Transcriptome Analysis Reveals Novel Inflammatory Signalings to Glaesserella parasuis Infection.

Glaesserella parasuis (GPS) can cause severe systemic inflammation in pigs, resulting in huge economic losses to the pig industry. At present, no effective method is available for the prevention and control of GPS infection. Molecular breeding for disease resistance is imminent, but disease-resistance genes have not been identified. To study the mechanism of systemic acute inflammation caused by GPS, we established three in vitro infection models (3D4/21 cells, PK15 cells, and PAVEC cells) according to its infection path. There was no significant difference in apoptosis among the three kinds of cells after 12 h of continuous GPS stimulation, while inflammatory factors were significantly upregulated. Subsequent transcriptome analysis revealed 1969, 1207, and 3564 differentially expressed genes (DEGs) in 3D4/21 cells, PK15 cells, and PAVEC cells, respectively, after GPS infection. Many of the DEGs were predicted to be associated with inflammatory responses (C3, CD44, etc.); cell proliferation, growth and apoptosis; gene expression; and protein phosphorylation. Key signaling pathways, including S100 family signaling, bacteria and virus recognition, and pathogen-induced cytokine storm signaling, were enriched based on Ingenuity Pathway Analysis (IPA). Furthermore, a total of three putative transmembrane receptors and two putative G-protein-coupled receptors, namely F3, ICAM1, PLAUR, ACKR3, and GPRC5A, were identified by IPA among the three types of cells. ACKR3 and GPRC5A play pivotal roles in bacterial adhesion, invasion, host immune response and inflammatory response through the S100 family signaling pathway. Our findings provide new insights into the pathological mechanisms underlying systemic inflammation caused by GPS infection in pigs, and they lay a foundation for further research on disease-resistance breeding to GPS.

HbpA from Glaesserella parasuis induces an inflammatory response in 3D4/21 cells by activating the MAPK and NF-κB signalling pathways and protects mice against G. parasuis when used as an immunogen.

Glaesserella parasuis is usually a benign swine commensal in the upper respiratory tract, but virulent strains can cause systemic infection characterized by pneumonia, meningitis, and fibrinous polyserositis. The intensive pulmonary inflammatory response following G. parasuis infection is the main cause of lung injury and death in pigs. Vaccination has failed to control the disease due to the lack of extended cross-protection. Accumulating evidence indicates that the heme-binding protein A (HbpA) is a potential virulence determinant and a promising antigen candidate for the development of a broader range of vaccines. However, it is not yet known whether HbpA contributes to G. parasuis virulence or has any potential immune protective effects against G. parasuis. Here, we show that HbpA can induce the transcription and secretion of proinflammatory cytokines (IL-6, TNF-α, and MCP-1) in porcine alveolar macrophages (PAM, 3D4/31). The HbpA protein is recognized by Toll-like receptors 2 and 4 on 3D4/21 macrophages, resulting in the activation of MAP kinase and NF-κB signalling cascades and the transcription and secretion of proinflammatory cytokines. HbpA contributes to virulence and bacterial pulmonary colonization in C57BL/6 mice and plays a role in adhesion to host cells and evasion of the bactericidal effect of pulmonary macrophages. In addition, mice immunized with HbpA were partially protected against challenge by G. parasuis SC1401. The results suggest that HbpA plays an important role in the pathogenesis of disease caused by G. parasuis and lay a foundation for the development of a subunit or chimeric anti-G. parasuis vaccine.

The role of cytolethal distending toxin in Glaesserella parasuis JS0135 strain infection: Cytotoxicity, phagocytic resistance and pathogenicity.

Glaesserella parasuis is an important porcine pathogen that commonly colonizes the upper respiratory tract of pigs and is prone to causing Glässer's disease under complex conditions. As yet, the disease has led to serious economic losses to the swine industry worldwide. Studies so far have found that several virulence factors are associated with the pathogenicity of G. parasuis, but the pathogenic mechanism is still not fully understood. Cytolethal distending toxin (CDT), a potential virulence factor in G. parasuis, is involved in cytotoxicity, serum resistance, adherence to and invasion of host cells in vitro. Here, to further investigate the pathogenic role of CDT during G. parasuis infection in vitro and in vivo, a double cdt1 and cdt2 deletion mutant (Δcdt1Δcdt2) without selectable marker was first generated in G. parasuis JS0135 strain by continuous natural transformations and replica plating. Morphological observation and lactate dehydrogenase assay showed that the Δcdt1Δcdt2 mutant was defective in cytotoxicity. Additionally, the Δcdt1Δcdt2 mutant was more susceptible to phagocytosis caused by 3D4/2 macrophages compared to the wild-type JS0135 strain. Moreover, by focusing on clinical signs, necropsy, bacterial recovery and pathological observation, we found that the deletion of cdt1 and cdt2 genes led to a significant attenuation of virulence in G. parasuis. Taken together, these findings suggest that as an important virulence factor, CDT can significantly affect the pathogenicity of G. parasuis.

Tolerance to Haemophilus influenzae infection in human epithelial cells: Insights from a primary cell-based model.

Haemophilus influenzae is a human respiratory pathogen and inhabits the human respiratory tract as its only niche. Despite this, the molecular mechanisms that allow H. influenzae to establish persistent infections of human epithelia are not well understood. Here, we have investigated how H. influenzae adapts to the host environment and triggers the host immune response using a human primary cell-based infection model that closely resembles human nasal epithelia (NHNE). Physiological assays combined with dualRNAseq revealed that NHNE from five healthy donors all responded to H. influenzae infection with an initial, 'unproductive' inflammatory response that included a strong hypoxia signature but did not produce pro-inflammatory cytokines. Subsequently, an apparent tolerance to large extracellular and intraepithelial burdens of H. influenzae developed, with NHNE transcriptional profiles resembling the pre-infection state. This occurred in parallel with the development of intraepithelial bacterial populations, and appears to involve interruption of NFκB signalling. This is the first time that large-scale, persistence-promoting immunomodulatory effects of H. influenzae during infection have been observed, and we were able to demonstrate that only infections with live, but not heat-killed H. influenzae led to immunomodulation and reduced expression of NFκB-controlled cytokines such as IL-1ß, IL-36γ and TNFα. Interestingly, NHNE were able to re-activate pro-inflammatory responses towards the end of the 14-day infection, resulting in release of IL-8 and TNFα. In addition to providing first molecular insights into mechanisms enabling persistence of H. influenzae in the host, our data further indicate the presence of infection stage-specific gene expression modules, highlighting fundamental similarities between immune responses in NHNE and canonical immune cells, which merit further investigation.

Expression profiles of miRNAs in the lung tissue of piglets infected with Glaesserella parasuis and the roles of ssc-miR-135 and ssc-miR-155-3p in the regulation of inflammation.

MicroRNAs (miRNAs) have been shown to play an important regulatory role in the process of pathogenic infection. However, the miRNAs that regulate the pathogenic process of G. parasuis and their functions are still unknown. Here, high-throughput sequencing was used to quantify the expression of miRNA in piglet lung tissue after G. parasuis XX0306 strain infection. A total of 25 differentially expressed microRNAs (DEmiRNAs) were identified. GO and KEGG pathway enrichment analysis showed that many of the functions of genes that may be regulated by DEmiRNA are related to inflammatory response and immune regulation. Further studies found that ssc-miR-135 may promote the expression of inflammatory factors through NF-κB signaling pathway. Whereas, ssc-miR-155-3p inhibited the inflammatory response induced by G. parasuis, and its regulatory mechanism remains to be further investigated. This study provides a valuable reference for revealing the regulatory effects of miRNAs on the pathogenesis of G. parasuis. DATA AVAILABILITY: The datasets generated during the current study are not publicly available due to this study is currently in the ongoing research stage, and some of the data cannot be made public sooner yet, but are available from the corresponding author on reasonable request.

Activation of the NLRP3-CASP-1 inflammasome is restrained by controlling autophagy during Glaesserella parasuis infection.

Infection with Glaesserella parasuis, the primary pathogen behind Glässer's disease, is often associated with diverse clinical symptoms, including serofibrinous polyserositis, arthritis, and meningitis. Autophagy plays a dual role in bacterial infections, exerting either antagonistic or synergistic effects depending on the nature of the pathogen. Our previous studies have demonstrated that autophagy serves as a defense mechanism, combating inflammation and invasion caused by infection of highly virulent G. parasuis. However, the precise mechanisms remain to be elucidated. Pathogens exhibit distinct interactions with inflammasomes and autophagy processes. Herein, we explored the effect of autophagy on inflammasomes during G. parasuis infection. We found that G. parasuis infection triggers NLRP3-dependent pro-CASP-1-IL-18/IL-1ß processing and maturation pathway, resulting in increased release of IL-1ß and IL-18. Inhibition of autophagy enhances NLRP3 inflammasome activity, whereas stimulation of autophagy restricts it during G. parasuis infection. Furthermore, assembled NLRP3 inflammasomes undergo ubiquitination and recruit the autophagic adaptor, p62, facilitating their sequestration into autophagosomes during G. parasuis infection. These results suggest that the induction of autophagy mitigates inflammation by eliminating overactive NLRP3 inflammasomes during G. parasuis infection. Our research uncovers a mechanism whereby G. parasuis infection initiates inflammatory responses by promoting the assembly of the NLRP3 inflammasomes and activating NLRP3-CASP-1, both of which processes are downregulated by autophagy. This suggests that pharmacological manipulation of autophagy could be a promising approach to modulate G. parasuis-induced inflammatory responses.

Substitutions in the nonactive site of the passenger domain on the activity of Haemophilus influenzae immunoglobulin A1 protease.

Immunoglobulin A1 (IgA1) protease is a critical virulence factor of Haemophilus influenzae that facilitates bacterial mucosal infection. This study investigates the effect of iga gene polymorphism on the enzymatic activity of H. influenzae IgA1 protease. The IgA1 protease activity was examined in the H. influenzae Rd KW20 strain and 51 isolates. Genetic variations in iga and deduced amino acid substitutions affecting IgA1 protease activity were assessed. Machine learning tools and functional complementation assays were used to analyze the effects of identified substitutions on the stability and activity of IgA1 protease, respectively. All 51 isolates exhibited similar iga expression levels. No igaB expression was detected. According to comparisons with the reference Rd KW20 strain, four substitutions in the protease domain, 26 in the nonprotease passenger domain, and two in the ß-barrel domain were associated with the change in IgA1 protease activity. No substitutions in the catalytic site of IgA1 protease were observed. Logistic regression, receiver operating characteristic curves, Venn diagrams, and protein stability analyses revealed that the substitutions Asn352Lys, Pro353Ala, Lys356Asn, Gln916Lys, and Gly917Ser, which were located in the nonactive site of the passenger domain, were associated with decreases in IgA1 protease activity and stability, whereas Asn914Lys was associated with an increase in these events. Functional complementation assays revealed that the Asn914Lys substitution increased IgA1 protease activity in the Rd KW20 strain. This study identified substitutions in the nonactive site of the passenger domain that affect both the activity and stability of H. influenzae IgA1 protease.

Semisynthetic Glycoconjugates as Potential Vaccine Candidates Against Haemophilus influenzae Type a.

Glycoconjugate vaccines are based on chemical conjugation of pathogen-associated carbohydrates with immunogenic carrier proteins and are considered a very cost-effective way to prevent infections. Most of the licensed glycoconjugate vaccines are composed of saccharide antigens extracted from bacterial sources. However, synthetic oligosaccharide antigens have become a promising alternative to natural polysaccharides with the advantage of being well-defined structures providing homogeneous conjugates. Haemophilus influenzae (Hi) is responsible for a number of severe diseases. In recent years, an increasing rate of invasive infections caused by Hi serotype a (Hia) raised some concern, because no vaccine targeting Hia is currently available. The capsular polysaccharide (CPS) of Hia is constituted by phosphodiester-linked 4-ß-d-glucose-(1→4)-d-ribitol-5-(PO4→) repeating units and is the antigen for protein-conjugated polysaccharide vaccines. To investigate the antigenic potential of the CPS from Hia, we synthesized related saccharide fragments containing up to five repeating units. Following the synthetic optimization of the needed disaccharide building blocks, they were assembled using the phosphoramidite approach for the installation of the phosphodiester linkages. The resulting CPS-based Hia oligomers were conjugated to CRM197 carrier protein and evaluated in vivo for their immunogenic potential, showing that all glycoconjugates were capable of raising antibodies recognizing Hia synthetic fragments.

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.

Antibody persistence to diphtheria toxoid, tetanus toxoid, Bordetella pertussis antigens, and Haemophilus influenzae type b following primary and first booster with pentavalent versus hexavalent vaccines.

Thailand has incorporated the whole-cell (wP) pertussis vaccine into the expanded program on immunization since 1977 and has offered the acellular pertussis (aP) vaccine as an optional vaccine for infants since 2001. We followed healthy children from a clinical trial (ClinicalTrials.gov NCT02408926) in which children were randomly assigned to receive either pentavalent (DTwP-HB-Hib) or hexavalent (DTaP-IPV-HB-Hib) vaccines for their primary series (administered at 2, 4, and 6 months) and first booster vaccination (18 months). Both groups received Tdap-IPV as a second booster at the age of 4 y. Blood samples were collected for evaluation of antibody persistence to diphtheria toxoid (DT), tetanus toxoid (TT), and Bordetella pertussis (B. pertussis) between 2 and 6 y of age annually, and for the immunogenicity study of Tdap-IPV at 1 month after the second booster. Antibody persistence to Haemophilus influenzae type b (Hib) was followed until 3 y of age. A total of 105 hexavalent-vaccinated children and 91 pentavalent-vaccinated children completed this study. Both pentavalent and hexavalent groups demonstrated increased antibody levels against DT, TT, and B. pertussis antigens following the second booster with Tdap-IPV. All children achieved a seroprotective concentration for anti-DT and anti-TT IgG at 1 month post booster. The hexavalent group possessed significantly higher anti-pertactin IgG (adjusted p = .023), whereas the pentavalent group possessed significantly higher anti-pertussis toxin IgG (adjusted p < .001) after the second booster. Despite declining levels post-second booster, a greater number of children sustained protective levels of anti-DT and anti-TT IgG compared to those after the first booster.

Designing of a multi-epitopes based vaccine against Haemophilius parainfluenzae and its validation through integrated computational approaches.

Haemophilus parainfluenzae is a Gram-negative opportunist pathogen within the mucus of the nose and mouth without significant symptoms and has an ability to cause various infections ranging from ear, eye, and sinus to pneumonia. A concerning development is the increasing resistance of H. parainfluenzae to beta-lactam antibiotics, with the potential to cause dental infections or abscesses. The principal objective of this investigation is to utilize bioinformatics and immuno-informatic methodologies in the development of a candidate multi-epitope Vaccine. The investigation focuses on identifying potential epitopes for both B cells (B lymphocytes) and T cells (helper T lymphocytes and cytotoxic T lymphocytes) based on high non-toxic and non-allergenic characteristics. The selection process involves identifying human leukocyte antigen alleles demonstrating strong associations with recognized antigenic and overlapping epitopes. Notably, the chosen alleles aim to provide coverage for 90% of the global population. Multi-epitope constructs were designed by using suitable linker sequences. To enhance the immunological potential, an adjuvant sequence was incorporated using the EAAAK linker. The final vaccine construct, comprising 344 amino acids, was achieved after the addition of adjuvants and linkers. This multi-epitope Vaccine demonstrates notable antigenicity and possesses favorable physiochemical characteristics. The three-dimensional conformation underwent modeling and refinement, validated through in-silico methods. Additionally, a protein-protein molecular docking analysis was conducted to predict effective binding poses between the multi-epitope Vaccine and the Toll-like receptor 4 protein. The Molecular Dynamics (MD) investigation of the docked TLR4-vaccine complex demonstrated consistent stability over the simulation period, primarily attributed to electrostatic energy. The docked complex displayed minimal deformation and enhanced rigidity in the motion of residues during the dynamic simulation. Furthermore, codon translational optimization and computational cloning was performed to ensure the reliability and proper expression of the multi-Epitope Vaccine. It is crucial to emphasize that despite these computational validations, experimental research in the laboratory is imperative to demonstrate the immunogenicity and protective efficacy of the developed vaccine. This would involve practical assessments to ascertain the real-world effectiveness of the multi-epitope Vaccine.

Lung mucosal immunity to NTHi vaccine antigens: Antibodies in sputum of chronic obstructive pulmonary disease patients.

Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory illness in older adults. A major cause of COPD-related morbidity and mortality is acute exacerbation of COPD (AECOPD). Bacteria in the lungs play a role in exacerbation development, and the most common pathogen is non-typeable Haemophilus influenzae (NTHi). A vaccine to prevent AECOPD containing NTHi surface antigens was tested in a clinical trial. This study measured IgG and IgA against NTHi vaccine antigens in sputum. Sputum samples from 40 COPD patients vaccinated with the NTHi vaccine were collected at baseline and 30 days after the second dose. IgG and IgA antibodies against the target antigens and albumin were analyzed in the sputum. We compared antibody signals before and after vaccination, analyzed correlation with disease severity and between sputum and serum samples, and assessed transudation. Antigen-specific IgG were absent before vaccination and present with high titers after vaccination. Antigen-specific IgA before and after vaccination were low but significantly different for two antigens. IgG correlated between sputum and serum, and between sputum and disease severity. Sputum albumin was higher in patients with severe COPD than in those with moderate COPD, suggesting changes in transudation played a role. We demonstrated that immunization with the NTHi vaccine induces antigen-specific antibodies in sputum. The correlation between IgG from sputum and serum and the presence of albumin in the sputum of severe COPD patients suggested transudation of antibodies from the serum to the lungs, although local IgG production could not be excluded.Clinical Trial Registration: NCT02075541.

What is the context? Chronic obstructive pulmonary disease (COPD) is the most common chronic respiratory illness in older adults and the third leading cause of death worldwide.One bacterium in the lungs, non-typeable Haemophilus influenzae (NTHi), is responsible for acute exacerbation of the disease, characterized by an increase in airway wall inflammation and symptoms, leading to high morbidity and mortality.A vaccine targeting NTHi was previously developed but did not show efficacy in reducing exacerbations in COPD patients, probably because the vaccine did not elicit an immune response in the lung mucosae, where the bacteria are located.What is the impact? Parenteral immunization with new vaccines targeting NTHi is able to elicit immune defense at the level of lung mucosae.Now that antibodies can be measured in sputum, new vaccines against COPD exacerbations or other lung infections can be tested for efficacy in the actual target tissue.Also, lung immunity against specific pathogens can now be tested.What is new? We determined that antigen-specific antibodies were present in the lungs after vaccination; these were assessed in sputum after vaccination with NTHi surface antigens.NTHi-specific IgG were present in the lungs and appeared to have arrived there primarily by transudation, a type of leakage from the serum to the lung mucosae.Transudation appeared to be stronger in severe than in moderate COPD patients.

Disparate kinetics in immune response of two different Haemophilus influenzae type b conjugate vaccines: Immunogenicity and safety observations from a randomized controlled phase IV study in healthy infants and toddlers using a 2+1 schedule.

Since the introduction of Haemophilus Influenzae type b (Hib) conjugate vaccines, invasive Hib disease has strongly declined worldwide, yet continued control of Hib disease remains important. In Europe, currently three different hexavalent combination vaccines containing Hib conjugates are marketed. In this phase IV, single-blind, randomized, controlled, multi-country study (NCT04535037), we aimed to compare, in a 2 + 1 vaccination schedule, the immunogenicity and safety and show non-inferiority, as well as superiority, of DTPa-HBV-IPV/Hib (Ih group) versus DTaP5-HB-IPV-Hib (Va group) in terms of anti-polyribosylribitol phosphate (PRP) antibody geometric mean concentrations (GMCs) and proportion of participants reaching anti-PRP antibody concentrations greater than or equal to a threshold of 5 µg/mL. One month after the booster vaccination, the anti-PRP antibody GMC ratio (Ih group/Va group) was 0.917 (95% CI: 0.710-1.185), meeting the non-inferiority criteria. The difference in percentage of participants (Ih group - Va group) reaching GMCs ≥5 µg/mL was -6.3% (95% CI: -14.1% to 1.5%), not reaching the predefined non-inferiority threshold. Interestingly, a slightly higher post-booster antibody avidity was observed in the Ih group versus the Va group. Both vaccines were well tolerated, and no safety concerns were raised. This study illustrates the different kinetics of the anti-PRP antibody response post-primary and post-booster using the two vaccines containing different Hib conjugates and indicates a potential differential impact of concomitant vaccinations on the anti-PRP responses. The clinical implications of these differences should be further studied.

Vaccination against Haemophilus influenzae type b (Hib) is included in the majority of national immunization programs worldwide and has shown to be effective in preventing Hib disease. In Europe, different vaccines containing Hib components are marketed. We compared the immune response and safety of 2 of these (DTPa-HBV-IPV/Hib, Ih group) and DTaP5-HB-IPV-Hib, Va group) in infants and toddlers, when used in a 2 + 1 schedule, i.e. two primary vaccination doses (at 2 and 4 months of age of the infant), followed by one booster dose at the age of one year. One month after the booster vaccination, the antibody concentration ratio between both groups (Ih group/Va group) was 0.917 (95% CI: 0.710­1.185) showing the DTPa-HBV-IPV/Hib vaccine was non-inferior to the DTaP5-HB-IPV-Hib vaccine; the difference in percentage of participants (Ih group ­ Va group) with antibody concentrations above 5 µg/mL was -6.3% (95% CI: −14.1% to 1.5%), which did not meet the pre-defined criterion for non-inferiority. In the Ih group, the quality of antibodies produced was somewhat higher versus the Va group. Both vaccines were well tolerated, and no safety concerns were raised. The kinetics of the immune response are different between the 2 vaccines. Since both vaccines contain different additional components (conjugated proteins), a possible effect of concomitant (simultaneously administered) vaccines was studied. Further investigations to confirm our findings are needed.

17ß-estradiol Attenuates the Middle Ear Inflammatory Response to Nontypeable Haemophilus influenzae.

OBJECTIVES: 17ß-estradiol (E2) is a steroidal hormone with immunomodulatory functions that play a role in infectious and inflammatory diseases. E2 was recently identified as the leading upstream regulator of differentially expressed genes in a comparative RNA sequencing study of pediatric patients with otitis media (OM) versus OM-free counterparts and may therefore play a role in the inflammatory response to bacterial otopathogens during pediatric OM. This study examined the effect of E2 on bacterial-induced inflammatory cytokine expression in an in vitro pediatric OM model. METHODS: An immortalized middle ear (ME) epithelial cell line, ROM-SV40, was developed from a pediatric recurrent OM patient. The culture was exposed to E2 at physiological levels for 1-48 h prior to 6 h-stimulation with nontypeable Haemophilus influenzae (NTHi) whole cell lysate. TNFA, IL1B, IL6, and IL8 were assayed by qPCR and ELISA. RESULTS: E2 pretreatment (24 h) abrogated NTHi induction of IL6; a longer pretreatment (1-10 nM, 48 h) abrogated IL1B induction (p < 0.05). E2 pretreatment (5 nM, 48 h) abrogated NTHi-induced IL8 secretion (p = 0.017). CONCLUSION: E2 pretreatment partially rescued NTHi-induced cytokine production by ME epithelia. These data support a role for E2 in moderating the excessive inflammatory response to middle ear infection that contributes to OM pathophysiology. LEVELS OF EVIDENCE: NA Laryngoscope, 134:3815-3819, 2024.

Biofilm Formation by Nontypeable Haemophilus Influenzae and Resistance to Complement-Mediated Clearance.

Biofilm formation has been suggested to be associated with phenotype changes compared with the planktonic form. We screened 1092 Haemophilus influenzae isolates for their genetic relationships and then selected 29 isolates from different genotypes and phenotypes and tested their ability to form biofilm. Our data showed a higher capacity of nontypable isolates, particularly isolates from respiratory and genital infections to form biofilm, compared with typable isolates. This ability to form biofilm was also correlated with reduced deposition of the complement component C3b on biofilm-involved bacteria. These data suggest that the biofilm formation contributes to the virulence of nontypable H. influenzae.

Evaluation of the indirect impact of the 10-valent pneumococcal Haemophilus influenzae protein D conjugate vaccine in a cluster-randomised trial.

BACKGROUND: In the nation-wide double-blind cluster-randomised Finnish Invasive Pneumococcal disease trial (FinIP, ClinicalTrials.gov NCT00861380, NCT00839254), we assessed the indirect impact of the 10-valent pneumococcal Haemophilus influenzae protein D conjugate vaccine (PHiD-CV10) against five pneumococcal disease syndromes. METHODS: Children 6 weeks to 18 months received PHiD-CV10 in 48 clusters or hepatitis B/A-vaccine as control in 24 clusters according to infant 3+1/2+1 or catch-up schedules in years 2009-2011. Outcome data were collected from national health registers and included laboratory-confirmed and clinically suspected invasive pneumococcal disease (IPD), hospital-diagnosed pneumonia, tympanostomy tube placements (TTP) and outpatient antimicrobial prescriptions. Incidence rates in the unvaccinated population in years 2010-2015 were compared between PHiD-CV10 and control clusters in age groups <5 and ≥5 years (5-7 years for TTP and outpatient antimicrobial prescriptions), and in infants <3 months. PHiD-CV10 was introduced into the Finnish National Vaccination Programme (PCV-NVP) for 3-month-old infants without catch-up in 9/2010. RESULTS: From 2/2009 to 10/2010, 45398 children were enrolled. Vaccination coverage varied from 29 to 61% in PHiD-CV10 clusters. We detected no clear differences in the incidence rates between the unvaccinated cohorts of the treatment arms, except in single years. For example, the rates of vaccine-type IPD, non-laboratory-confirmed IPD and empyema were lower in PHiD-CV10 clusters compared to control clusters in 2012, 2015 and 2011, respectively, in the age-group ≥5 years. CONCLUSIONS: This is the first report from a clinical trial evaluating the indirect impact of a PCV against clinical outcomes in an unvaccinated population. We did not observe consistent indirect effects in the PHiD-CV10 clusters compared to the control clusters. We consider that the sub-optimal trial vaccination coverage did not allow the development of detectable indirect effects and that the supervening PCV-NVP significantly diminished the differences in PHiD-CV10 vaccination coverage between the treatment arms.

Transcriptome profiling identifies immune response genes against porcine reproductive and respiratory syndrome virus and Haemophilus parasuis co-infection in the lungs of piglets.

BACKGROUND: Co-infections of the porcine reproductive and respiratory syndrome virus (PRRSV) and the Haemophilus parasuis (HPS) are severe in Chinese pigs, but the immune response genes against co-infected with 2 pathogens in the lungs have not been reported. OBJECTIVES: To understand the effect of PRRSV and/or HPS infection on the genes expression associated with lung immune function. METHODS: The expression of the immune-related genes was analyzed using RNA-sequencing and bioinformatics. Differentially expressed genes (DEGs) were detected and identified by quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC) and western blotting assays. RESULTS: All experimental pigs showed clinical symptoms and lung lesions. RNA-seq analysis showed that 922 DEGs in co-challenged pigs were more than in the HPS group (709 DEGs) and the PRRSV group (676 DEGs). Eleven DEGs validated by qRT-PCR were consistent with the RNA sequencing results. Eleven common Kyoto Encyclopedia of Genes and Genomes pathways related to infection and immune were found in single-infected and co-challenged pigs, including autophagy, cytokine-cytokine receptor interaction, and antigen processing and presentation, involving different DEGs. A model of immune response to infection with PRRSV and HPS was predicted among the DEGs in the co-challenged pigs. Dual oxidase 1 (DUOX1) and interleukin-21 (IL21) were detected by IHC and western blot and showed significant differences between the co-challenged pigs and the controls. CONCLUSIONS: These findings elucidated the transcriptome changes in the lungs after PRRSV and/or HPS infections, providing ideas for further study to inhibit ROS production and promote pulmonary fibrosis caused by co-challenging with PRRSV and HPS.

Nontypeable Haemophilus influenzae P5 Binds Human C4b-Binding Protein, Promoting Serum Resistance.

Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes infections mainly in the upper and lower respiratory tract. The bacterium is associated with bronchitis and exacerbations in patients suffering from chronic obstructive pulmonary disease and frequently causes acute otitis media in preschool children. We have previously demonstrated that the binding of C4b binding protein (C4BP) is important for NTHi complement evasion. In this study, we identified outer membrane protein 5 (P5) of NTHi as a novel ligand of C4BP. Importantly, we observed significantly lower C4BP binding and decreased serum resistance in P5-deficient NTHi mutants. Surface expression of recombinant P5 on Escherichia coli conferred C4BP binding and consequently increased serum resistance. Moreover, P5 expression was positively correlated with C4BP binding in a series of clinical isolates. We revealed higher levels of P5 surface expression and consequently more C4BP binding in isolates from the lower respiratory tract of chronic obstructive pulmonary disease patients and tonsil specimens compared with isolates from the upper respiratory tract and the bloodstream (invasive strains). Our results highlight P5 as an important protein for protecting NTHi against complement-mediated killing.

Differences in Pneumococcal and Haemophilus influenzae Natural Antibody Development in Papua New Guinean Children in the First Year of Life.

Background: Development of vaccines to prevent disease and death from Streptococcus pneumoniae, and nontypeable Haemophilus influenzae (NTHi), the main pathogens that cause otitis media, pneumonia, meningitis and sepsis, are a global priority. Children living in low and lower-middle income settings are at the highest risk of contracting and dying from these diseases. Improved vaccines with broader coverage are required. Data on the natural development of antibodies to putative vaccine antigens, especially in high-risk settings, can inform the rational selection of the best antigens for vaccine development. Methods: Serum IgG titres to four pneumococcal proteins (PspA1, PspA2, CbpA, and Ply) and five NTHi antigens (P4, P6, OMP26, rsPilA and ChimV4) were measured in sera collected from 101 Papua New Guinean children at 1, 4, 9, 10, 23 and 24 months of age using multiplexed bead-based immunoassays. Carriage density of S. pneumoniae and H. influenzae were assessed by quantitative PCR on genomic DNA extracted from nasopharyngeal swabs using species-specific primers and probes. All data were log-transformed for analysis using Student's unpaired t-tests with geometric mean titre (GMT) or density (GMD) calculated with 95% confidence intervals (CI). Results: Serum -pneumococcal protein-specific IgG titres followed a "U" shaped pattern, with a decrease in presumably maternally-derived IgG titres between 1 and 4 months of age and returning to similar levels as those measured at 1 month of age by 24 months of age. In contrast, NTHi protein-specific IgG titres steadily increased with age. There was no correlation between antibody titres and carriage density for either pathogen. Conclusion: This longitudinal study indicates that the waning of maternally- derived antibodies that is usually observed in infants, after infants does not occur for NTHi antigens in Papua New Guinean infants. Whether NTHi antigen IgG can be transferred maternally remains to be determined. Vaccines that are designed to specifically increase the presence of protective NTHi antibodies in the first few months of life may be most effective in reducing NTHi disease. Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT01619462.

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.