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Secretory (S) IgA antibodies against severe acute respiratory syndrome (SARS)-CoV-2 are induced in saliva and upper respiratory tract (URT) secretions by natural infection and may be critical in determining the outcome of initial infection. Secretory IgA1 (SIgA1) is the predominant isotype of antibodies in these secretions. Neutralization of SARS-CoV-2 is most effectively accomplished by polymeric antibodies such as SIgA. We hypothesize that cleavage of SIgA1 antibodies against SARS-CoV-2 by unique bacterial IgA1 proteases to univalent Fabα antibody fragments with diminished virus neutralizing activity would facilitate the descent of the virus into the lungs to cause serious disease and also enhance its airborne transmission to others. Recent studies of the nasopharyngeal microbiota of patients with SARS-CoV-2 infection have revealed significant increases in the proportions of IgA1 protease-producing bacteria in comparison with healthy subjects. Similar considerations might apply also to other respiratory viral infections including influenza, possibly explaining the original attribution of influenza to Haemophilus influenzae , which produces IgA1 protease., Competing Interests: The authors declare no conflict of interest.

Background: Recurrent respiratory tract infections (rRTIs) are a common reason for immunodiagnostic testing in children, which relies on serum antibody level measurements. However, because RTIs predominantly affect the respiratory mucosa, serum antibodies may inaccurately reflect local immune defences. We investigated antibody responses in saliva and their interplay with the respiratory microbiota in relation to RTI severity and burden in young children with rRTIs., Methods: We conducted a prospective cohort study including 100 children aged <10 years with rRTIs, their family members and healthy healthcare professionals. Total and polyreactive antibody concentrations were determined in serum and saliva (ELISA); respiratory microbiota composition (16S rRNA sequencing) and respiratory viruses (quantitative PCR) were characterised in nasopharyngeal swabs. Proteomic analysis (Olink) was performed on saliva and serum samples. RTI symptoms were monitored with a daily mobile phone application and assessed using latent class analysis and negative binomial mixed models., Results: Serum antibody levels were not associated with RTI severity. Strikingly, 28% of salivary antibodies and only 2% of serum antibodies displayed polyreactivity (p<0.001). Salivary polyreactive IgA was negatively associated with recurrent lower RTIs (adjusted OR 0.80, 95% CI 0.67-0.94) and detection of multiple respiratory viruses (adjusted OR 0.76, 95% CI 0.61-0.96). Haemophilus influenzae abundance was positively associated with RTI symptom burden (regression coefficient 0.05, 95% CI 0.02-0.08)., Conclusion: These results highlight the importance of mucosal immunity in RTI severity and burden, and suggest that the level of salivary polyreactive IgA and H. influenzae abundance may serve as indicators of infection severity and burden in young children with rRTIs., Competing Interests: Conflict of interest: D. Bogaert reports grants from GSK and OM Pharma, speaker fees from MedImmune, and a leadership role as Treasurer of the International Society of Pneumonia and Pneumococcal Diseases, all outside the submitted work. The authors have no other conflicts of interest to disclose., (Copyright ©The authors 2024.)

Objective: In a previous study we have shown that, in the presence of interleukin (IL)-33, repeated, per-nasal challenge of murine airways with Streptococcus pneumoniae ( S. pneumoniae ) organisms induces human asthma-like airways inflammation. It is not clear, however, whether this effect is unique or manifest in response to other common respiratory pathogens. Methods: To explore this, airways of BALB/c mice were repeatedly challenged per-nasally with formaldehyde-inactivated bacterial bodies in the presence or absence of murine recombinant IL-33. Serum concentrations of S.pneumoniae , Moraxella catarrhalis ( M.catarrhalis ) and Haemophilus influenzae ( H.influenzae ) lysates-specific IgE were measured in patients with asthma and control subjects. Results: We showed that in the presence of IL-33, repeated, per-nasal airways exposure to the bodies of these bacteria induced airways hyperresponsiveness (AHR) in the experimental mice. This was accompanied by cellular infiltration into bronchoalveolar lavage fluid (BALF), eosinophilic infiltration and mucous hypertrophy of the lung tissue, with elevated local expression of some type 2 cytokines and elevated, specific IgG and IgE in the serum. The precise characteristics of the inflammation evoked by exposure to each bacterial species were distinguishable. Conclusions: These results suggest that in the certain circumstances, inhaled or commensal bacterial body antigens of both Gram-positive ( S. pneumoniae ) and Gram-negative ( M. catarrhalis and H. influenzae ) respiratory tract bacteria may initiate type 2 inflammation typical of asthma in the airways. In addition, we demonstrated that human asthmatic patients manifest elevated serum concentrations of M.catarrhalis- and H.influenzae- specific IgE.

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-β-d-glucose-(1→4)-d-ribitol-5-(PO 4 →) 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 CRM 197 carrier protein and evaluated in vivo for their immunogenic potential, showing that all glycoconjugates were capable of raising antibodies recognizing Hia synthetic fragments., (© 2024 Wiley-VCH GmbH.)

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-1β, 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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kappler et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Antibodies from sera of a multiple sclerosis (MS) patient subpopulation preferentially recognize the hyperglucosylated adhesin protein HMW1ct(Glc) of the pathogen Haemophilus influenzae. This protein is the first example of an N-glucosylated native antigen candidate, potentially triggering pathogenic antibodies in MS. Specific antibodies in patients' sera can be isolated exploiting their biospecific interaction with antigens by affinity chromatography. Herein, the proteins HMW1ct and HMW1ct(Glc) were first immobilized on appropriately functionalized supports and further used to purify antibodies directly from MS patients sera. We describe a protocol to obtain an antibody fraction specifically recognizing the glusosylated residues on the HMW1ct(Glc) adhesin protein depleting antibodies to the unglucosylated HMW1ct sequence. Different elution solutions have been tested to recover the purified antibody fraction, strongly bound to the immobilized HMW1ct(Glc) adhesin protein., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Australian First Nations children are at very high risk of early, recurrent, and persistent bacterial otitis media and respiratory tract infection. With the PREVIX randomised controlled trials, we aimed to evaluate the immunogenicity of novel pneumococcal conjugate vaccine (PCV) schedules., Methods: PREVIX&#95;BOOST was a parallel, open-label, outcome-assessor-blinded, randomised controlled trial. Aboriginal children living in remote communities of the Northern Territory of Australia were eligible if they had previously completed the three-arm PREVIX&#95;COMBO randomised controlled trial of the following vaccine schedules: three doses of a 13-valent PCV (PCV13; PPP) or a ten-valent pneumococcal Haemophilus influenzae protein D conjugate vaccine (PHiD-CV10; SSS) given at 2, 4, and 6 months, or SSS given at 1, 2, and 4 months followed by PCV13 at 6 months (SSSP). At age 12 months, eligible children were randomly assigned by a computer-generated random sequence (1:1, stratified by primary group allocation) to receive either a PCV13 booster or a PHiD-CV10 booster. Analyses used intention-to-treat principles. Co-primary outcomes were immunogenicity against protein D and serotypes 3, 6A, and 19A. Immunogenicity measures were geometric mean concentrations (GMC) and proportion of children with IgG concentrations of 0·35 μg/mL or higher (threshold for invasive pneumococcal disease), and GMCs and proportion of children with antibody levels of 100 EU/mL or higher against protein D. Standardised assessments of otitis media, hearing impairment, nasopharyngeal carriage, and developmental outcomes are reported. These trials are registered with ClinicalTrials.gov (NCT01735084 and NCT01174849)., Findings: Between April 10, 2013, and Sept 4, 2018, 261 children were randomly allocated to receive a PCV13 booster (n=131) or PHiD-CV10 booster (n=130). Adequate serum samples for pneumococcal serology were obtained from 127 (95%) children in the PCV13 booster group and 126 (97%) in the PHiD-CV10 booster group; for protein D, adequate samples were obtained from 126 (96%) children in the PCV13 booster group and 123 (95%) in the PHiD-CV10 booster group. The proportions of children with IgG concentrations above standard thresholds in PCV13 booster versus PHiD-CV10 booster groups were the following: 71 (56%) of 126 versus 81 (66%) of 123 against protein D (difference 10%, 95% CI -2 to 22), 85 (67%) of 127 versus 59 (47%) of 126 against serotype 3 (-20%, -32 to -8), 119 (94%) of 127 versus 91 (72%) of 126 against serotype 6A (-22%, -31 to -13), and 116 (91%) of 127 versus 108 (86%) of 126 against serotype 19A (-5%, -13 to 3). Infant PCV13 priming mitigated differences between PCV13 and PHiD-CV10 boosters. In both groups, we observed a high prevalence of otitis media (about 90%), hearing impairment (about 75%), nasopharyngeal carriage of pneumococcus (about 66%), and non-typeable H influenzae (about 57%). Of 66 serious adverse events, none were vaccine related., Interpretation: Low antibody concentrations 6 months post-booster might indicate increased risk of pneumococcal infection. The preferred booster was PCV13 if priming did not have PCV13, otherwise either PCV13 or PHiD-CV10 boosters provided similar immunogenicity. Mixed schedules offer flexibility to regional priorities. Non-PCV13 serotypes and non-typeable H influenzae continue to cause substantial disease and disability in Australian First Nation's children., Funding: National Health and Medical Research Council (NHMRC)., Competing Interests: Declaration of interests ABC served as advisor on an unlicensed vaccine (GlaxoSmithKline) and an unlicensed monoclonal antibody (AstraZeneca), was an adviser on an unlicensed molecule for chronic cough (Merck); and has various project grants and a Centre of Research Excellence relating to various aspects of bronchiectasis in children from the National Health and Medical Research Council. PM served on a data safety and monitoring board for the Novavax COVID-19 vaccine. JB provided a report to MSD Australia. All other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: In chronic obstructive pulmonary disease (COPD), exacerbations cause acute inflammatory flare-ups and increase the risk for hospitalization and mortality. Exacerbations are common in all disease stages and are often caused by bacterial infections e.g., non-typeable Heamophilus influenzae (NTHi). Accumulating evidence also associates vitamin D deficiency with the severity of COPD and exacerbation frequency. However, it is still unclear whether vitamin D deficiency when combined with cigarette smoking would worsen and prolong exacerbations caused by repeated infections with the same bacterial strain., Methods: Vitamin D sufficient (VDS) and deficient (VDD) mice were exposed to nose-only cigarette smoke (CS) for 14 weeks and oropharyngeally instilled with NTHi at week 6, 10 and 14. Three days after the last instillation, mice were assessed for lung function, tissue remodeling, inflammation and immunity. The impact of VDD and CS on inflammatory cells and immunoglobulin (Ig) production was also assessed in non-infected animals while serum Ig production against NTHi and dsDNA was measured in COPD patients before and 1 year after supplementation with Vitamin D3., Results: VDD enhanced NTHi eradication, independently of CS and complete eradication was reflected by decreased anti-NTHi Ig's within the lung. In addition, VDD led to an increase in total lung capacity (TLC), lung compliance (Cchord), MMP12/TIMP1 ratio with a rise in serum Ig titers and anti-dsDNA Ig's. Interestingly, in non-infected animals, VDD exacerbated the CS-induced anti-NTHi Ig's, anti-dsDNA Ig's and inflammatory cells within the lung. In COPD patients, serum Ig production was not affected by vitamin D status but anti-NTHi IgG increased after vitamin D3 supplementation in patients who were Vitamin D insufficient before treatment., Conclusion: During repeated infections, VDD facilitated NTHi eradication and resolution of local lung inflammation through production of anti-NTHi Ig, independently of CS whilst it also promoted autoantibodies. In COPD patients, vitamin D supplementation could be protective against NTHi infections in vitamin D insufficient patients. Future research is needed to decipher the determinants of dual effects of VDD on adaptive immunity., Trail Registration: ClinicalTrials, NCT00666367. Registered 23 April 2008, https://www.clinicaltrials.gov/ct2/show/study/NCT00666367 ., (© 2022. The Author(s).)

The mucosal immune system prevents microorganism invasion through mucosal surfaces and consists of inductive and effector sites. Nasopharynx-associated lymphoid tissue (NALT) functions as an inductive site, inducing mucosal immune responses in the upper respiratory tract. It follows that intranasal vaccines may prevent upper respiratory infections. To induce and enhance the immune response by administering inactivated antigens intranasally, mucosal adjuvants have been developed, including mutant cholera toxin and cationic cholesteryl pullulan nanogel, which do not accumulate in the central nervous system. Moreover, multivalent pneumococcal polysaccharide conjugate vaccines are used to prevent invasive pneumococcal infections and otitis media, although they only provide moderate protection against acute otitis media because non-vaccine serotypes of Streptococcus pneumoniae and Haemophilus influenzae also cause this infection. To address this problem, pneumococcal surface protein A of S. pneumoniae and P6 of H. influenzae are used as broad-spectrum vaccine antigens. Alternatively, phosphorylcholine (PC) is present in the cell walls of both gram-positive and gram-negative bacteria and induces immune responses through antigenic activity. The significant effects of PC as a mucosal vaccine have been demonstrated through intranasal and sublingual immunization in mice. Furthermore, intranasal administration of PC reverses increases in IgE levels and prevents allergic rhinitis. After immunization with pneumococcal polysaccharide conjugate vaccine, intranasal immunization with PC boosts immune responses to vaccine strains and to PC itself. Thus, PC may be useful as a mucosal vaccine to prevent upper respiratory infections and allergic rhinitis, and it could be used as a booster to the currently used pneumococcal vaccine as it protects against non-vaccine strains., (Copyright © 2021. Published by Elsevier B.V.)

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., Competing Interests: The Finnish Institute for Health and Welfare (THL) received funding for the conduct of the FinIP study from the GSK group of companies. AAP, HN, ER, and JJ are employees of THL. HR-K was an employee of THL at the time of study conduct. DB and LS are employees of the GSK group of companies; MM was an employee of the GSK group of companies. MM, DB and LS have shares in the GSK group of companies. We note that several authors have an affiliation to the commercial funder of this research study: GlaxoSmithKline Biologicals SA. This does not alter our adherence to PLOS ONE policies on sharing data and materials.