Vaccination with SARS-CoV-2 spike protein lacking glycan shields elicits enhanced protective responses in animal models.

A major challenge to end the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is to develop a broadly protective vaccine that elicits long-term immunity. As the key immunogen, the viral surface spike (S) protein is frequently mutated, and conserved epitopes are shielded by glycans. Here, we revealed that S protein glycosylation has site-differential effects on viral infectivity. We found that S protein generated by lung epithelial cells has glycoforms associated with increased infectivity. Compared to the fully glycosylated S protein, immunization of S protein with N-glycans trimmed to the mono-GlcNAc-decorated state (SMG) elicited stronger immune responses and better protection for human angiotensin-converting enzyme 2 (hACE2) transgenic mice against variants of concern (VOCs). In addition, a broadly neutralizing monoclonal antibody was identified from SMG-immunized mice that could neutralize wild-type SARS-CoV-2 and VOCs with subpicomolar potency. Together, these results demonstrate that removal of glycan shields to better expose the conserved sequences has the potential to be an effective and simple approach for developing a broadly protective SARS-CoV-2 vaccine.

Alteration of Gut Microbial Metabolites in the Systemic Circulation of Patients with Parkinson's Disease.

BACKGROUND: Emerging evidence suggests that gut dysbiosis contributes to Parkinson's disease (PD) by signaling through microbial metabolites. Hippuric acid (HA), indole derivatives, and secondary bile acids are among the most common gut metabolites. OBJECTIVE: To examine the relationship of systemic concentrations of these microbial metabolites associated with changes of gut microbiota, PD status, and severity of PD. METHODS: We enrolled 56 patients with PD and 43 age- and sex-matched healthy participants. Motor and cognitive severity were assessed with Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) part III motor score and the Mini-Mental State Examination (MMSE), respectively. Plasma concentrations of targeted gut metabolites were measured with liquid chromatography-tandem mass spectrometry. Gut microbiota was analyzed with shotgun metagenomic sequencing. RESULTS: Compared with controls, PD patients had significantly higher plasma levels of HA, indole-3-propionic acid (IPA), deoxycholic acid (DCA), and glycodeoxycholic acid (GDCA). After adjustment for age and sex in a multivariate logistic regression analysis, plasma levels of HA (odds ratio [OR] 3.21, p < 0.001), IPA (OR 2.59, p = 0.031), and GDCA (OR 2.82, p = 0.036) were associated with positive PD status. Concentrations of these gut metabolites did not correlate with MDS-UPDRS part III score or MMSE after adjustment for confounders. Microbial metabolite levels were associated with the relative abundance of pro-inflammatory gut bacteria. CONCLUSION: Aberrant gut microbial metabolites of HA, indole derivatives and secondary bile acids associated with specific gut microbiota changes were observed in patients with PD.

Association of Fecal and Plasma Levels of Short-Chain Fatty Acids With Gut Microbiota and Clinical Severity in Patients With Parkinson Disease.

BACKGROUND AND OBJECTIVES: Short-chain fatty acids (SCFAs) are gut microbial metabolites that promote the disease process in a rodent model of Parkinson disease (PD), but fecal levels of SCFAs in patients with PD are reduced. Simultaneous assessments of fecal and plasma SCFA levels, and their interrelationships with the PD disease process, are scarce. We aimed to compare fecal and plasma levels of different SCFA subtypes in patients with PD and healthy controls to delineate their interrelations and link to gut microbiota changes and clinical severity of PD. METHODS: A cohort of 96 patients with PD and 85 controls were recruited from National Taiwan University Hospital. Fecal and plasma concentrations of SCFAs were measured using chromatography and mass spectrometry. Gut microbiota was analyzed using metagenomic shotgun sequencing. Body mass index and medical comorbidities were evaluated and dietary information was obtained using a food frequency questionnaire. To assess motor and cognitive impairment, we used the Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) and the Mini-Mental Status Examination (MMSE). RESULTS: Compared with controls, patients with PD had lower fecal but higher plasma concentrations of acetate, propionate, and butyrate. After adjustment for age, sex, disease duration, and anti-PD medication dosage, MDS-UPDRS part III motor scores correlated with reduced fecal levels of acetate (ρ = -0.37, p = 0.012), propionate (ρ = -0.32, p = 0.036), and butyrate (ρ = -0.40, p = 0.004) and with increased plasma propionate concentrations (ρ = 0.26, p = 0.042) in patients with PD. MMSE scores negatively correlated with plasma levels of butyrate (ρ = -0.09, p = 0.027) and valerate (ρ = -0.032, p = 0.033) after adjustment for confounders. SCFAs-producing gut bacteria correlated positively with fecal levels of SCFAs in healthy controls but revealed no association in patients with PD. In the PD patient group, the abundance of proinflammatory microbes, such as Clostridiales bacterium NK3B98 and Ruminococcus sp AM07-15, significantly correlated with decreased fecal levels and increased plasma levels of SCFAs, especially propionic acid. DISCUSSION: Reductions in fecal SCFAs but increased plasma SCFAs were observed in patients with PD and corelated to specific gut microbiota changes and the clinical severity of PD. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that gut metabolite SCFAs distinguish between patients with PD and controls and are associated with disease severity in patients with PD.

A non-neutralizing antibody broadly protects against influenza virus infection by engaging effector cells.

Hemagglutinin (HA) is the immunodominant protein of the influenza virus. We previously showed that mice injected with a monoglycosylated influenza A HA (HAmg) produced cross-strain-reactive antibodies and were better protected than mice injected with a fully glycosylated HA (HAfg) during lethal dose challenge. We employed a single B-cell screening platform to isolate the cross-protective monoclonal antibody (mAb) 651 from mice immunized with the HAmg of A/Brisbane/59/2007 (H1N1) influenza virus (Bris/07). The mAb 651 recognized the head domain of a broad spectrum of HAs from groups 1 and 2 influenza A viruses and offered prophylactic and therapeutic efficacy against A/California/07/2009 (H1N1) (Cal/09) and Bris/07 infections in mice. The antibody did not possess neutralizing activity; however, antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis mediated by natural killer cells and alveolar macrophages were important in the protective efficacy of mAb 651. Together, this study highlighted the significance of effector functions for non-neutralizing antibodies to exhibit protection against influenza virus infection.

Development of an Efficient and Sensitive Chemical Derivatization-Based LC-MS/MS Method for Quantifying Gut Microbiota-Derived Metabolites in Human Plasma and Its Application in Studying Cardiovascular Disease.

Recently, the gut microbiota has been found to be associated with many diseases, such as inflammatory bowel disease, depression, Parkinson's disease, cancer, metabolic syndrome, and cardiovascular disease (CVD). Among various gut microbiota-derived metabolites (GMs), short-chain fatty acids (SCFAs), bile acids (BAs), and tryptophan (TRP) metabolites are the most frequently discussed metabolites. LC-MS/MS shows advantages in quantifying the levels of metabolites with good sensitivity and selectivity; however, the poor ionization efficiency and polar characteristics of SCFAs make their analysis challenging, especially when analyzing plasma samples with low SCFA concentrations. Moreover, without characteristic fragment ions for unconjugated BAs and different detection ion modes for TRP metabolites and BAs, GM analysis is complex and time-consuming. To overcome these problems, we developed a derivatization method combined with LC-MS/MS to enhance the sensitivity and LC retention of GMs. Through derivatization with 3-nitrophenylhydrazine (3-NPH), 7 SCFAs, 9 bile acids, and 6 tryptophan metabolites can be simultaneously analyzed via separation within 14 min on a reversed-phase C18 column. For accurate quantification, 13C6-3NPH-labeled standards were used as one-to-one internal standards. This derivatization approach was optimized and then validated. We further applied this method to investigate the targeted GM profile in patients with CVD. The results showed a significant reduction in plasma butyrate levels in CVD patients compared with healthy controls, suggesting its potentially protective role in CVD. In summary, this work provides a sensitive and effective LC-MS/MS method for simultaneously quantifying gut microbiota-related metabolites in human plasma, which could benefit various future gut microbiota-related studies.

Chimeric hemagglutinin vaccine elicits broadly protective CD4 and CD8 T cell responses against multiple influenza strains and subtypes.

Vaccination has been used to control the spread of seasonal flu; however, the virus continues to evolve and escape from host immune response through mutation and increasing glycosylation. Efforts have been directed toward development of a universal vaccine with broadly protective activity against multiple influenza strains and subtypes. Here we report the design and evaluation of various chimeric vaccines based on the most common avian influenza H5 and human influenza H1 sequences. Of these constructs, the chimeric HA (cHA) vaccine with consensus H5 as globular head and consensus H1 as stem was shown to elicit broadly protective CD4+ and CD8+ T cell responses. Interestingly, the monoglycosylated cHA (cHAmg) vaccine with GlcNAc on each glycosite induced more stem-specific antibodies, with higher antibody-dependent cellular cytotoxicity (ADCC), and better neutralizing and stronger cross-protection activities against H1, H3, H5, and H7 strains and subtypes. Moreover, the cHAmg vaccine combined with a glycolipid adjuvant designed for class switch further enhanced the vaccine efficacy with more IFN-γ, IL-4, and CD8+ memory T cells produced.

Elevated Platelet Galectin-3 and Rho-Associated Protein Kinase Activity Are Associated with Hemodialysis Arteriovenous Shunt Dysfunction among Subjects with Diabetes Mellitus.

INTRODUCTION: Hyperglycemia is a major factor in influencing the patency rate of arteriovenous shunts, potentially associated with the RhoA/Rho-associated protein kinase (ROCK) pathway. Besides, galectin-3 mediates thrombotic mechanisms in venous thrombosis and peripheral artery disease. We hypothesized that high ROCK activity and galectin-3 levels are associated with arteriovenous shunt dysfunction. METHODS: We prospectively enrolled 38 patients diagnosed with arteriovenous shunt dysfunction. 29 patients received a complete follow-up and each provided two blood samples, which were collected at the first visit for occluded status of arteriovenous shunts and 1 month later for patent status. A Western blot assay for a myosin phosphatase target subunit (MYPT) was performed to examine Rho-kinase activity. A Western blot assay for platelet galectin-3 and enzyme-linked immunosorbent assay (ELISA) for circulating galectin-3 were completed. RESULTS: Higher platelet MYPT ratios and galectin-3 levels were identified at occluded arteriovenous shunts (MYPT ratio: 0.5 [0.3-1.4] vs. 0.4 [0.3-0.6], p = 0.01; galectin-3: 1.2 [0.4-1.6] vs. 0.7 [0.1-1.2], p = 0.0004). The plasma galectin-3 binding protein ELISA was also higher at occluded arteriovenous shunts (8.4 [6.0-9.7] µg/mL vs. 7.1 [4.5-9.1] µg/mL, p = 0.009). Biomarker ratios (occluded/patent status) trended high in patients with poorly controlled diabetes (MYPT ratio: 1.7 [1.0-3.0] vs. 1.1 [0.7-1.3], p = 0.06; galectin-3: 1.6 [1.3-3.4] vs. 1.1 [0.8-1.9], p = 0.05). CONCLUSION: High platelet ROCK activity and galectin-3 levels are associated with increased risk in arteriovenous shunt dysfunction, especially in patients with poorly controlled diabetes.

Development of a universal influenza vaccine using hemagglutinin stem protein produced from Pichia pastoris.

The development of a universal influenza vaccine has become a major effort to combat the high mutation rate of influenza. To explore the use of the highly conserved stem region of hemagglutinin (HA) as a universal vaccine, we produced HA-stem-based protein using yeast expression systems. The glycosylation effects on the immunogenicity and protection activities were investigated. The yield of the A/Brisbane/59/2007 HA stem produced from Pichia pastoris reached 100 mg/l. The immunogenicity of HA stem proteins in various glycoforms was further investigated and compared. All glycoforms of the HA stem protein can induce cross-reactive antibody responses, antibody-dependent cellular cytotoxicity (ADCC)-mediated protection as well as T-cell responses, with broad protection in mice. The monoglycosylated form of the A/Brisbane/59/2007 HA stem produced in yeast, together with the glycolipid C34 as the adjuvant, can elicit greater ADCC responses, better neutralizing activities against heterologous strains, and broader protection in mice.

Broadly neutralizing DNA vaccine with specific mutation alters the antigenicity and sugar-binding activities of influenza hemagglutinin.

The rapid genetic drift of influenza virus hemagglutinin is an obstacle to vaccine efficacy. Previously, we found that the consensus hemagglutinin DNA vaccine (pCHA5) can only elicit moderate neutralization activities toward the H5N1 clade 2.1 and clade 2.3 viruses. Two approaches were thus taken to improve the protection broadness of CHA5. The first one was to include certain surface amino acids that are characteristic of clade 2.3 viruses to improve the protection profiles. When we immunized mice with CHA5 harboring individual mutations, the antibodies elicited by CHA5 containing P157S elicited higher neutralizing activity against the clade 2.3 viruses. Likewise, the viruses pseudotyped with hemagglutinin containing 157S became more susceptible to neutralization. The second approach was to update the consensus sequence with more recent H5N1 strains, generating a second-generation DNA vaccine pCHA5II. We showed that pCHA5II was able to elicit higher cross-neutralization activities against all H5N1 viruses. Comparison of the neutralization profiles of CHA5 and CHA5II, and the animal challenge studies, revealed that CHA5II induced the broadest protection profile. We concluded that CHA5II combined with electroporation delivery is a promising strategy to induce antibodies with broad cross-reactivities against divergent H5N1 influenza viruses.

Differential receptor binding affinities of influenza hemagglutinins on glycan arrays.

A library of 27 sialosides, including seventeen 2,3-linked and ten 2,6-linked glycans, has been prepared to construct a glycan array and used to profile the binding specificity of different influenza hemagglutinins (HA) subtypes, especially from the 2009 swine-originated H1N1 and seasonal influenza viruses. It was found that the HAs from the 2009 H1N1 and the seasonal Brisbane strain share similar binding profiles yet different binding affinities toward various α2,6 sialosides. Analysis of the binding profiles of different HA subtypes indicate that a minimum set of 5 oligosaccharides can be used to differentiate influenza H1, H3, H5, H7, and H9 subtypes. In addition, the glycan array was used to profile the binding pattern of different influenza viruses. It was found that most binding patterns of viruses and HA proteins are similar and that glycosylation at Asn27 is essential for receptor binding.

Domain requirement of moenomycin binding to bifunctional transglycosylases and development of high-throughput discovery of antibiotics.

Moenomycin inhibits bacterial growth by blocking the transglycosylase activity of class A penicillin-binding proteins (PBPs), which are key enzymes in bacterial cell wall synthesis. We compared the binding affinities of moenomycin A with various truncated PBPs by using surface plasmon resonance analysis and found that the transmembrane domain is important for moenomycin binding. Full-length class A PBPs from 16 bacterial species were produced, and their binding activities showed a correlation with the antimicrobial activity of moenomycin against Enterococcus faecalis and Staphylococcus aureus. On the basis of these findings, a fluorescence anisotropy-based high-throughput assay was developed and used successfully for identification of transglycosylase inhibitors.