Understanding the Chemistry and Biology of Glycosylation with Glycan Synthesis.

Glycoscience research has been significantly impeded by the complex compositions of the glycans present in biological molecules and the lack of convenient tools suitable for studying the glycosylation process and its function. Polysaccharides and glycoconjugates are not encoded directly by genes; instead, their biosynthesis relies on the differential expression of carbohydrate enzymes, resulting in heterogeneous mixtures of glycoforms, each with a distinct physiological activity. Access to well-defined structures is required for functional study, and this has been provided by chemical and enzymatic synthesis and by the engineering of glycosylation pathways. This review covers general methods for preparing glycans commonly found in mammalian systems and applying them to the synthesis of therapeutically significant glycoconjugates (glycosaminoglycans, glycoproteins, glycolipids, glycosylphosphatidylinositol-anchored proteins) and the development of carbohydrate-based vaccines.

A multi-enzyme machine polymerizes the Haemophilus influenzae type b capsule.

Bacterial capsules have critical roles in host-pathogen interactions. They provide a protective envelope against host recognition, leading to immune evasion and bacterial survival. Here we define the capsule biosynthesis pathway of Haemophilus influenzae serotype b (Hib), a Gram-negative bacterium that causes severe infections in infants and children. Reconstitution of this pathway enabled the fermentation-free production of Hib vaccine antigens starting from widely available precursors and detailed characterization of the enzymatic machinery. The X-ray crystal structure of the capsule polymerase Bcs3 reveals a multi-enzyme machine adopting a basket-like shape that creates a protected environment for the synthesis of the complex Hib polymer. This architecture is commonly exploited for surface glycan synthesis by both Gram-negative and Gram-positive pathogens. Supported by biochemical studies and comprehensive 2D nuclear magnetic resonance, our data explain how the ribofuranosyltransferase CriT, the phosphatase CrpP, the ribitol-phosphate transferase CroT and a polymer-binding domain function as a unique multi-enzyme assembly.

Expression conditions and characterization of a novelly constructed lipoprotein intended as a vaccine to prevent human Haemophilus influenzae infections.

Bacterial lipoproteins are structurally divided into two groups, based on their lipid moieties: diacylated (present in Gram-positive bacteria) and triacylated (present in some Gram-positive and most Gram-negative bacteria). Diacylated and triacylated lipid moieties differ by a single amide-linked fatty acid chain. Lipoproteins induce host innate immune responses by the mammalian Toll-like receptor 2 (TLR2). In this study, we added a lipid moiety to recombinant OMP26, a native nonlipidated (NL) membrane protein of Haemophilus influenzae, and characterized it extensively under different expression conditions using flow cytometry, LC/MS, and MALDI-TOF. We also investigated the ability of NL and lipidated (L) OMP26 to induce in vitro stimulation of HEK Blue-hTLR2-TR1 and hTLR-TLR6 cells. Our L-OMP26 was predominantly expressed in diacylated form, so we employed an additional gene copy of apolipoprotein N-acetyltransferase enzyme (Lnt)-rich Escherichia coli strain that further acylates the diacyl lipoproteins to enhance the production of triacylated L-OMP26. The diacyl and triacyl versions of L-OMP26, intended as a vaccine for use in humans, were characterized and evaluated as protein vaccine components in a mouse model. We found that the diacyl and triacyl L-OMP26 protein formulations differed markedly in their immune-stimulatory activity, with diacylated L-OMP26 stimulating higher adaptive immune responses compared with triacylated L-OMP26 and both stimulating higher adaptive immune response compared to NL-OMP26. We also constructed and characterized an L-OMP26φNL-P6 fusion protein, where NL-P6 protein (a commonly studied H. influenzae vaccine candidate) was recombinantly fused to L-OMP26. We observed a similar pattern of lipidation (predominantly diacylated) in the L-OMP26φNL-P6 fusion protein.

Preparation and immunogenicity evaluation of C-HapS-P6 fusion protein vaccine against nontypeable Haemophilus influenzae in mice.

Nontypeable Haemophilus influenzae (NTHi) is the dominant pathogen in several infectious diseases. Currently the use of antibiotics is the main intervention to prevent NTHi infections, however with the emergence of drug resistant strains, it has compromised the treatment of respiratory infections with antibiotics. Therefore there is an urgent need to develop a safe and effective vaccine to prevent NTHi infections. We investigate the potential of C-HapS-P6 fusion protein as a vaccine for treating NTHi in murine models. PGEX-6P2/C-HapS-P6 fusion gene was constructed using overlap extension polymerase chain reaction. The recombined plasmid was transformed into Escherichia coli for protein expression. The mice were subjected to intraperitoneal immunization using purified antigens. Immunoglobulin (Ig) G in serum samples and IgA in nasal and lung lavage fluids were analyzed using enzyme-linked immunosorbent assay. Cytokine release and proliferation capacity of splenic lymphocytes in response to antigens were measured in vitro. The protective effect of the C-HapS-P6 protein against NTHi infection was evaluated by NTHi count and histological examination. The data showed that the C-HapS-P6 fusion protein increased significantly the levels of serum IgG and nasal and lung IgA, and promoted the release of interleukin (IL)-2, interferon-ϒ, IL-4, IL-5, and IL-17 and the proliferation of splenic lymphocytes compared with C-HapS or P6 protein treatment alone. Moreover, C-HapS-P6 effectively reduced the NTHi colonization in the nasopharynx and lungs of mice. In conclusion, our results demonstrated that the C-HapS-P6 fusion protein vaccine can significantly enhance humoral and cell immune responses and effectively prevent against NTHi infection in the respiratory tract in murine models.

Evaluation of immunoregulation and immunoprotective efficacy of Glaesserella parasuis histidine kinase QseC.

QseC is a membrane sensor kinase that enables bacteria to perceive autoinducers -3, adrenaline, and norepinephrine to initiate downstream gene transcription. In this study, we found that the QseC protein of Glaesserella parasuis can serve as an effective antigen to activate the host's immune response. Therefore, we investigated the immunogenicity and host protective effect of this protein. ELISA and indirect immunofluorescence results showed that QseC protein can induce high titer levels of humoral immunity in mice and regularly generate specific serum antibodies. We used MTS reagents to detect lymphocyte proliferation levels and found that QseC protein can cause splenic lymphocyte proliferation with memory and specificity. Further immunological analysis of the spleen cell supernatant revealed significant upregulation of levels of IL-1ß, IL-4 and IFN-γ in the QseC + adjuvant group. In the mouse challenge experiment, it was found that QseC + adjuvant can provide effective protection. The results of this study demonstrate that QseC protein provides effective protection in a mouse model and has the potential to serve as a candidate antigen for a novel subunit vaccine for further research.

Immunization with HMW1 and HMW2 adhesins protects against colonization by heterologous strains of nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is a common cause of localized respiratory tract disease and results in significant morbidity. The pathogenesis of NTHi disease begins with nasopharyngeal colonization, and therefore, the prevention of colonization represents a strategy to prevent disease. The NTHi HMW1 and HMW2 proteins are a family of conserved adhesins that are present in 75 to 80% of strains and have been demonstrated to play a critical role in colonization of the upper respiratory tract in rhesus macaques. In this study, we examined the vaccine potential of HMW1 and HMW2 using a mouse model of nasopharyngeal colonization. Immunization with HMW1 and HMW2 by either the subcutaneous or the intranasal route resulted in a strain-specific antibody response associated with agglutination of bacteria and restriction of bacterial adherence. Despite the specificity of the antibody response, immunization resulted in protection against colonization by both the parent NTHi strain and heterologous strains expressing distinct HMW1 and HMW2 proteins. Pretreatment with antibody against IL-17A eliminated protection against heterologous strains, indicating that heterologous protection is IL-17A dependent. This work demonstrates the vaccine potential of the HMW1 and HMW2 proteins and highlights the importance of IL-17A in protection against diverse NTHi strains.

Pili Subunit PilA Contributes to the Cytoadhesion of Glaesserella Parasuis to Host Cells and Provides Immunoprotection.

Glaesserella parasuis (G. parasuis) is commonly located in the upper respiratory tract of pigs as an opportunistic pathogen. It can cause Glässer's disease, which leads to serious economic losses in the swine industry. The occurrence of the disease is often linked with the adhesion and colonization of the pathogen. The PilA pilus subunit is important for adhesion to the host, twitching motility, and biofilm formation in many bacteria. However, no research has focused on the function of PilA in G. parasuis. To further reveal the pathogenesis of G. parasuis and to search for subunit vaccine candidates, we investigated whether PilA could adhere to cells and provide immune protection. A bioinformatic analysis showed that the protein secondary structure of the G. parasuis PilA was similar to that of Haemophilus influenzae (HI). Cell adhesion, ELISA, and far-Western blotting showed that rPilA could bind porcine-derived, porcine kidney-15 (PK-15) cells, swine tracheal epithelial cells (STECs), and the extracellular matrix components fibronectin (FN) and laminin (LN). An immunogenicity analysis showed that recombinant PilA (rPilA) reacted specifically with convalescent and hyperimmune serum. Importantly, purified rPilA elicited a strong immune response and conferred robust protection against challenges with serovar 5 G. parasuis in mice. These results suggested that the PilA protein might help G. parasuis adhere to host cells by binding to FN and LN, and its immunogenicity establishes it as a promising, novel subunit vaccine candidate against infections with G. parasuis. IMPORTANCE G. parasuis is one of the most prevalent bacterial infections in swine production and can lead to huge economic losses around the world. A full understanding of colonization and immunity with G. parasuis infections will be essential in disease control. In this study, the PilA protein, which is a common virulence factor in other bacteria that mediates adherence to the host, was assessed. The results suggested that the PilA protein of G. parasuis can mediate adhesion to host cells through FN and LN, which provides a new idea for the study of the pathogenicity of G. parasuis. Furthermore, fimbriae usually have high immunogenicity. Immunogenicity and protective capacity results showed that the use of this recombinant PilA antigen might be a promising candidate vaccine antigen with which to prevent G. parasuis infections.

Vaginal carriage of Haemophilus influenzae in a non-pregnant reproductive-age population.

BACKGROUND: Haemophilus influenzae (Hi) is an emerging cause of early onset neonatal sepsis, but mechanisms of transmission are not well understood. We aimed to determine the prevalence of vaginal carriage of Hi in reproductive age women and to examine behavioral and demographic characteristics associated with its carriage. METHODS: We performed a secondary analysis of stored vaginal lavage specimens from a prospective cohort study of nonpregnant reproductive-age women. After extraction of bacterial genomic DNA, samples were tested for the presence of the gene encoding Haemophilus protein d (hpd) by quantitative real-time polymerase chain reaction (PCR) using validated primers and probe. PCR for the V3-V4 region of the 16 S rRNA gene (positive control) assessed sample quality. Samples with cycle threshold (CT) value < 35 were defined as positive. Sanger sequencing confirmed the presence of hpd. Behavioral and demographic characteristics associated with vaginal carriage of Hi were examined. RESULTS: 415 samples were available. 315 (75.9%) had sufficient bacterial DNA and were included. 14 (4.4%) were positive for hpd. There were no demographic or behavioral differences between the women with Hi vaginal carriage and those without. There was no difference in history of bacterial vaginosis, vaginal microbiome community state type, or presence of Group B Streptococcus in women with and without vaginal carriage of Hi. CONCLUSION: Hi was present in vaginal lavage specimens of 4.4% of this cohort. Hi presence was unrelated to clinical or demographic characteristics, though the relatively small number of positive samples may have limited power to detect such differences.

Secondary Cases of Invasive Disease Caused by Encapsulated and Nontypeable Haemophilus influenzae - 10 U.S. Jurisdictions, 2011-2018.

Haemophilus influenzae (Hi) can cause meningitis and other serious invasive disease. Encapsulated Hi is classified into six serotypes (a-f) based on chemical composition of the polysaccharide capsule; unencapsulated strains are termed nontypeable Hi (NTHi). Hi serotype b (Hib) was the most common cause of bacterial meningitis in children in the pre-Hib vaccine era, and secondary transmission of Hi among children (e.g., to household contacts and in child care facilities) (1,2) led to the Advisory Committee on Immunization Practices (ACIP) recommendation for antibiotic chemoprophylaxis to prevent Hib disease in certain circumstances.* High Hib vaccination coverage since the 1990s has substantially reduced Hib disease, and other serotypes now account for most Hi-associated invasive disease in the United States (3). Nevertheless, CDC does not currently recommend chemoprophylaxis for contacts of persons with invasive disease caused by serotypes other than Hib and by NTHi (non-b Hi). Given this changing epidemiology, U.S. surveillance data were reviewed to investigate secondary cases of invasive disease caused by Hi. The estimated prevalence of secondary transmission was 0.32% among persons with encapsulated Hi disease (≤60 days of one another) and 0.12% among persons with NTHi disease (≤14 days of one another). Isolates from all Hi case pairs were genetically closely related, and all patients with potential secondary infection had underlying medical conditions. These results strongly suggest that secondary transmission of non-b Hi occurs. Expansion of Hi chemoprophylaxis recommendations might be warranted to control invasive Hi disease in certain populations in the United States, but further analysis is needed to evaluate the potential benefits against the risks, such as increased antibiotic use.

Epidemiology of invasive Haemophilus influenzae disease in northwestern Ontario: comparison of invasive and noninvasive H. influenzae clinical isolates.

In the post-Haemophilus influenzae type b (Hib) vaccine era, invasive H. influenzae type a (Hia) disease emerged in North American Indigenous populations. The role of Hia in noninvasive disease is uncertain; it is unknown whether noninvasive Hia infections are prevalent in populations with a high incidence of invasive disease, and whether invasive and noninvasive Hia isolates have different characteristics. We analyzed all invasive and noninvasive clinical H. influenzae isolates collected in a northwestern Ontario hospital serving 82% Indigenous population over 5.5 years. Serotyping, clonal analysis, and antimicrobial sensitivity testing were conducted on 233 noninvasive and 20 invasive isolates. Among noninvasive isolates, 91% were nontypeable (NTHi) and 3% were Hia; Hia was the most frequent invasive isolate (60%). Incidence rates of invasive H. influenzae disease (12.5/100 000/year) greatly exceeded average provincial data, with the highest found in <6-year-old children (63.9/100 000/year); the proportion of Hia among invasive isolates was seven times larger than in Ontario. No difference in clonal characteristics between invasive and noninvasive Hia isolates was found. Antibiotic resistance was more common among NTHi than among encapsulated isolates, without differences between invasive and noninvasive isolates. Considering the significance of Hia in Indigenous populations, pediatric immunization against Hia will be useful to prevent serious infections in young Indigenous children.

Invasive Haemophilus influenzae Infections after 3 Decades of Hib Protein Conjugate Vaccine Use.

Haemophilus influenzae serotype b (Hib) was previously the most common cause of bacterial meningitis and an important etiologic agent of pneumonia in children aged <5 years. Its major virulence factor is the polyribosyl ribitol phosphate (PRP) polysaccharide capsule. In the 1980s, PRP-protein conjugate Hib vaccines were developed and are now included in almost all national immunization programs, achieving a sustained decline in invasive Hib infections. However, invasive Hib disease has not yet been eliminated in countries with low vaccine coverage, and sporadic outbreaks of Hib infection still occur occasionally in countries with high vaccine coverage. Over the past 2 decades, other capsulated serotypes have been recognized increasingly as causing invasive infections. H. influenzae serotype a (Hia) is now a major cause of invasive infection in Indigenous communities of North America, prompting a possible requirement for an Hia conjugate vaccine. H. influenzae serotypes e and f are now more common than serotype b in Europe. Significant year-to-year increases in nontypeable H. influenzae invasive infections have occurred in many regions of the world. Invasive H. influenzae infections are now seen predominantly in patients at the extremes of life and those with underlying comorbidities. This review provides a comprehensive and critical overview of the current global epidemiology of invasive H. influenzae infections in different geographic regions of the world. It discusses those now at risk of invasive Hib disease, describes the emergence of other severe invasive H. influenzae infections, and emphasizes the importance of long-term, comprehensive, clinical and microbiologic surveillance to monitor a vaccine's impact.

SEROPROTECTION RATE OF HAEMOPHILUS INFLUENZAE TYPE B CONJUGATE VACCINE AND ASSOCIATED CLINICAL OUTCOMES AMONG NIGERIAN CHILDREN AGED 6 TO 23 MONTHS.

Introduction: Haemophilus influenzae type b (Hib) causes invasive infections almost exclusively in under- fives with those aged 6-23 months being the most vulnerable. In Nigeria, it is estimated to cause nearly 400,000 annual infections and another 30,000 under-five mortality attributable to pneumonia and meningitis alone. The Hib Conjugate Vaccine (HCV) is in widespread use to combat these devastating infections. Data on its impact in Nigeria is grossly scanty. This study evaluated the seroprotection rates (SPR) of HCV and associated clinical outcomes among children aged 6-23 months in Obi L.G.A. of Nasarawa State, Nigeria. Methods: A cross-sectional study of 267 children aged 6-23 months who had completed three doses of HCV. They were enrolled via a two-staged household-level cluster sampling. Relevant sociodemographic and clinical data were obtained using structured questionnaires and serum samples collected were analysed serologically for antipolyribosylribitol phosphate (anti-PRP) antibodies using ELISA. Results: The overall SPRs against invasive Hib disease and Hib nasopharyngeal colonization were 74.2% and 26.2%, respectively. The overall geometric mean titre (GMT) of anti-PRP was 1.85 µg/mL (95%CI: 1.60-2.14) and across age groups, GMTs were >1 µg/mL-the threshold for long-term protection against invasive Hib disease. Rates/duration of healthcare admissions and average episodes of probable Hib disease syndromes were lower in seroprotected but not statistically different from non-seroprotected children. Conclusion: The demonstrated anti-PRP titres and Seroprotection Rates infer a very good HCV efficacy in Nigerian children. The lack of significant difference in clinical outcomes may be attributable to nonspecificity.

Laboratory surveillance of invasive Haemophilus influenzae disease in Argentina, 2011-2019.

The incorporation of Haemophilus influenzae type b (Hib) vaccine into the Argentine National Immunization Program in 1998 resulted in a dramatic decrease in the incidence of invasive disease due to this serotype. We assessed 1405 H. influenzae (Hi) isolates causing invasive infections referred to the National Reference Laboratory between 2011 and 2019. Non-encapsulated Hi were the most common strains (44.5%), followed by types b (41.1%) and a (10.0%). Significant increase in the proportion of type b was observed, from 31.2% in 2011, to 50% in 2015, correlating with the peak incidence rate, later decreasing to 33.6% by 2019. We compared the genetic relationship between clones circulating during the period of increased Hib incidence (2011-2015) and those of the prevaccination-transition period (1997-1998). Four pulsotypes predominated in both periods, G, M, P and K, G being the most common. Multi-locus sequence typing revealed that the 4 pulsotypes belonged to ST6, or one of its simple or double locus variants. Isolates from fully vaccinated individuals did not differ from those of the rest of the population studied. After ruling out aspects associated with emergence of specific clones, we concluded that factors such as low booster coverage rates, delayed vaccination schedules and use of different vaccines may have contributed to the reemergence of Hib infections.

Lipidation of Haemophilus influenzae Antigens P6 and OMP26 Improves Immunogenicity and Protection against Nasopharyngeal Colonization and Ear Infection.

Nontypeable Haemophilus influenzae (NTHi) causes respiratory infections that lead to high morbidity and mortality worldwide, encouraging development of effective vaccines. To achieve a protective impact on nasopharyngeal (NP) colonization by NTHi, enhanced immunogenicity beyond that achievable with recombinant-protein antigens is likely to be necessary. Adding a lipid moiety to a recombinant protein would enhance immunogenicity through Toll-like receptor 2 signaling of antigen-presenting cells and Th17 cell response in the nasal-associated lymphoid tissue (NALT). We investigated effects of lipidation (L) of recombinant proteins P6 and OMP26 compared to nonlipidated (NL) P6 and OMP26 and as fusion constructs (L-OMP26ϕNL-P6 and L-P6ϕNL-OMP26) in a mouse model. After intraperitoneal or intranasal vaccination, antibody responses were compared and protection from NP colonization and middle ear infection were assessed. L-P6 and L-OMP26 induced approximately 10- to 100-fold-higher IgG antibody levels than NL-P6 and NL-OMP26. Fusion constructs significantly increased IgG antibody to both target proteins, even though only one of the proteins was lipidated. NP colonization and middle ear bullae NTHi density was 1 to 4 logs lower following vaccination with L-P6 and L-OMP26 than with NL-P6 and NL-OMP26. Fusion constructs also resulted in a 1- to 3-log-lower NTHi density following vaccination. NALT cells from mice vaccinated with lipidated protein constructs had higher levels of interleukin-17 (IL-17), IL-22, and CD4+ T-cell memory. Passive transfer of sera from L-OMP26ϕNL-P6-vaccinated mice to recipient infant mice reduced NP colonization and ear bulla NTHi density. We conclude that L-P6, L-OMP26, and fusion constructs generate enhanced antibody responses and protection from NP colonization and middle ear infection by NTHi in mice.

Epidemiology and burden of Haemophilus influenzae disease in Thai children before implementation of the routine immunisation programme: A National Health Data Analysis.

OBJECTIVES: To conduct the first pre-Haemophilus influenzae (Hi) type b (Hib) immunisation programme-based epidemiological study using national health data. METHODS: We analysed National Health Security Office data, which cover 72% of the Thai population. The study population included children aged <18 years admitted for Hi disease from 2015 to 2019. Hi disease diagnosis and death were based on the International Statistical Classification of Diseases and Related Health Problems (10th revision) hospital discharge summary codes. We estimated the hospital cost per admission using diagnosis-related grouping with a global budget. RESULTS: A total of 1125 children aged <18 years were admitted for Hi disease. During the 5-year-study, the annual incidence of Hi disease varied from 1.5 to 1.9 per 100,000 children, with an overall case fatality rate (CFR) of 2%. Pneumonia was the most common clinical form, followed by meningitis and sepsis. The incidence, clinical forms and severity of Hi disease were age specific. Infant CFR was higher than that of other age groups. The incidence of Hi disease in children aged <5 years was 4.9 per 100,000 (CFR = 2.0%). Sepsis was the primary cause of infant death, whereas pneumonia was the cause of death in children aged >5 years. The hospital cost ranged from 25,000 to 30,000 THB per admission. CONCLUSIONS: This analysis provided epidemiological data of Hi in Thai children before the Hib routine immunisation programme. The incidence of Hi disease was lower than that previously speculated. Our results could facilitate an assessment of the impact of Hib immunisation programme in Thailand.

Anti-glycan antibodies: roles in human disease.

Carbohydrate-binding antibodies play diverse and critical roles in human health. Endogenous carbohydrate-binding antibodies that recognize bacterial, fungal, and other microbial carbohydrates prevent systemic infections and help maintain microbiome homeostasis. Anti-glycan antibodies can have both beneficial and detrimental effects. For example, alloantibodies to ABO blood group carbohydrates can help reduce the spread of some infectious diseases, but they also impose limitations for blood transfusions. Antibodies that recognize self-glycans can contribute to autoimmune diseases, such as Guillain-Barre syndrome. In addition to endogenous antibodies that arise through natural processes, a variety of vaccines induce anti-glycan antibodies as a primary mechanism of protection. Some examples of approved carbohydrate-based vaccines that have had a major impact on human health are against pneumococcus, Haemophilus influeanza type b, and Neisseria meningitidis. Monoclonal antibodies specifically targeting pathogen associated or tumor associated carbohydrate antigens (TACAs) are used clinically for both diagnostic and therapeutic purposes. This review aims to highlight some of the well-studied and critically important applications of anti-carbohydrate antibodies.

Hib Vaccines: Their Impact on Haemophilus influenzae Type b Disease.

Haemophilus influenzae serotype b (Hib) is an important cause of serious, invasive infections, particularly in young children. Since 1985, a series of vaccines composed of the type b capsular polysaccharide polyribosylribitol phosphate (PRP), followed by PRP conjugated to various proteins, have been licensed for use in the United States and worldwide. The conjugated vaccines offer increased immunogenicity and prolonged durability of immune protection compared to the plain PRP vaccine and increasingly are combined with other childhood vaccines for decreased cost and increased ease of vaccination. Hib vaccines have a very favorable safety profile, have been found to be either cost-saving or cost-effective by many public health agencies, and, in most countries, are initiated during early infancy as part of routine childhood immunization programs. As a result of widespread use of the vaccines, the incidence of Hib infections, and their associated morbidity and mortality, has fallen dramatically across the globe. Yet, many children remain unimmunized or underimmunized against Hib, particularly in limited-resource countries. Future efforts to further reduce the disease burden of Hib infections remain a high priority.

Impact of Vaccination on Haemophilus influenzae Type b Carriage in Healthy Children Less Than 5 Years of Age in an Urban Population in Nepal.

BACKGROUND: Reduction in detection of asymptomatic carriage of Haemophilus influenzae type b (Hib) can be used to assess vaccine impact. In Nepal, routine vaccination against Hib in children at 6, 10, and 14 weeks of age was introduced in 2009. Before vaccine introduction, Hib carriage was estimated at 5.0% among children aged <13 years in Nepal, with higher rates among children under 5. Large-scale evaluation of Hib carriage in children has not been investigated since the introduction of the pentavalent diphtheria-tetanus-pertussis/Hib/hepatitis B (DTP-Hib-HepB) vaccine in Nepal. METHODS: A total of 666 oropharyngeal swabs were collected between August and December 2018 from healthy children between 6 months and 5 years of age attending the vaccination clinic at Patan Hospital, Kathmandu, Nepal. Of these 666 swabs, 528 (79.3%) were tested for Hib by culture. Demographic and vaccination data were collected. RESULTS: Among 528 swabs tested for Hib, 100% came from fully vaccinated children. No swabs were positive for Hib (95% confidence interval, .0-.7). The absence of Hib in 2018 suggests vaccine-induced protection against Hib carriage 9 years after vaccine introduction. CONCLUSIONS: Following 3 doses of pentavalent DTP-Hib-HepB vaccine, Hib carriage in children under the age of 5 years in Nepal is no longer common. Ongoing high coverage with Hib vaccine in early childhood is expected to maintain protection against Hib disease in Nepal.

Clinical Characteristics and Adverse Clinical Outcomes of Invasive Haemophilus influenzae Serotype a Cases-United States, 2011-2015.

BACKGROUND: Incidence of invasive disease due to Haemophilus influenzae serotype a (Hia) increased an average of 13% annually from 2002 through 2015. We describe clinical characteristics and adverse clinical outcomes of US invasive Hia cases detected through multistate surveillance during 2011-2015. METHODS: Medical record data were abstracted for cases reported in 8 jurisdictions conducting active population- and laboratory-based surveillance for invasive Hia disease across the United States. Isolates from sterile sites were serotyped using real-time polymerase chain reaction. Adverse clinical outcomes were defined as any possible complication of meningitis, bacteremic pneumonia, or bacteremia (including hearing loss and developmental delay, but excluding death) and were assessed at hospital discharge and one-year post-disease onset. RESULTS: During 2011-2015, 190 Hia cases were reported to the 8 participating sites; 169 (88.9%) had data abstracted. Many patients were aged <5 years (42.6%). Meningitis was the most common clinical presentation among those aged <1 year (71.4%); bacteremic pneumonia was the most common presentation among persons aged ≥50 years (78.7%). Overall, 95.9% of patients were hospitalized. Among those hospitalized, 47.5% were admitted to an intensive care unit and 6.2% died during hospitalization. At hospital discharge and one-year post-disease onset, adverse outcomes were identified in 17.7% and 17.8% of patients overall and in 43.9% and 48.5% of patients with meningitis (primarily children). CONCLUSIONS: Hia infection can cause severe disease that requires hospitalization and may also cause short- and long-term adverse clinical outcomes, especially among children. Novel vaccines could prevent morbidity and mortality.

A heme-binding protein produced by Haemophilus haemolyticus inhibits non-typeable Haemophilus influenzae.

Commensal bacteria serve as an important line of defense against colonisation by opportunisitic pathogens, but the underlying molecular mechanisms remain poorly explored. Here, we show that strains of a commensal bacterium, Haemophilus haemolyticus, make hemophilin, a heme-binding protein that inhibits growth of the opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) in culture. We purified the NTHi-inhibitory protein from H. haemolyticus and identified the hemophilin gene using proteomics and a gene knockout. An x-ray crystal structure of recombinant hemophilin shows that the protein does not belong to any of the known heme-binding protein folds, suggesting that it evolved independently. Biochemical characterisation shows that heme can be captured in the ferrous or ferric state, and with a variety of small heme-ligands bound, suggesting that hemophilin could function under a range of physiological conditions. Hemophilin knockout bacteria show a limited capacity to utilise free heme for growth. Our data suggest that hemophilin is a hemophore and that inhibition of NTHi occurs by heme starvation, raising the possibility that competition from hemophilin-producing H. haemolyticus could antagonise NTHi colonisation in the respiratory tract.