity was eliminated by treating the toxoids with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide. Mass spectrometry and amino acid analysis of the EDC-inactivated toxoids identified crosslinks, glycine adducts, and ß-alanine adducts. Surface plasmon resonance analysis demonstrated that modifications resulting from the chemical treatment did not appreciably affect recognition of epitopes by both toxin A- and B-specific neutralizing monoclonal antibodies. Compared to formaldehyde-inactivated toxoids, the EDC/N-hydroxysuccinimide-inactivated toxoids exhibited superior stability in solution with respect to reversion of cytotoxic activity.

Investigation of the biosynthesis of the pipecolate moiety of neuroprotective polyketide meridamycin.

Biogenesis of the pipecolate moiety of neuroprotective agent meridamycin in Streptomyces sp. NRRL30748 was investigated in feeding studies using lysine specifically labeled with (15)N at the α-amino or the ε-amino nitrogen position. Fourier transform mass spectrometry analysis with ultra-high mass resolving power and accurate mass measurement capability was employed to resolve the (15)N peak of labeled meridamycin from the (13)C peak of unlabeled meridamycin, allowing the precise calculation of labeling contents under each condition. The relative enrichment of (15)N-labeled meridamycin was ~43% with L-[α-(15)N]-lysine feeding and ~14% with L-[α-(15)N]-lysine feeding, suggesting two distinguishable pathways, with concomitant loss of either the ε-amino group or the α-amino group of lysine, were involved in the generation of the pipecolate moiety of meridamycin in this bacterium. PCR cloning using degenerate primers identified a proC gene encoding a putative pyrroline-5-carboxylate reductase, which was expected to catalyze the conversion of piperideine-6-carboxylate to pipecolate. However, inactivation of this locus did not significantly affect the incorporation of α-(15)N- or ε-(15)N-labeled lysine into meridamycin, indicating the existence of an alternative route for the last step of the lysine ε-transamination pathway. This work revealed the diversity and complexity of the biosynthetic pathways for pipecolate synthesis in the meridamycin producing bacterium Streptomyces sp. NRRL30748.

PWT-458, a novel pegylated-17-hydroxywortmannin, inhibits phosphatidylinositol 3-kinase signaling and suppresses growth of solid tumors.

Deregulated phosphatidylinositol 3-kinase (PI3K) signaling pathway is widely implicated in tumor growth and resistance to chemotherapy. While a strong rationale exists for pharmacological targeting of PI3K, only a few proof-of-principle in vivo efficacy studies are currently available. PWT-458, pegylated-17-hydroxywortmannin, is a novel and highly potent inhibitor of PI3K in animal models. Upon in vivo cleavage of its poly(ethyleneglycol) (PEG), PWT-458 releases its active moiety 17-hydroxywortmannin (17-HWT), the most potent inhibitor in its class. Here we show that a single intravenous injection of PWT-458 rapidly inhibited PI3K signaling, as measured by a complete loss of AKT (Ser-473) phosphorylation in xenograft tumors grown in nude mice. Following a daily X5 dosing regimen, PWT-458 demonstrated single-agent antitumor activity in nude mouse xenograft models of U87MG glioma, nonsmall cell lung cancer (NSCLC) A549, and renal cell carcinoma (RCC) A498. Efficacious doses ranged from 0.5 mg/kg to 10 mg/kg, achieving a superior therapeutic index over 17-HWT. PWT-458 augmented anticancer efficacy of a suboptimal dose of paclitaxel against A549 and U87MG tumors. Combination treatment of PWT-458 and an mTOR inhibitor, Pegylated-Rapamycin (Peg-Rapa), resulted in an enhanced antitumor efficacy in U87MG. Finally, PWT-458 in combination with interferon-alpha (Intron-A) caused a dramatic regression of RCC A498, which was not achieved by either agent alone. These studies identify PWT-458 as an effective anticancer agent and provide strong proof-of-principle for targeting the PI3K pathway as novel anticancer therapy.

FTMS structure elucidation of natural products: application to muraymycin antibiotics using ESI multi-CHEF SORI-CID FTMS(n), the top-down/bottom-up approach, and HPLC ESI capillary-skimmer CID FTMS.

The molecular formulas for the structures and substructures of muraymycin antibiotics A1 (C52H90N14O19, MW 1214) and B1 (C49H83N11O18, MW 1113) were determined using electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). The muraymycin A1 and B1 structures were elucidated by utilizing capillary-skimmer fragmentation with up to five stages of mass spectrometry (MS5). Multi-CHEF, a multiple ion isolation method, was used at each stage of MS(n) to isolate a parent ion and up to four reference ions, for exact-mass calibration. The parent ions were fragmented by SORI-CID and the product ions internally calibrated with average absolute mass errors less than 1 ppm at each stage in the fragmentation processes. Using the top-down/bottom-up approach, the molecular formulas for the antibiotics were determined by summing the elemental formulas of the neutral losses, obtained by measuring the mass differences (<500 Da) between the genetically related sequential parent ion masses in the MS(n) spectra, with the unique elemental formula of the lowest parent ion mass (<500 Da). The structures of 12 additional compounds in the muraymycin complex were elucidated using HPLC ESI capillary-skimmer CID FTMS by correlating their fragmentation patterns with those of muraymycins A1 and B1. Sequential neutral losses of an aminosugar, a valine, a uridine, and an ester fatty acid from the muraymycin parent ions provided diagnostic fragments for characterization.

Structures of the muraymycins, novel peptidoglycan biosynthesis inhibitors.

The muraymycins, a family of nucleoside-lipopeptide antibiotics, were purified from the extract of Streptomyces sp. LL-AA896. The antibiotics were purified by chromatographic methods and characterized by NMR spectroscopy, degradation studies, and mass spectrometry. The structures of 19 compounds were established. The muraymycins constitute a new antibiotic family whose core structure contains a glycosylated uronic acid derivative joined by an aminopropane group to a hexahydro-2-imino-4-pyrimidylglycyl residue (epicapreomycidine) containing dipeptide that is further extended by a urea-valine moiety. Members of this family show broad-spectrum in vitro antimicrobial activity against a variety of clinical isolates (MIC 2 to >64 mug/mL). The muraymycins inhibited peptidoglycan biosynthesis. The fatty acid substituent and the presence or absence of the amino sugar play important roles in biological activity. One of the most active compounds, muraymycin A1, demonstrated protection in vivo against Staphylococcus aureus infection in mice (ED50 1.1 mg/kg).