Dynamic tracing of sugar metabolism reveals the mechanisms of action of synthetic sugar analogs.

Synthetic sugar analogs are widely applied in metabolic oligosaccharide engineering (MOE) and as novel drugs to interfere with glycoconjugate biosynthesis. However, mechanistic insights on their exact cellular metabolism over time are mostly lacking. We combined ion-pair ultrahigh performance liquid chromatography-triple quadrupole mass spectrometry mass spectrometry using tributyl- and triethylamine buffers for sensitive analysis of sugar metabolites in cells and organisms and identified low abundant nucleotide sugars, such as UDP-arabinose in human cell lines and CMP-sialic acid (CMP-NeuNAc) in Drosophila. Furthermore, MOE revealed that propargyloxycarbonyl (Poc)-labeled ManNPoc was metabolized to both CMP-NeuNPoc and UDP-GlcNPoc. Finally, time-course analysis of the effect of antitumor compound 3Fax-NeuNAc by incubation of B16-F10 melanoma cells with N-acetyl-D-[UL-13C6]glucosamine revealed full depletion of endogenous ManNAc 6-phosphate and CMP-NeuNAc within 24 h. Thus, dynamic tracing of sugar metabolic pathways provides a general approach to reveal time-dependent insights into the metabolism of synthetic sugars, which is important for the rational design of analogs with optimized effects.

Endocarditis Caused by Nontypeable Streptococcus pneumoniae.

The Streptococcus pneumoniae capsule is regarded as indispensable in bacteremia. We report an infant with a ventricular septal defect and infective endocarditis caused by nontypeable S. pneumoniae. In-depth investigation confirmed a deficient capsule yet favored pneumococcal fitness for causing infective endocarditis, rather than a host immune disorder, as the cause of infective endocarditis in this case.

Nasopharyngeal colonisation dynamics of bacterial pathogens in patients with fever in rural Burkina Faso: an observational study.

BACKGROUND: Nasopharyngeal colonisation with clinically relevant bacterial pathogens is a risk factor for severe infections, such as pneumonia and bacteraemia. In this study, we investigated the determinants of nasopharyngeal carriage in febrile patients in rural Burkina Faso. METHODS: From March 2016 to June 2017, we recruited 924 paediatric and adult patients presenting with fever, hypothermia or suspicion of severe infection to the Centre Medical avec Antenne Chirurgicale Saint Camille de Nanoro, Burkina Faso. We recorded a broad range of clinical data, collected nasopharyngeal swabs and tested them for the presence of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and Klebsiella pneumoniae by quantitative polymerase chain reaction. Using logistic regression, we investigated the determinants of carriage and aimed to find correlations with clinical outcome. RESULTS: Nasopharyngeal colonisation with S. pneumoniae, H. influenzae and M. catarrhalis was highly prevalent and strongly dependent on age and season. Females were less likely to be colonised with S. pneumoniae (OR 0.71, p = 0.022, 95% CI 0.53-0.95) and M. catarrhalis (OR 0.73, p = 0.044, 95% CI 0.54-0.99) than males. Colonisation rates were highest in the age groups < 1 year and 1-2 years of age and declined with increasing age. Colonisation also declined towards the end of the rainy season and rose again during the beginning of the dry season. K. pneumoniae prevalence was low and not significantly correlated with age or season. For S. pneumoniae and H. influenzae, we found a positive association between nasopharyngeal carriage and clinical pneumonia [OR 1.75, p = 0.008, 95% CI 1.16-2.63 (S. pneumoniae) and OR 1.90, p = 0.004, 95% CI 1.23-2.92 (H. influenzae)]. S. aureus carriage was correlated with mortality (OR 4.01, p < 0.001, 95% CI 2.06-7.83), independent of bacteraemia caused by this bacterium. CONCLUSIONS: Age, sex and season are important determinants of nasopharyngeal colonisation with S. pneumoniae, H. influenzae and M. catarrhalis in patients with fever in Burkina Faso. S. pneumoniae and H. influenzae carriage is associated with clinical pneumonia and S. aureus carriage is associated with mortality in patients with fever. These findings may help to understand the dynamics of colonisation and the associated transmission of these pathogens. Furthermore, understanding the determinants of nasopharyngeal colonisation and the association with disease could potentially improve the diagnosis of febrile patients.

Immunomodulatory aged neutrophils are augmented in blood and skin of psoriasis patients.

BACKGROUND: Neutrophil accumulation in the skin is a hallmark of psoriasis. Novel insights on neutrophil phenotypic and functional heterogeneity raise the question to what extent these cells contribute to the sustained inflammatory skin reaction. OBJECTIVE: We sought to examine the phenotype and functional properties of neutrophils in blood and skin of patients with psoriasis, and the effect of TNF-α and p40(IL-12/IL-23) antibody therapy on circulating neutrophils. METHODS: Thirty-two patients with psoriasis were enrolled in an observational study performed in 2 university hospitals. We evaluated neutrophil phenotype and function using in vitro (co)culture stimulation assays, flow cytometry, multiplex immunohistochemistry, and multispectral imaging of patient-derived blood and skin samples. RESULTS: Cluster of differentiation (CD)10pos and CD10neg neutrophils were increased in peripheral blood of patients with psoriasis. In CD10neg neutrophils, different maturation stages were observed, including a subset resembling aged neutrophils that was 3 times more abundant than in healthy individuals. These aged neutrophils displayed suboptimal canonical neutrophil functions and induced IL-17 and IFN-γ production by T cells in vitro, mediated by neutrophil extracellular trap formation. Also, mature and aged neutrophils were present in psoriatic skin and were found in the vicinity of T cells. Upon antibody therapy, numbers of these cells in circulation decreased. CONCLUSIONS: Patients with psoriasis reveal a unique neutrophil profile in circulation, and 2 distinct neutrophil subsets are present in psoriatic skin. Targeted biological treatment may aid in the containment of sustained neutrophil-mediated inflammation.

Structure-Activity Relationship of Fluorinated Sialic Acid Inhibitors for Bacterial Sialylation.

Bacterial pathogens such as Nontypeable Haemophilus influenzae (NTHi) can evade the immune system by taking up and presenting host-derived sialic acids. Herein, we report a detailed structure-activity relationship of sialic acid-based inhibitors that prevent the transfer of host sialic acids to NTHi. We report the synthesis and biological evaluation of C-5, C-8, and C-9 derivatives of the parent compound 3-fluorosialic acid (SiaNFAc). Small modifications are tolerated at the C-5 and C-9 positions, while the C-8 position does not allow for modification. These structure-activity relationships define the chemical space available to develop selective bacterial sialylation inhibitors.

Antibody Binding and Complement-Mediated Killing of Invasive Haemophilus influenzae Isolates from Spain, Portugal, and the Netherlands.

Haemophilus influenzae is a Gram-negative bacterium that can be classified into typeable (types a through f) and nontypeable (NTHi) groups. This opportunistic pathogen asymptomatically colonizes the mucosal epithelium of the upper respiratory tract, from where it spreads to other neighboring regions, potentially leading to disease. Infection with NTHi can cause otitis media, sinusitis, conjunctivitis, exacerbations of chronic obstructive pulmonary disease, and pneumonia, but it is increasingly causing invasive disease, including bacteremia and meningitis. Invasive NTHi strains are more resistant to complement-mediated killing. However, the mechanisms of complement resistance have never been studied in large numbers of invasive NTHi strains. In this study, we determined the relationship between binding of IgG or IgM and the bacterial survival in normal human serum for 267 invasive H. influenzae strains from Spain, Portugal, and the Netherlands, of which the majority (200 [75%]) were NTHi. NTHi bacteria opsonized with high levels of IgM had the lowest survival in human serum. IgM binding to the bacterial surface, but not IgG binding, was shown to be associated with complement-mediated killing of NTHi strains. We conclude that evasion of IgM binding by NTHi strains increases survival in blood, thereby potentially contributing to their ability to cause severe invasive diseases.

Nasal Delivery of a Commensal Pasteurellaceae Species Inhibits Nontypeable Haemophilus influenzae Colonization and Delays Onset of Otitis Media in Mice.

Nasopharyngeal colonization with nontypeable Haemophilus influenzae (NTHi) is a prerequisite for developing NTHi-associated infections, including otitis media. Therapies that block NTHi colonization may prevent disease development. We previously demonstrated that Haemophilus haemolyticus, a closely related human commensal, can inhibit NTHi colonization and infection of human respiratory epithelium in vitro We have now assessed whether Muribacter muris (a rodent commensal from the same family) can prevent NTHi colonization and disease in vivo using a murine NTHi otitis media model. Otitis media was modeled in BALB/c mice using coinfection with 1 × 104.5 PFU of influenza A virus MEM H3N2, followed by intranasal challenge with 5 × 107 CFU of NTHi R2866 Specr Mice were pretreated or not with an intranasal inoculation of 5 × 107 CFU M. muris 24 h before coinfection. NTHi and M. muris viable counts and inflammatory mediators (gamma interferon [IFN-γ], interleukin-1ß [IL-1ß], IL-6, keratinocyte chemoattractant [KC], and IL-10) were measured in nasal washes and middle ear tissue homogenate. M. muris pretreatment decreased the median colonization density of NTHi from 6 × 105 CFU/ml to 9 × 103 CFU/ml (P = 0.0004). Only 1/12 M. muris-pretreated mice developed otitis media on day 5 compared to 8/15 mice with no pretreatment (8% versus 53%, P = 0.0192). Inflammation, clinical score, and weight loss were also lower in M. muris-pretreated mice. We have demonstrated that a single dose of a closely related commensal can delay onset of NTHi otitis media in vivo Human challenge studies investigating prevention of NTHi colonization are warranted to reduce the global burden of otitis media and other NTHi diseases.

Interacting, Nonspecific, Immunological Effects of Bacille Calmette-Guérin and Tetanus-diphtheria-pertussis Inactivated Polio Vaccinations: An Explorative, Randomized Trial.

BACKGROUND: Certain vaccines, such as Bacille Calmette-Guérin (BCG), have nonspecific effects, which modulate innate immune responses and lead to protection against mortality from unrelated infections (trained immunity). In contrast, in spite of the disease-specific effects, an enhanced overall mortality has been described after diphtheria-tetanus-pertussis (DTP) vaccination in females. This randomized trial aimed to investigate the nonspecific immunological effects of BCG and DTP-containing vaccines on the immune response to unrelated pathogens. METHODS: We randomized 75 healthy, female, adult volunteers to receive either BCG, followed by a booster dose of tetanus-diphtheria-pertussis inactivated polio vaccine (Tdap) 3 months later; BCG and Tdap combined; or Tdap followed by BCG 3 months later. Blood was collected before vaccination, as well as at 1 day, 4 days, 2 weeks, and 3 months after the first vaccination(s), plus 2 weeks after the second vaccination. Ex vivo leukocyte responses to unrelated stimuli and pathogens were assessed. RESULTS: Tdap vaccination led to short-term potentiation and long-term repression of monocyte-derived cytokine responses, and short-term as well as long-term repression of T-cell reactivity to unrelated pathogens. BCG led to short-term and long-term potentiation of monocyte-derived cytokine responses. When given together with Tdap or after Tdap, BCG abrogated the immunosuppressive effects of Tdap vaccination. CONCLUSIONS: Tdap induces immunotolerance to unrelated antigens, which is partially restored by concurrent or subsequent BCG vaccination. These data indicate that the modulation of heterologous immune responses is induced by vaccination with Tdap and BCG, and more studies are warranted to investigate whether this is involved in the nonspecific effects of vaccines on mortality. CLINICAL TRIALS REGISTRATION: NCT02771782.

Unraveling Haemophilus influenzae virulence mechanisms enable discovery of new targets for antimicrobials and vaccines.

PURPOSE OF REVIEW: The human upper respiratory tract is colonized with a variety of bacterial microorganisms including Haemophilus influenzae. The species H. influenzae consists of typeable and nontypeable H. influenzae (NTHi) variants. Typeable H. influenzae are subdivided into types a through f, based on the polysaccharide capsule, whereas the NTHi strains do not express a polysaccharide capsule. In this review, we highlight the current advances in the field of H. influenzae, with the focus on bacterial virulence mechanisms that facilitate bacterial colonization and disease, particularly for NTHi. RECENT FINDINGS: In the past decade, it has become apparent that NTHi has the ability to cause invasive infections. Recently, a number of adhesins have been shown to be crucial for bacterial colonization and invasion and these proteins were investigated as vaccine antigens. Although NTHi lacks a polysaccharide capsule, it expresses lipooligosaccharide that contribute to adhesion and evasion of complement-mediated killing, both contributing to bacterial virulence, which could potentially be targeted by novel antimicrobial drugs or vaccines. SUMMARY: The unraveling of H. influenzae virulence mechanisms resulted in the identification of promising targets for novel antimicrobials and vaccine antigens aiming to prevent or treat both typeable and nontypeable H. influenzae infections.

Serum IgM and C-Reactive Protein Binding to Phosphorylcholine of Nontypeable Haemophilus influenzae Increases Complement-Mediated Killing.

Nontypeable Haemophilus influenzae (NTHi) colonizes the human upper respiratory tract without causing disease symptoms, but it is also a major cause of upper and lower respiratory tract infections in children and elderly, respectively. NTHi synthesizes various molecules to decorate its lipooligosaccharide (LOS), which modulates the level of virulence. The presence of phosphorylcholine (PCho) on NTHi LOS increases adhesion to epithelial cells, which is an advantage for the bacterium enabling nasopharyngeal colonization. However, when PCho is incorporated on the LOS of NTHi, it is recognized by the acute-phase C-reactive protein (CRP) and PCho-specific antibodies, both potent initiators of the classical pathway of complement activation. We determined the presence of PCho and binding of IgG and IgM to the bacterial surface for 319 NTHi strains collected from the nasopharynx/oropharynx, middle ear, and lower respiratory tract. PCho detection was higher for NTHi strains collected from the nasopharynx/oropharynx, which was associated with increased binding of IgM and IgG to the bacterial surface. Binding of CRP and IgM to the bacterial surface of PChohigh NTHi strains increased complement-mediated killing, which was largely dependent on PCho-specific IgM. The levels of PCho-specific IgM varied in sera from 12 healthy individuals, and higher PCho-specific IgM levels were associated with increased complement-mediated killing of a PChohigh NTHi strain. In conclusion, incorporation of PCho on the LOS of NTHi marks the bacterium for binding of CRP and IgM, resulting in complement-mediated killing. Therefore, having a lower PCho might be beneficial in situations where sufficient PCho-specific antibodies and complement are present.

Uptake of Sialic Acid by Nontypeable Haemophilus influenzae Increases Complement Resistance through Decreasing IgM-Dependent Complement Activation.

Although nontypeable Haemophilus influenzae (NTHi) is a human-specific nasopharyngeal commensal bacterium, it also causes upper respiratory tract infections in children and lower respiratory tract infections in the elderly, resulting in frequent antibiotic use. The transition from symbiotic colonizing bacterium to opportunistic pathogen is not completely understood. Incorporation of sialic acids into lipooligosaccharides is thought to play an important role in bacterial virulence. It has been known for more than 25 years that sialic acids increase resistance to complement-mediated killing; however, the mechanism of action has not been elucidated thus far. Here, we provide evidence that growth of NTHi in the presence of sialic acids Neu5Ac and Neu5Gc decreases complement-mediated killing through abrogating the classical pathway of complement activation by preventing mainly IgM antibody binding to the bacterial surface. Therefore, strategies that interfere with uptake or incorporation of sialic acids into the lipooligosaccharide, such as novel antibiotics and vaccines, might be worth exploring to prevent or treat NTHi infections.

Nontypeable Haemophilus influenzae Invasive Blood Isolates Are Mainly Phosphorylcholine Negative and Show Decreased Complement-Mediated Killing That Is Associated with Lower Binding of IgM and CRP in Comparison to Colonizing Isolates from the Oropharynx.

Nontypeable Haemophilus influenzae (NTHi) bacteria express various molecules that contribute to their virulence. The presence of phosphocholine (PCho) on NTHi lipooligosaccharide increases adhesion to epithelial cells and is an advantage for the bacterium, enabling nasopharyngeal colonization, as measured in humans and animal models. However, when PCho is expressed on the lipooligosaccharide, it is also recognized by the acute-phase protein C-reactive protein (CRP) and PCho-specific antibodies, both of which are potent initiators of the classical pathway of complement activation. In this study, we show that blood isolates, which are exposed to CRP and PCho-specific antibodies in the bloodstream, have a higher survival in serum than oropharyngeal isolates, which was associated with a decreased presence of PCho. PCholow strains showed decreased IgM, CRP, and complement C3 deposition, which was associated with increased survival in human serum. Consistent with the case for the PCholow strains, removal of PCho expression by licA gene deletion decreased IgM, CRP, and complement C3 deposition, which increased survival in human serum. Complement-mediated killing of PChohigh strains was mainly dependent on binding of IgM to the bacterial surface. These data support the hypothesis that a PCholow phenotype was selected in blood during invasive disease, which increased resistance to serum killing, mainly due to lowered IgM and CRP binding to the bacterial surface.

Plasma therapy leads to an increase in functional IgA and IgM concentration in the blood and saliva of a patient with X-linked agammaglobulinemia.

BACKGROUND: Patients with X-linked agammaglobulinemia (XLA) are protected against invasive bacterial infections due to IgG replacement therapy, but are still at higher risk for mucosal infections of the gut and respiratory tract. This might be explained by to the lack of IgA and IgM, as these antibodies are especially important for protection against invading bacterial pathogens on the mucosal surface. METHODS: In an attempt to eliminate a chronic norovirus infection in a patient with X-linked agammaglobulinemia, fresh frozen plasma (FFP) was given two times a week for 3 weeks. At each visit, pre- and post-FFP infusion serum and saliva was collected to determine IgG-, IgA- and IgM-concentrations and serum half-life was calculated. Functionality of the immunoglobulins pre- and post-FFP infusion in both serum and saliva was tested by measuring complement activation, agglutination and killing of non-typeable Haemophilus influenzae (NTHi). RESULTS: Administration of FFP failed to eradicate the chronic norovirus infection. Serum IgA and IgM half-life was 4.2 ± 0.3 and 3.8 ± 0.3 days, respectively. The presence of serum IgM was associated with increased complement binding and complement-mediated killing of NTHi. IgA in saliva was detectable post-FFP and was associated with increased agglutination of NTHi. IgM in saliva was not detectable. CONCLUSIONS: We conclude that FFP treatment, although ineffective in clearing a chronic norovirus infection in this single patient, might be beneficial to prevent or eliminate bacterial infections in XLA patients by increasing IgM dependent complement-mediated killing in serum and IgA dependent bacterial agglutination on the mucosal surface.

Desialylation of Platelets by Pneumococcal Neuraminidase A Induces ADP-Dependent Platelet Hyperreactivity.

Platelets are increasingly recognized to play a role in the complications of Streptococcus pneumoniae infections. S. pneumoniae expresses neuraminidases, which may alter glycans on the platelet surface. In the present study, we investigated the capability of pneumococcal neuraminidase A (NanA) to remove sialic acid (desialylation) from the platelet surface, the consequences for the platelet activation status and reactivity, and the ability of neuraminidase inhibitors to prevent these effects. Our results show that soluble NanA induces platelet desialylation. Whereas desialylation itself did not induce platelet activation (P-selectin expression and platelet fibrinogen binding), platelets became hyperreactive to ex vivo stimulation by ADP and cross-linked collagen-related peptide (CRP-XL). Platelet aggregation with leukocytes also increased. These processes were dependent on the ADP pathway, as inhibitors of the pathway (apyrase and ticagrelor) abrogated platelet hyperreactivity. Inhibition of NanA-induced platelet desialylation by neuraminidase inhibitors (e.g., oseltamivir acid) also prevented the platelet effects of NanA. Collectively, our findings show that soluble NanA can desialylate platelets, leading to platelet hyperreactivity, which can be prevented by neuraminidase inhibitors.

Streptococcus pneumoniae PspC Subgroup Prevalence in Invasive Disease and Differences in Contribution to Complement Evasion.

The pneumococcal capsular serotype is an important determinant of complement resistance and invasive disease potential, but other virulence factors have also been found to contribute. Pneumococcal surface protein C (PspC), a highly variable virulence protein that binds complement factor H to evade C3 opsonization, is divided into two subgroups: choline-bound subgroup I and LPxTG-anchored subgroup II. The prevalence of different PspC subgroups in invasive pneumococcal disease (IPD) and functional differences in complement evasion are unknown. The prevalence of PspC subgroups in IPD isolates was determined in a collection of 349 sequenced strains of Streptococcus pneumoniae isolated from adult patients. pspC deletion mutants and isogenic pspC switch mutants were constructed to study differences in factor H binding and complement evasion in relation to capsule thickness. Subgroup I pspC was far more prevalent in IPD isolates than subgroup II pspC The presence of capsule was associated with a greater ability of bound factor H to reduce complement opsonization. Pneumococcal subgroup I PspC bound significantly more factor H and showed more effective complement evasion than subgroup II PspC in isogenic encapsulated pneumococci. We conclude that variation in the PspC subgroups, independent of capsule serotypes, affects pneumococcal factor H binding and its ability to evade complement deposition.

The Complement System Contributes to Functional Antibody-Mediated Responses Induced by Immunization with Plasmodium falciparum Malaria Sporozoites.

Long-lasting and sterile homologous protection against malaria can be achieved by the exposure of malaria-naive volunteers under chemoprophylaxis to Plasmodium falciparum-infected mosquitoes (chemoprophylaxis and sporozoite [CPS] immunization). While CPS-induced antibodies neutralize sporozoite infectivity in vitro and in vivo, antibody-mediated effector mechanisms are still poorly understood. Here, we investigated whether complement contributes to CPS-induced preerythrocytic immunity. Sera collected before and after CPS immunization in the presence of active or inactive complement were assessed for the recognition of homologous NF54 and heterologous NF135.C10 sporozoites, complement fixation, sporozoite lysis, and possible subsequent effects on in vitro sporozoite infectivity in human hepatocytes. CPS immunization induced sporozoite-specific IgM (P < 0.0001) and IgG (P = 0.001) antibodies with complement-fixing capacities (P < 0.0001). Sporozoite lysis (P = 0.017), traversal (P < 0.0001), and hepatocyte invasion inhibition (P < 0.0001) by CPS-induced antibodies were strongly enhanced in the presence of active complement. Complement-mediated invasion inhibition in the presence of CPS-induced antibodies negatively correlated with cumulative parasitemia during CPS immunizations (P = 0.013). While IgG antibodies similarly recognized homologous and heterologous sporozoites, IgM binding to heterologous sporozoites was reduced (P = 0.023). Although CPS-induced antibodies did not differ in their abilities to fix complement, lyse sporozoites, or inhibit the traversal of homologous and heterologous sporozoites, heterologous sporozoite invasion was more strongly inhibited in the presence of active complement (P = 0.008). These findings demonstrate that CPS-induced antibodies have complement-fixing activity, thereby significantly further enhancing the functional inhibition of homologous and heterologous sporozoite infectivity in vitro The combined data highlight the importance of complement as an additional immune effector mechanism in preerythrocytic immunity after whole-parasite immunization against Plasmodium falciparum malaria.

IgM Augments Complement Bactericidal Activity with Serum from a Patient with a Novel CD79a Mutation.

Antibody replacement therapy for patients with antibody deficiencies contains only IgG. As a result, concurrent IgM and IgA deficiency present in a large proportion of antibody deficient patients persists. Especially patients with IgM deficiency remain at risk for recurrent infections of the gastrointestinal and respiratory tract. The lack of IgM in the current IgG replacement therapy is likely to contribute to the persistence of these mucosal infections because this antibody class is especially important for complement activation on the mucosal surface. We evaluated whether supplementation with IgM increased serum bactericidal capacity in vitro. Serum was collected from a patient with agammaglobulinemia and supplemented with purified serum IgM to normal levels. Antibody and complement deposition on the bacterial surface was determined by multi-color flow cytometry. Bacterial survival in serum was determined by colony-forming unit counts. We present a patient previously diagnosed with agammaglobulinemia due to CD79A (Igα) deficiency revealing a novel pathogenic insertion variant in the CD79a gene (NM_001783.3:c.353_354insT). Despite IgG replacement therapy and antibiotic prophylaxis, this patient developed a Campylobacter jejuni spondylodiscitis of lumbar vertebrae L4-L5. We found that serum IgM significantly contributes to complement activation on the bacterial surface of C. jejuni. Furthermore, supplementation of serum IgM augmented serum bactericidal activity significantly. In conclusion, supplementation of intravenous IgG replacement therapy with IgM may potentially offer greater protection against bacterial infections, also in the context of increasing antibiotic resistance.

Th17-Mediated Cross Protection against Pneumococcal Carriage by Vaccination with a Variable Antigen.

Serotype-specific protection against Streptococcus pneumoniae is an important limitation of the current polysaccharide-based vaccines. To prevent serotype replacement, reduce transmission, and limit the emergence of new variants, it is essential to induce broad protection and restrict pneumococcal colonization. In this study, we used a prototype vaccine formulation consisting of lipopolysaccharide (LPS)-detoxified outer membrane vesicles (OMVs) from Salmonella enterica serovar Typhimurium displaying the variable N terminus of PspA (α1α2) for intranasal vaccination, which induced strong Th17 immunity associated with a substantial reduction of pneumococcal colonization. Despite the variable nature of this protein, a common major histocompatibility complex class (MHC-II) epitope was identified, based on in silico prediction combined with ex vivo screening, and was essential for interleukin-17 A (IL-17A)-mediated cross-reactivity and associated with cross protection. Based on 1,352 PspA sequences derived from a pneumococcal carriage cohort, this OMV-based vaccine formulation containing a single α1α2 type was estimated to cover 19.1% of strains, illustrating the potential of Th17-mediated cross protection.

Metabolic Oligosaccharide Engineering with Alkyne Sialic Acids Confers Neuraminidase Resistance and Inhibits Influenza Reproduction.

Metabolic incorporation of azide- or alkyne-modified sialic acids into the cellular glycosylation pathway enables the study of sialoglycan expression, localization, and trafficking via bioorthogonal chemistry. Herein, we report that such modifications of the sialic acid sugar can have a profound influence on their hydrolysis by neuraminidases (sialidase). Azidoacetyl (Az)-modified sialic acids were prone to neuraminidase cleavage, whereas propargyloxycarbonyl (Poc)-modified sialic acids were largely resistant to cleavage. Because the influenza virus infection cycle depends on the hydrolysis of host-cell-surface sialic acids, influenza cell-to-cell transmission was strongly reduced in Poc sialic acid glycoengineered host cells. The use of Poc sialic acids may disturb biological processes involving neuraminidase cleavage but also provides perspective for use in applications in which sialic acid hydrolysis is not desired, such as antibody modification, viral infection, etc.