Chemical Synthesis of Truncated Capsular Oligosaccharide of Serotypes 6C and 6D of Streptococcus pneumoniae with Their Immunological Studies.

Serotypes 6C and 6D of Streptococcus pneumoniae are two major variants that cause invasive pneumococcal disease (IPD) in serogroup 6 alongside serotypes 6A and 6B. Since the introduction of the pneumococcal conjugate vaccines PCV7 and PCV13, the number of cases of IPD caused by pneumococcus in children and the elderly population has greatly decreased. However, with the widespread use of vaccines, a replacement effect has recently been observed among different serotypes and lowered the effectiveness of the vaccines. To investigate protection against the original serotypes and to explore protection against variants and replacement serotypes, we created a library of oligosaccharide fragments derived from the repeating units of the capsular polysaccharides of serotypes 6A, 6B, 6C, and 6D through chemical synthesis. The library includes nine pseudosaccharides with or without exposed terminal phosphate groups and four pseudotetrasaccharides bridged by phosphate groups. Six carbohydrate antigens related to 6C and 6D were prepared as glycoprotein vaccines for immunogenicity studies. Two 6A and two 6B glycoconjugate vaccines from previous studies were included in immunogenicity studies. We found that the conjugates containing four phosphate-bridged pseudotetrasaccharides were able to induce good immune antibodies and cross-immunogenicity by showing superior activity and broad cross-protective activity in OPKA bactericidal experiments.

Acute traumatic coma awakening by right median nerve electrical stimulation: a randomised controlled trial.

PURPOSE: Severe traumatic brain injury (TBI) leads to acute coma and may result in prolonged disorder of consciousness (pDOC). We aimed to determine whether right median nerve electrical stimulation is a safe and effective treatment for accelerating emergence from coma after TBI. METHODS: This randomised controlled trial was performed in 22 centres in China. Participants with acute coma at 7-14 days after TBI were randomly assigned (1:1) to either routine therapy and right median nerve electrical stimulation (RMNS group) or routine treatment (control group). The RMNS group received 20 mA, 300 µs, 40 Hz stimulation pulses, lasting 20 s per minutes, 8 h per day, for 2 weeks. The primary outcome was the proportion of patients who regained consciousness 6 months post-injury. The secondary endpoints were Glasgow Coma Scale (GCS), Full Outline of Unresponsiveness scale (FOUR), Coma Recovery Scale-Revised (CRS-R), Disability Rating Scale (DRS) and Glasgow Outcome Scale Extended (GOSE) scores reported as medians on day 28, 3 months and 6 months after injury, and GCS and FOUR scores on day 1 and day 7 during stimulation. Primary analyses were based on the intention-to-treat set. RESULTS: Between March 26, 2016, and October 18, 2020, 329 participants were recruited, of whom 167 were randomised to the RMNS group and 162 to the control group. At 6 months post-injury, a higher proportion of patients in the RMNS group regained consciousness compared with the control group (72.5%, n = 121, 95% confidence interval (CI) 65.2-78.7% vs. 56.8%, n = 92, 95% CI 49.1-64.2%, p = 0.004). GOSE at 3 months and 6 months (5 [interquartile range (IQR) 3-7] vs. 4 [IQR 2-6], p = 0.002; 6 [IQR 3-7] vs. 4 [IQR 2-7], p = 0.0005) and FOUR at 28 days (15 [IQR 13-16] vs. 13 [interquartile range (IQR) 11-16], p = 0.002) were significantly increased in the RMNS group compared with the control group. Trajectory analysis showed that significantly more patients in the RMNS group had faster GCS, CRS-R and DRS improvement (p = 0.01, 0.004 and 0.04, respectively). Adverse events were similar in both groups. No serious adverse events were associated with the stimulation device. CONCLUSION: Right median nerve electrical stimulation is a possible effective treatment for patients with acute traumatic coma, that will require validation in a confirmatory trial.

Mechanism of Antigen Presentation and Specificity of Antibody Cross-Reactivity Elicited by an Oligosaccharide-Conjugate Cancer Vaccine.

Polysaccharides have been successfully used as immunogens for the development of vaccines against bacterial infection; however, there are no oligosaccharide-based vaccines available to date and no previous studies of their processing and presentation. We reported here the intracellular enzymatic processing and antigen presentation of an oligosaccharide-conjugate cancer vaccine prepared from the glycan of Globo-H (GH), a globo-series glycosphingolipid (GSL). This oligosaccharide-conjugate vaccine was shown to elicit antibodies against the glycan moieties of all three globo-series GSLs that are exclusively expressed on many types of cancer and their stem cells. To understand the specificity and origin of cross-reactivity of the antibodies elicited by the vaccine, we found that the vaccine is first processed by fucosidase 1 in the early endosome of dendritic cells to generate a common glycan antigen of the GSLs along with GH for MHC class II presentation. This work represents the first study of oligosaccharide processing and presentation and is expected to facilitate the design and development of glycoconjugate vaccines based on oligosaccharide antigens.

Ganglioside-focused Glycan Array Reveals Abnormal Anti-GD1b Auto-antibody in Plasma of Preclinical Huntington's Disease.

Huntington's disease (HD) is a progressive and devastating neurodegenerative disease marked by inheritable CAG nucleotide expansion. For offspring of HD patients carrying abnormal CAG expansion, biomarkers that predict disease onset are crucially important but still lacking. Alteration of brain ganglioside patterns has been observed in the pathology of patients carrying HD. Here, by using a novel and sensitive ganglioside-focused glycan array, we examined the potential of anti-glycan auto-antibodies for HD. In this study, we collected plasma from 97 participants including 42 control (NC), 16 pre-manifest HD (pre-HD), and 39 HD cases and measured the anti-glycan auto-antibodies by a novel ganglioside-focused glycan array. The association between plasma anti-glycan auto-antibodies and disease progression was analyzed using univariate and multivariate logistic regression. The disease-predictive capacity of anti-glycan auto-antibodies was further investigated by receiver operating characteristic (ROC) analysis. We found that anti-glycan auto-antibodies were generally higher in the pre-HD group when compared to the NC and HD groups. Specifically, anti-GD1b auto-antibody demonstrated the potential for distinguishing between pre-HD and control groups. Moreover, in combination with age and the number of CAG repeat, the level of anti-GD1b antibody showed excellent predictability with an area under the ROC curve (AUC) of 0.95 to discriminate between pre-HD carriers and HD patients. With glycan array technology, this study demonstrated abnormal auto-antibody responses that showed temporal changes from pre-HD to HD.

Development of Klebsiella pneumoniae Capsule Polysaccharide-Conjugated Vaccine Candidates Using Phage Depolymerases.

Klebsiella pneumoniae is an important pathogen associated with nosocomial infection and has developed increasing resistance to antibiotics such as extended-spectrum ß-lactams and carbapenem. In recent years, K. pneumoniae isolates have emerged as a major cause of global community-acquired infections such as pneumonia and pyogenic liver abscess. Although serotypes K1 and K2 have been identified as the predominant capsular types associated with invasive infections, no K. pneumoniae vaccine is commercially available, probably due to immunogenicity loss in the traditional depolymerization method to obtain capsule polysaccharide (CPS) for the preparation of conjugated vaccine. In this study, we successfully retained immunogenicity by using K1 (K1-ORF34) and K2 (K2-ORF16) CPS depolymerases that were identified from phages to cleave K1 and K2 CPSs into intact structural units of oligosaccharides with intact modifications. The obtained K1 and K2 oligosaccharides were separately conjugated with CRM197 carrier protein to generate CPS-conjugated vaccines. Immunization experiments of mice showed both K1 and K2 CPS-conjugated vaccines induced anti-CPS antibodies with 128-fold and 64-fold increases of bactericidal activities, respectively, compare to mice without vaccinations. Challenge tests indicated that K1 or K2 CPS-conjugated vaccine and divalent vaccine (a mixture of K1 and K2 CPS-conjugated vaccines) protected mice from subsequent infection of K. pneumoniae by the respective capsular type. Thus, we demonstrated K1 and K2 CPS-conjugated vaccines prepared by CPS depolymerases is a promising candidate for developing vaccines against human K. pneumoniae infections.

Synthesis of Azido-Globo H Analogs for Immunogenicity Evaluation.

Globo H (GH) is a tumor-associated carbohydrate antigen (TACA), and GH conjugations have been evaluated as potential cancer vaccines. However, like all carbohydrate-based vaccines, low immunogenicity is a major issue. Modifications of the TACA increase its immunogenicity, but the systemic modification on GH is challenging and the synthesis is cumbersome. In this study, we synthesized several azido-GH analogs for evaluation, using galactose oxidase to selectively oxidize C6-OH of the terminal galactose or N-acetylgalactosamine on lactose, Gb3, Gb4, and SSEA3 into C6 aldehyde, which was then transformed chemically to the azido group. The azido-derivatives were further glycosylated to azido-GH analogs by glycosyltransferases coupled with sugar nucleotide regeneration. These azido-GH analogs and native GH were conjugated to diphtheria toxoid cross-reactive material CRM197 for vaccination with C34 adjuvant in mice. Glycan array analysis of antisera indicated that the azido-GH glycoconjugate with azide at Gal-C6 of Lac (1-CRM197) elicited the highest antibody response not only to GH, SSEA3, and SSEA4, which share the common SSEA3 epitope, but also to MCF-7 cancer cells, which express these Globo-series glycans.

Synthetic Library of Oligosaccharides Derived from the Capsular Polysaccharide of Streptococcus pneumoniae Serotypes 6A and 6B and Their Immunological Studies.

Streptococcus pneumoniae serotypes 6A and 6B are two of the common causes of invasive pneumococcal diseases. Although capsular polysaccharide conjugates of these two serotypes are included in the leading 13-valent pneumococcal conjugate vaccine, its low immunogenicity and high threshold for manufacturing technology indicated the need for vaccine improvement. Structurally defined synthetic immunogens have potential in dealing with these problems. To this end, we built a library of capsular polysaccharide fragments through convergent chemical synthesis in [2 + 2], [4 + 4], [4 + 3], [4 + 2], and [4 + 1] coupling manners. The library is comprised of 18 glycan antigens from trisaccharides to pseudo-octasaccharides, derived from the capsular repeating phosphorylated pseudo-tetrasaccharide with or without phosphate. Eight of them were selected for mouse immunization and further immunological studies. Four pseudo-tetrasaccharides with terminal or bridging phosphate elicited opsonic antibodies, which exhibited bactericidal activities and moderate cross-reactivities.

Immunogenicity Evaluation of N-Glycans Recognized by HIV Broadly Neutralizing Antibodies.

While the improved treatment of human immunodeficiency virus type 1 (HIV-1) infection is available, the development of an effective and safe prophylactic vaccine against HIV-1 is still an unrealized goal. Encouragingly, the discovery of broadly neutralizing antibodies (bNAbs) from HIV-1 positive patients that are capable of neutralizing a broad spectrum of HIV-1 isolates of various clades has accelerated the progress of vaccine development in the past few years. Some of these bNAbs recognize the N-glycans on the viral surface gp120 glycoprotein. We have been interested in using the glycan epitopes recognized by bNAbs for the development of vaccines to elicit bNAb-like antibodies with broadly neutralizing activities. Toward this goal, we have identified novel hybrid-type structures with subnanomolar avidity toward several bNAbs including PG16, PGT121, PGT128-3C, 2G12, VRC13, VRC-PG05, VRC26.25, VRC26.09, PGDM1400, 35O22, and 10-1074. Here, we report the immunogenicity evaluation of a novel hybrid glycan conjugated to carrier DTCRM197, a nontoxic mutant of the diphtheria toxin, for immunization in mice. Our results indicated that the IgG response was mainly against the chitobiose motif with nonspecific binding to a panel of N-glycans with reducing end GlcNAc-GlcNAc (chitobiose) printed on the glass slides. However, the IgM response was mainly toward the reducing end GlcNAc moiety. We further used the glycoconjugates of Man3GlcNAc2, Man5GlcNAc2, and Man9GlcNAc2 glycans for immunization, and a similar specificity pattern was observed. These findings suggest that the immunogenicity of chitobiose may interfere with the outcome of N-glycan-based vaccines, and modification may be necessary to increase the immunogenicity of the entire N-glycan epitope.

A Synthetic Carbohydrate-Protein Conjugate Vaccine Candidate against Klebsiella pneumoniae Serotype K2.

Klebsiella pneumoniae causes pneumonia and liver abscesses in humans worldwide and contains virulence factor capsular polysaccharides and lipopolysaccharides linked to the cell wall. Although capsular polysaccharides are good antigens for vaccine production and capsular oligosaccharides conjugate vaccines are proven effective against infections caused by encapsulated pathogens, there is still no Klebsiella pneumoniae vaccine available. One obstacle is that the capsular polysaccharide of a dominated Klebsiella pneumoniae serotype K2 is difficult to synthesize chemically due to the three 1,2-cis linkages in its structure. In this study, we successfully synthesized K2 capsular polysaccharides from tetra- to octasaccharides in highly a stereoselective manner. Subsequently, three synthesized glycans were conjugated to DT protein to provide glycoconjugate vaccine candidates (DT-Hexa, DT-Hepta, and DT-Octa) that were used in in vivo immunization experiments in mice. The results of immunized studies showed all three glycoconjugates elicited antibodies that recognized all of the synthetic glycans at 1:200-fold dilution. Particularly, the DT-Hepta conjugate elicited a higher level of antibodies that can recognize longer glycan (octasaccharide) even at 1:12800-fold dilution and exhibited good bactericidal activity. Our results concluded that heptasaccharide is the minimal epitope and a potential candidate for the vaccine against the K2 sero group of Klebsiella pneumoniae.

Synthesis of Asymmetric N-Glycans as Common Core Substrates for Structural Diversification through Selective Enzymatic Glycosylation.

N-glycans on the cell surface provide distinct signatures that are recognized by different glycan-binding proteins (GBPs) and pathogens. Most glycans in humans are asymmetric and isomeric, yet their biological functions are not well understood due to their lack of availability for studies. In this work, we have developed an improved strategy for asymmetric N-glycan assembly and diversification using designed common core substrates prepared chemically for selective enzymatic fucosylation and sialylation. The resulting 26 well-defined glycans that carry the sialic acid residue on different antennae were used in a microarray as a representative application to profile the binding specificity of hemagglutinin (HA) from the avian influenza virus (H5N2). We found distinct binding affinity for the Neu5Ac-Gal epitope linked to the N-acetylglucosamine (GlcNAc) of different branches and only a minor effect in binding for the terminal galactose on different branches. Overall, the microarray analysis showed branch-biased and context-based recognition patterns.

Unprecedented Role of Hybrid N-Glycans as Ligands for HIV-1 Broadly Neutralizing Antibodies.

The development of an HIV vaccine has been hampered by the extraordinary mutability and genetic diversity of the virus, particularly the substantial sequence diversity of gp120 and gp 41 envelope glycoproteins existing in more than 2000 HIV variants. The highly diverse glycans on HIV spikes are commonly considered as immunologically silent self-antigens; however, the discovery of highly potent broadly neutralizing antibodies (bNAbs) from HIV patients targeting the viral surface glycans has raised a major question about the origin of their antigens. Recent epitope mapping studies of the bNAb PG9 indicated a requirement of a properly spaced high mannose and a complex type glycan connected by a short peptide spacer. We have recently discovered that a 1:1 mixture of Man5 and sialyl biantennary glycan with well-defined distance and without the peptide spacer is well recognized by PG9 with high avidity and, thus, proposed that a hybrid glycan with oligomannose and complex-type arm could be the proper ligand of PG9. To verify this proposition, we first designed and chemo-enzymatically synthesized a series of unusual hybrid-type N-glycan structures, which may exist on HIV surface glycoproteins through the host-guided N-glycosylation pathway. The synthetic hybrid glycans were then used to prepare glycan arrays for the binding studies of PG9 and several other highly potent bNAbs, including PG16, PGT121, PGT128-3C, 2G12, VRC13, VRC-PG05, VRC26.25, VRC26.09, PGDM1400, 35O22, and 10-1074. Our results demonstrated that PG9 and some other bNAbs bind with strong avidity (subnanomolar Kd) to certain hybrid structures, suggesting that these unusual glycans may serve as epitopes for the design of vaccines against HIV.

Modular synthesis of N-glycans and arrays for the hetero-ligand binding analysis of HIV antibodies.

A new class of broadly neutralizing antibodies (bNAbs) from HIV donors has been reported to target the glycans on gp120--a glycoprotein found on the surface of the virus envelope--thus renewing hope of developing carbohydrate-based HIV vaccines. However, the version of gp120 used in previous studies was not from human T cells and so the glycosylation pattern could be somewhat different to that found in the native system. Moreover, some antibodies recognized two different glycans simultaneously and this cannot be detected with the commonly used glycan microarrays on glass slides. Here, we have developed a glycan microarray on an aluminium-oxide-coated glass slide containing a diverse set of glycans, including homo- and mixed N-glycans (high-mannose, hybrid and complex types) that were prepared by modular chemo-enzymatic methods to detect the presence of hetero-glycan binding behaviours. This new approach allows rapid screening and identification of optimal glycans recognized by neutralizing antibodies, and could speed up the development of HIV-1 vaccines targeting cell surface glycans.

Immunogenicity study of Globo H analogues with modification at the reducing or nonreducing end of the tumor antigen.

Globo H-based therapeutic cancer vaccines have been tested in clinical trials for the treatment of late stage breast, ovarian, and prostate cancers. In this study, we explored Globo H analogue antigens with an attempt to enhance the antigenic properties in vaccine design. The Globo H analogues with modification at the reducing or nonreducing end were synthesized using chemoenzymatic methods, and these modified Globo H antigens were then conjugated with the carrier protein diphtheria toxoid cross-reactive material (CRM) 197 (DT), and combined with a glycolipid C34 as an adjuvant designed to induce a class switch to form the vaccine candidates. After Balb/c mice injection, the immune response was studied by a glycan array and the results showed that modification at the C-6 position of reducing end glucose of Globo H with the fluoro, azido, or phenyl group elicited IgG antibody response to specifically recognize Globo H (GH) and the GH-related epitopes, stage-specific embryonic antigen 3 (SSEA3) (also called Gb5) and stage-specific embryonic antigen 4 (SSEA4). However, only the modification of Globo H with the azido group at the C-6 position of the nonreducing end fucose could elicit a strong IgG immune response. Moreover, the antibodies induced by these vaccines were shown to recognize GH expressing tumor cells (MCF-7) and mediate the complement-dependent cell cytotoxicity against tumor cells. Our data suggest a new potential approach to cancer vaccine development.

Immunization of fucose-containing polysaccharides from Reishi mushroom induces antibodies to tumor-associated Globo H-series epitopes.

Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galß1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, D-galactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent d-mannose and d-xylose, respectively), underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycans.