Variability of Vaccine Responsiveness in Young Children.

BACKGROUND: Variability in vaccine responsiveness among young children is poorly understood. METHODS: Nasopharyngeal secretions were collected in the first weeks of life for measurement of cytokines/chemokines seeking a biomarker, and blood samples collected at age one year to identify vaccine responsiveness status, defined as low vaccine responder (LVR), normal (NVR) and high (HVR), to test for vaccine antigen-induced immune memory, and for antigen presenting cell (APC) function. RESULTS: Significantly lower specific cytokine/chemokine levels as biosignatures, measurable in nasopharyngeal secretions at infant age 1-3 weeks old, predicted LVR status compared to NVR and HVR children. Antibiotic exposures were correlated with increased occurrence of LVR. At age 1 year old, LVRs had fewer CD4+ T-helper-1 and T-helper-2 memory cells responsive to specific vaccine antigens. APC responses observed among LVRs, both at rest and in response to TLR7/8 stimulation by R848 were suboptimal, suggesting that altered innate immunity may contribute to immune deficiency in LVRs. CONCLUSION: Cytokine biosignatures in the first weeks of life may predict vaccine responsiveness in children during the first year of life. Antibiotic exposure associates with LVR in children. CD4+ T-cell memory induction and APC deficiencies occur in LVR children.

Variability of vaccine responsiveness in early life.

Vaccinations in early life elicit variable antibody and cellular immune responses, sometimes leaving fully vaccinated children unprotected against life-threatening infectious diseases. Specific immune cell populations and immune networks may have a critical period of development and calibration in a window of opportunity occurring during the first 100 days of early life. Among the early life determinants of vaccine responses, this review will focus on modifiable factors involving development of the infant microbiota and metabolome: antibiotic exposure, breast versus formula feeding, and Caesarian section versus vaginal delivery of newborns. How microbiota may serve as natural adjuvants for vaccine responses and how microbiota-derived metabolites influence vaccine responses are also reviewed. Early life poor vaccine responsiveness can be linked to increased infection susceptibility because both phenotypes share similar immunity dysregulation profiles. An early life pre-vaccination endotype, when interventions have the highest potential for success, should be sought that predicts vaccine response trajectories.

Very early life microbiome and metabolome correlates with primary vaccination variability in children.

IMPORTANCE: We show that simultaneous study of stool and nasopharyngeal microbiome reveals divergent timing and patterns of maturation, suggesting that local mucosal factors may influence microbiome composition in the gut and respiratory system. Antibiotic exposure in early life as occurs commonly, may have an adverse effect on vaccine responsiveness. Abundance of gut and/or nasopharyngeal bacteria with the machinery to produce lipopolysaccharide-a toll-like receptor 4 agonist-may positively affect future vaccine protection, potentially by acting as a natural adjuvant. The increased levels of serum phenylpyruvic acid in infants with lower vaccine-induced antibody levels suggest an increased abundance of hydrogen peroxide, leading to more oxidative stress in low vaccine-responding infants.

Window of Susceptibility to Acute Otitis Media Infection.

BACKGROUND: Contemporary, quantitative data are needed to inform recommendations and decision-making regarding referral and surgeon endorsement of tympanostomy tube placement in young children with recurrent acute otitis media (AOM). METHODS: A prospective, observational cohort study of 286 children in a primary care pediatric practice setting, who had at least 1 AOM (range 1-8). Children were followed longitudinally from 6 to 36 months old. AOMs were microbiologically confirmed by tympanocentesis for diagnostic accuracy. A window of susceptibility (WOS) was defined as AOMs closely spaced in time with no gap in occurrence >6 months. For prediction of total number of AOMs, we used a quasi-poisson generalized linear model. RESULTS: Eighty percent of AOMs occurred during child age 6 to 21 months old. Seventy two percent of WOS intervals were <5 months and 97% were <10 months. Clinically applicable models were developed to predict which children would benefit most from tympanostomy tubes. Significant predictors were child age at the first AOM (P < .001) and daycare attendance (P = .03). The age of a child when 2, 3, or 4 AOMs had occurred allowed prediction of the number of additional AOMs that might occur. After insertion of tympanostomy tubes, 16 (52%) of 31 children had no additional AOMs. CONCLUSIONS: Recurrent AOM occurs in a narrow WOS and number of AOMs can be predicted at time of AOM based on child age and daycare attendance. Insertion of tympanostomy tubes likely occurs in many children after the WOS to recurrent AOM has passed or only 1 more AOM may be prevented at most.

Vaccine target and carrier molecule nontypeable Haemophilus influenzae protein D dimerizes like the close Escherichia coli GlpQ homolog but unlike other known homolog dimers.

We have determined the 1.8 Å X-ray crystal structure of nonlipidated (i.e., N-terminally truncated) nontypeable Haemophilus influenzae (NTHi; H. influenzae) protein D. Protein D exists on outer membranes of H. influenzae strains and acts as a virulence factor that helps invade human cells. Protein D is a proven successful antigen in animal models to treat obstructive pulmonary disease (COPD) and otitis media (OM), and when conjugated to polysaccharides also has been used as a carrier molecule for human vaccines, for example in GlaxoSmithKline Synflorix™. NTHi protein D shares high sequence and structural identify to the Escherichia coli (E. coli) glpQ gene product (GlpQ). E. coli GlpQ is a glycerophosphodiester phosphodiesterase (GDPD) with a known dimeric structure in the Protein Structural Database, albeit without an associated publication. We show here that both structures exhibit similar homodimer organization despite slightly different crystal lattices. Additionally, we have observed both the presence of weak dimerization and the lack of dimerization in solution during size exclusion chromatography (SEC) experiments yet have distinctly observed dimerization in native mass spectrometry analyses. Comparison of NTHi protein D and E. coli GlpQ with other homologous homodimers and monomers shows that the E. coli and NTHi homodimer interfaces are distinct. Despite this distinction, NTHi protein D and E. coli GlpQ possess a triose-phosphate isomerase (TIM) barrel domain seen in many of the other homologs. The active site of NTHi protein D is located near the center of this TIM barrel. A putative glycerol moiety was modeled in two different conformations (occupancies) in the active site of our NTHi protein D structure and we compared this to ligands modeled in homologous structures. Our structural analysis should aid in future efforts to determine structures of protein D bound to substrates, analog intermediates, and products, to fully appreciate this reaction scheme and aiding in future inhibitor design.

Antibiotic Use and Vaccine Antibody Levels.

BACKGROUND: The majority of children are prescribed antibiotics in the first 2 years of life while vaccine-induced immunity develops. Researchers have suggested a negative association of antibiotic use with vaccine-induced immunity in adults, but data are lacking in children. METHODS: From 2006 to 2016, children aged 6 to 24 months were observed in a cohort study. A retrospective, unplanned secondary analysis of the medical record regarding antibiotic prescriptions and vaccine antibody measurements was undertaken concurrently. Antibody measurements relative to diphtheria-tetanus-acellular pertussis (DTaP), inactivated polio (IPV), Haemophilus influenzae type b (Hib), and pneumococcal conjugate (PCV) vaccines were made. RESULTS: In total, 560 children were compared (342 with and 218 without antibiotic prescriptions). Vaccine-induced antibody levels to several DTaP and PCV antigens were lower (P < .05) in children given antibiotics. A higher frequency of vaccine-induced antibodies below protective levels in children given antibiotics occurred at 9 and 12 months of age (P < .05). Antibiotic courses over time was negatively associated with vaccine-induced antibody levels. For each antibiotic course the child received, prebooster antibody levels to DTaP antigens were reduced by 5.8%, Hib by 6.8%, IPV by 11.3%, and PCV by 10.4% (all P ≤ .05), and postbooster antibody levels to DTaP antigens were reduced by 18.1%, Hib by 21.3%, IPV by 18.9%, and PCV by 12.2% (all P < .05). CONCLUSIONS: Antibiotic use in children <2 years of age is associated with lower vaccine-induced antibody levels to several vaccines.

Dynamic changes in otopathogens colonizing the nasopharynx and causing acute otitis media in children after 13-valent (PCV13) pneumococcal conjugate vaccination during 2015-2019.

The otopathogens colonizing the nasopharynx (NP) and causing acute otitis media (AOM) have shown dynamic changes following introduction of pneumococcal conjugate vaccines. Five hundred eighty-nine children were prospectively enrolled, 2015-2019. Two thousand fifty-nine visits (1528 healthy, 393 AOM, and 138 AOM follow-up) were studied. Two thousand forty-two NP and 495 middle ear fluid (MEF) samples by tympanocentesis from 319 AOM cases were cultured for bacterial identification and antibiotic susceptibility. Streptococcus pneumoniae (Spn) isolates were serotyped by Quellung, and multi-locus sequence type (ST) determined by genomic analysis. Haemophilus influenzae (Hi) was the most common otopathogen cultured from MEF during AOM (34% in MEF) followed by Spn (24% in MEF), then Moraxella catarrhalis (Mcat) (15% in MEF). NP isolates during healthy visit were Mcat (39%), Spn (32%), Hi (12%). 48.6% of Hi isolates from MEF were beta-lactamase-producing. Spn non-susceptibility to penicillin and other antibiotics was high. The most common Spn serotypes associated with AOM (and colonizing the NP during healthy visits) were 35B, 23B, and 15B/C. ST558 and ST199 were the most common sequence types. During 2015-2019, Hi was the most common otopathogen cultured from MEF during AOM among young children. Pneumococcal AOM was most commonly caused by non-PCV13 serotypes of Spn, predominantly 35B, 23B, and 15B/C. Resistance to common antibiotics among Spn strains showed an increasing trend.

Nasopharyngeal microbiome composition associated with Streptococcus pneumoniae colonization suggests a protective role of Corynebacterium in young children.

Streptococcus pneumoniae (Spn) is a leading respiratory tract pathogen that colonizes the nasopharynx (NP) through adhesion to epithelial cells and immune evasion. Spn actively interacts with other microbiota in NP but the nature of these interactions are incompletely understood. Using 16S rRNA gene sequencing, we analyzed the microbiota composition in the NP of children with or without Spn colonization. 96 children were included in the study cohort. 74 NP samples were analyzed when children were 6 months old and 85 NP samples were analyzed when children were 12 months old. We found several genera that correlated negatively or positively with Spn colonization, and some of these correlations appeared to be influenced by daycare attendance or other confounding factors such as upper respiratory infection (URI) or Moraxella colonization. Among these genera, Corynebacterium showed a consistent inverse relationship with Spn colonization with little influence by daycare attendance or other factors. We isolated Corynebacterium propinquum and C. pseudodiphtheriticum and found that both inhibited the growth of Spn serotype 22F strain in vitro.

Pneumonia, Sinusitis, Influenza and Other Respiratory Illnesses in Acute Otitis Media-Prone Children.

BACKGROUND: Recurrent acute otitis media in the first years of life can be explained by immune dysfunction. Consequently, it would be expected that otitis-prone (OP) children would be more susceptible to other infectious diseases, especially respiratory infections, since a component of the immune problem involves nasopharyngeal innate immunity. DESIGN: Cohort study with prospective identification of all physician-diagnosed, medically attended respiratory illness visits in children 6 months to 5 years of age to determine the incidence of pneumonia, acute sinusitis, influenza and other bacterial and viral infections among OP compared with non-OP (NOP) children. Tympanocentesis to microbiologically confirm acute otitis media disease. RESULTS: Two hundred eighty-five children were studied. Thirty-nine met a standard definition of stringently defined OP (sOP) determined by tympanocentesis and 246 were NOP. sOP children had increased frequency of presumptive respiratory infections, pneumonia (6-fold higher, P < 0.001), sinusitis (2.1-fold higher, P = 0.026) and influenza (2.9-fold higher, P = 0.002), compared with NOP children. Demographic and risk factor covariate-adjusted fold difference between sOP and NOP children for all respiratory infection illness visits was 2.4-fold (P < 0.00001) at 6-18 months of age, 2.2-fold (P < 0.00001) at 18-30 months of age and at age and 2.4-fold (P = 0.035) higher at 30 to 42 months. For both sOP and NOP children, more frequent medically attended respiratory infection illness visits from 6-18 months of age predicted more frequent visits experienced from 18-60 months of age. CONCLUSIONS: Clinicians should be aware of a significant increased likelihood of bacterial and viral respiratory infection proneness among OP children.

Comparison of specific in-vitro virulence gene expression and innate host response in locally invasive vs colonizer strains of Streptococcus pneumoniae.

Among Rochester NY children, a dramatic increase in nasopharyngeal (NP) colonization by non-vaccine pneumococcal serotypes 35B and 15A occurred during years 2010-2015, after introduction of 13-valent pneumococcal conjugate vaccine (PCV13). In our population, serotype 35B strains colonized in the nasopharynx (NP) but infrequently caused acute otitis media (AOM) whereas serotype 15A strains displayed virulence, evidenced by causing AOM. To explain the virulence difference, virulence genes expression between 35B and 15A, as well as the host's immune response during asymptomatic colonization were analyzed. We investigated differences in regulation of 19 virulence genes for differences in virulence using RT-PCR in 20 35B and 14 15A strains and measured gene expression of 9 host innate cytokines in the NP to assess the mucosal inflammatory response during asymptomatic colonization. Comparing 35B versus 15A strains, genes for competence ComA and RrgC were upregulated; capsular (Cps2D) and virulence genes (PfbA, PcpA and PhtE) were downregulated among 35B strains. PavB, LytA, LytB, NanA, CiaR, PhtD, LuxS, PspA and pneumolysin (Ply) showed no difference. IL17 and IL23 gene expression were > tenfold higher during 35B compared to 15A strain asymptomatic colonization. Only IL23 showed significant difference. In the first 5 years after introduction of PCV13, serotype 35B strains emerged as asymptomatic colonizers and 15A strains emerged to cause AOM in young children. Various genes (PfbA, PcpA, Cps2D and PhtE) among tested in this analysis were downregulated in 35B whereas ComA and RrgC were significantly upregulated. For the host's cytokine response, IL23 proinflammatory response which is essential for the differentiation of Th17 lymphocytes in the NP of children with 35B strains was significantly higher than the response to 15A during asymptomatic colonization.

Nasopharyngeal microbiome analyses in otitis-prone and otitis-free children.

OBJECTIVES: About 10-15% children develop frequent acute otitis media (AOM) confirmed by tympanocentesis. These children are designated sOP (stringently defined otitis-prone) because all AOM episodes have been microbiologically confirmed. The cause of otitis-proneness in sOP children is multi-factorial, including frequent otopathogen nasopharyngeal (NP) colonization and deficiency in innate and adaptive immune responses. A largely unexplored contributor to otitis proneness is NP microbiome composition. Since the microbiome modulates otopathogen NP colonization and immune responses, we hypothesized that the NP microbiome composition in sOP children might be dysregulated. METHODS: We performed 16S rRNA sequencing to analyze microbiome composition in 157 NP samples from 28 sOP and 68 AOM-free children when they were 6 months or 12 months old and healthy. Bioinformatic approaches were employed to examine the composition difference between the two populations and its correlation with changes in levels of inflammatory cytokines. RESULTS: A different global microbiome profile and reduced alpha diversity was observed in the NP microbiome of sOP children when 6 months old, compared with that from AOM-free children of the same age. This difference was resolved when groups were compared at 12 months old. We found 4 bacterial genera-Bacillus, Veillonella, Gemella, and Prevotella-correlated with higher levels of pro-inflammatory cytokines in the NP. Those 4 bacterial genera were in lower abundance in sOP compared to AOM-free children. CONCLUSION: Dysbiosis occurs in the NP microbiome of sOP children at an early age even when they were healthy. This dysbiosis correlates with a lower inflammatory state in the NP of these children.

Rising Pneumococcal Antibiotic Resistance in the Post-13-Valent Pneumococcal Conjugate Vaccine Era in Pediatric Isolates From a Primary Care Setting.

BACKGROUND: Antibiotic-resistant Streptococcus pneumoniae strains may cause infections that fail to respond to antimicrobial therapy. Results reported from hospitalized patients with invasive, bacteremic infections may not be the same as those observed in a primary care setting where young children receive care for noninvasive infections. Young children experience the highest burden of pneumococcal disease. The aim of this study was to determine the antibiotic susceptibility of S. pneumoniae strains isolated from children in a primary care setting in the post-13-valent pneumococcal conjugate vaccine (PCV13) era. METHODS: This was a prospective collection of 1201 isolates of S. pneumoniae from 2006 through 2016 in a primary care setting. Antibiotic susceptibility testing to 16 different antibiotics of 10 classes was performed. Participants were children aged 6-36 months. Nasopharyngeal swabs were obtained from patients during acute otitis media (AOM) visits and routine healthy visits. Middle ear fluid was obtained by tympanocentesis. RESULTS: After introduction of PCV13, antibiotic susceptibility of pneumococci, especially to penicillin, initially improved largely due to disappearance of serotype 19A, included in PCV13. However, beginning in 2013, antibiotic susceptibility among pneumococcal strains began decreasing due to new serotypes not included in PCV13. In addition to reduced susceptibility to penicillin, the most recent isolates show reduced susceptibility to third-generation cephalosporins, fluoroquinolones, and carbapenems, antibiotics commonly used to treat life-threatening, invasive pneumococcal diseases. CONCLUSIONS: In recent years, pneumococcal nasopharyngeal and AOM isolates from children exhibit reduced susceptibility to penicillin, third-generation cephalosporin, fluoroquinolone, and carbapenem antibiotics. The new strains have a different profile of resistance compared to the pre-PCV13 era.

Nasopharyngeal colonization with pathobionts is associated with susceptibility to respiratory illnesses in young children.

Some children are more susceptible to viral and bacterial respiratory infections in the first few years of life than others. However, the factors contributing to this susceptibility are incompletely understood. In a retrospective analysis of clinical samples collected from a prospectively-enrolled cohort of 358 children we sought associations between physician-attended illness visits and bacterial colonization in the first five years of life. A subset of children was identified by unsupervised clustering analysis as infection and allergy prone (IAP). Several respiratory infection- and allergy-mediated illnesses co-occurred at higher rates in IAP children, while the rates of other illnesses were not significantly different between the groups. Analyses of nasopharyngeal (NP) pathobionts and microbiota commensals showed that early age of first colonization with pathobionts Streptococcus pneumonia, non-typeable Haemophilus influenzae, and Moraxella catarrhalis was associated with IAP children, and particularly Moraxella abundance was negatively associated with NP microbiome diversity. We conclude that mucosal pathobiont exposures in early life can influence susceptibility to respiratory illnesses in children.

Ampicillin triggers the release of Pal in toxic vesicles from Escherichia coli.

In addition to lipopolysaccharides (LPS), outer membrane proteins - Lpp, OmpA and peptidoglycan-associated lipoprotein (Pal) - are part of the outer membrane of Escherichia coli and are proposed to contribute to bacterial sepsis-related inflammation. This study showed that ampicillin (a ß-lactam antibiotic) enhances Pal's release from Escherichia coli to a greater extent than gentamicin and levofloxacin (aminoglycoside and quinolone antibiotics, respectively). It is proposed that the majority of Pal is released in outer membrane vesicles (OMVs), which also contain LPS and other outer membrane and periplasmic proteins. The OMVs were purified by ultracentrifugation and characterised by transmission electron microscopy and nanoparticle tracking analysis, and Pal and other E. coli proteins were detected by Western blot. It also proposed that sepsis treatments using certain ß-lactam antibiotics may further aggravate the over-exuberant inflammatory response by enhancing the release of Pal and LPS in OMVs.

Immune Network Modeling Predicts Specific Nasopharyngeal and Peripheral Immune Dysregulation in Otitis-Prone Children.

Acute otitis media (AOM) pathogenesis involves nasopharyngeal colonization by potential otopathogens and a viral co-infection. Stringently-defined otitis prone (sOP) children show characteristic patterns of immune dysfunction. We hypothesized that otitis proneness is largely a result of altered signaling between immune components that are otherwise competent, resulting in increased susceptibility to infection by bacterial otopathogens. To test this, we constructed a regulatory immune network model linking immune cells and signaling elements known to be involved in AOM and/or dysregulated in sOP children. The alignment of immune response mechanisms with data from in vivo and in vitro experimental observations produced 82 putative immune network models, each describing variants of immune regulatory networks consistent with available observations. Analysis of these models suggested that new measurements of serum levels of IL-4 and CXCL8 could refine competing models and resulted in the elimination of 38 of the models. Further analysis of the remaining 44 models suggested specific deviations in the predicted regulation of nasopharyngeal and peripheral immunity during response to AOM. Specifically, immune responses active in sOP children during AOM were characterized by early and constitutive activation of pro-inflammatory signaling in the nasopharynx and a Th2- and Treg-dominated profile in the periphery. We conclude that sOP children have altered regulation of key immune mediators during both health and pathogenesis. This altered regulation may be amenable to therapeutic intervention.

Comparison of pneumococcal conjugate vaccine (PCV-13) cellular immune responses after primary and booster doses of vaccine.

Since their widespread use, pneumococcal conjugate vaccines (PCVs) have proven effective at reducing both invasive and noninvasive pneumococcal diseases and nasopharyngeal carriage of Streptococcus pneumoniae (Spn). To establish this level of protection, a three-dose schedule with a single booster (3 + 1) was the immunization regime in the USA. Alternatively, WHO-approved schedules of 3 + 0 and 2 + 1 are now becoming adopted in many countries to reduce the cost of vaccination. Sustained protection from pneumococcal disease and carriage requires persisting levels of antibody and cellular immune memory. Although antibody responses to PCVs are well studied, less is known concerning the cellular response to the vaccine in young children. In this report, circulating PCV-13 serotype-specific B and T cell memory in paired blood samples from children before and after PCV13 dose 3 and booster immunizations was analyzed to determine changes in the adaptive immune response. No significant differences in memory B cell populations were detected comparing post dose 2 vs. post dose 3. However, the booster dose significantly increased the frequency of Spn-specific memory B cells compared to the pre-booster. Spn-specific memory T cells were not detected with the method used. These data suggest that booster vaccination increases Spn-specific memory B cells that may impact long-term protective antibody titers.

Synchrony in serum antibody response to conserved proteins of Moraxella catarrhalis in young children.

Conserved Moraxella catarrhalis (Mcat) proteins, oligopeptide permease (Opp)A, hemagglutinin (Hag), outer membrane protein (OMP) CD, Pilin A clade 2 (PilA2), and Moraxella surface protein (Msp) 22 have been studied as vaccine candidates. Children who experience frequent acute otitis media (AOM) confirmed with pathogen identification by tympanocentesis are referred to as stringently-defined otitis prone (sOP). Synchrony of serum antibody responses against 5 Mcat proteins, OppA, Hag, OMP CD, PilA2, and Msp22 resulting from nasopharyngeal colonization and AOM was studied for 85 non-otitis prone (NOP) children and 34 sOP children. Changes in serum IgG were quantitated with ELISA. Serum IgG antibody levels against OppA, Hag, OMP CD, and Msp22 rose in synchrony in NOP and sOP children; that is, the proteins appeared equally and highly immunogenic in children at age 6 to 22-25 months old and then leveled off in their rise at 22-25 to 30 months old. In contrast, rises of PilA2 were slow from 6 months old and kept constant and did not level off significantly before 30 months old. OppA, Hag, OMP CD, and Msp22 elicited a synchronous acquisition of naturally-induced serum antibody in young children. A multi-valent Mcat protein vaccine combining OppA, Hag, OMP CD, and Msp22 may exhibit less antigen competition when administered as a combination vaccine in young children.

Immunologic dysfunction contributes to the otitis prone condition.

Acute Otitis Media (AOM) is a multifactorial disease occurring mostly in young children who are immunologically naïve to AOM pathogens. This review focuses on work from Rochester NY, USA over the past 12 years among young children who had AOM infections microbiologically-confirmed by tympanocentesis, so called "stringently-defined". Among stringently-defined otitis prone children deficiencies in fundamental immune defense mechanisms have been identified that contribute to the propensity of young children to experience recurrent AOM. Dysfunction in innate immune responses that cause an immunopathological impact in the nasopharynx have been discovered including inadequate proinflammatory cytokine response and poor epithelial cell repair. Adaptive immunity defects in B cell function and immunologic memory resulting in low levels of antibody to otopathogen-specific antigens allows repeated infections. CD4+ and CD8+ T cell function and memory defects significantly contribute. The immune profile of an otitis prone child resembles that of a neonate through the first year of life. Immunologic deficits in otitis prone children cause them to be unusually vulnerable to viral upper respiratory infections and respond inadequately to routine pediatric vaccines.