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.

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.

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.

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.

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.

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.

Impaired Proinflammatory Response in Stringently Defined Otitis-prone Children During Viral Upper Respiratory Infections.

BACKGROUND: Viral upper respiratory infections (URIs) are common and often precipitate acute otitis media (AOM), caused by bacterial otopathogens, in young children. Acute inflammatory responses initiated in the early phase of viral URI contribute to preventing the development of AOM. Stringently-defined otitis-prone (sOP) children are susceptible to recurrent AOM. METHODS: We assessed proinflammatory cytokine and chemokine levels in the nasopharynxes during viral URIs, and examined the different nasopharyngeal responses between viral URI events and the following AOM episodes in both sOP and non-otitis-prone (NOP) children. RESULTS: The sOP children exhibited significantly more AOM episodes per child (8.86-fold higher), viral URIs (P < .0001), and viral URIs followed by AOMs (P < .0001) than the NOP children. The sOP children had lower nasal proinflammatory levels of interleukin (IL)-6 (P = .05), IL-10 (P = .001), tumor necrosis factor (TNF)-α (P = .004), and regulated on activation, normal T-cell-expressed and -secreted (RANTES; P = .002) than NOP children during viral URIs. NOP children had higher levels of IL-6 (P = .02), IL-10 (P = .02), interferon-γ (P = .003), TNF-α (P = .006), IL-1ß (P = .022), monocyte chemoattractant protein 1 (P = .028), RANTES (P = .005), IL-2 (P = .002), and IL-17 (P = .007) during viral URIs versus AOMs following the URIs, when compared to sOP children. CONCLUSIONS: We conclude that sOP children have more frequent viral URIs than NOP children, due to deficient antiviral nasopharyngeal proinflammatory cytokine and chemokine responses.

Development and Modification of an Outcome Measure to Follow Symptoms of Children with Sinusitis.

OBJECTIVE: To develop a parent-reported Pediatric Rhinosinusitis Symptom Scale (PRSS) that could be used to monitor symptoms of young children with acute sinusitis in response to therapy. STUDY DESIGN: We developed an 8-item symptom severity scale and evaluated its internal reliability, construct validity, and responsiveness in children 2-12 years of age with acute sinusitis. Parents of 258 children with acute sinusitis completed the PRSS at the time of diagnosis, as a diary at home, and at the follow-up visit at days 10-12. Based on psychometric results and additional parent feedback, we revised the scale. We evaluated the revised version in 185 children with acute sinusitis. RESULTS: Correlations between the scale and reference measures on the day of enrollment were in the expected direction and of the expected magnitude. PRSS scores at the time of presentation correlated with radiographic findings (P < .001), functional status (P < .001), and parental assessment of overall symptom severity (P < .001). Responsiveness (standardized response mean) and test-retest reliability of the revised scale were good (2.17 and 0.75, respectively). CONCLUSIONS: We have developed an outcome measure to track the symptoms of acute sinusitis. Data presented here support the use of the PRSS as a measure of change in symptom burden in clinical trials of children with acute sinusitis.

Reduced T-Helper 17 Responses to Streptococcus pneumoniae in Infection-Prone Children Can Be Rescued by Addition of Innate Cytokines.

Background: T-helper (Th) 17 cells are important in the control of Streptococcus pneumoniae. We sought to understand the mechanism of failure of Th17 immunity resulting in S. pneumoniae infections in children <2 years old. Methods: Peripheral blood mononuclear cells (PBMCs) from infection-prone (IP) and non-IP (NIP) children 9-18 months old were examined for their responses to heat-killed S. Pneumoniae, using flow cytometry, reverse-transcription polymerase chain reaction, and enzyme-linked immunoassay. We measured cytokine production, proliferation, and differentiation of Th17 cells and the expression of transcription factors in response to S. pneumoniae. Results: PBMCs of IP children stimulated with heat-killed S. pneumoniae had significantly reduced percentages of CD4+ Th1 (interleukin2, tumor necrosis factor α) and Th17 (interleukin 17A) cells compared with NIP children. Addition of exogenous Th17-promoting cytokines (interleukin 6, 1ß, and 23 and transforming growth factor ß) restored CD4+ Th17 cell function in cells from IP children to levels measured in NIP children. Conclusions: Reduced Th17 responses to S. pneumoniae in PBMCs of IP children can be rescued by addition of Th17-promoting cytokines.

Protection against Streptococcus pneumoniae Invasive Pathogenesis by a Protein-Based Vaccine Is Achieved by Suppression of Nasopharyngeal Bacterial Density during Influenza A Virus Coinfection.

An increase in Streptococcus pneumoniae nasopharynx (NP) colonization density during a viral coinfection initiates pathogenesis. To mimic natural S. pneumoniae pathogenesis, we commensally colonized the NPs of adult C57BL/6 mice with S. pneumoniae serotype (ST) 6A or 8 and then coinfected them with mouse-adapted H1N1 influenza A virus (PR/8/34). S. pneumoniae established effective commensal colonization, and influenza virus coinfection caused S. pneumoniae NP density to increase, resulting in bacteremia and mortality. We then studied histidine triad protein D (PhtD), an S. pneumoniae adhesin vaccine candidate, for its ability to prevent invasive S. pneumoniae disease in adult and infant mice. In adult mice, the efficacy of PhtD vaccination was compared with that of PCV13. Vaccination with PCV13 led to a greater reduction of S. pneumoniae NP density (>2.5 log units) than PhtD vaccination (∼1-log-unit reduction). However, no significant difference was observed with regard to the prevention of S. pneumoniae bacteremia, and there was no difference in mortality. Depletion of CD4+ T cells in PhtD-vaccinated adult mice, but not PCV13-vaccinated mice, caused a loss of vaccine-induced protection. In infant mice, passive transfer of antisera or CD4+ T cells from PhtD-vaccinated adult mice led to a nonsignificant reduction in NP colonization density, whereas passive transfer of antisera and CD4+ T cells was needed to cause a significant reduction in NP colonization density. For the first time, these data show an outcome with regard to prevention of invasive S. pneumoniae pathogenesis with a protein vaccine similar to that which occurs with a glycoconjugate vaccine despite a less robust reduction in NP bacterial density.

Decreased TNF family receptor expression on B-cells is associated with reduced humoral responses to Streptococcus pneumoniae infections in young children.

An underdeveloped or impaired immune response in young children is associated with increased susceptibility to Streptococcus pneumonia (Spn) infections. We determined serum antibody titers against 3 Spn vaccine candidate proteins and vaccine serotype polysaccharide antigens in a group of Spn infection prone 9-18months old and found lower IgG antibody titers to all tested antigens compared to age-matched non-infection-prone children. We also found the children had significantly reduced percentages of total memory B-cells, switched memory B-cells and plasma cells. We sought a mechanistic explanation for that result by examination of TNF family receptors (TNFRs) TACI, BCMA, and BAFFR receptor expression on B-cells and found significantly lower BAFFR and TACI expression; significantly lower proliferation of B-cells stimulated with exogenous BAFF; and diminished expression of co-stimulatory receptors B7-1 and B7-2 among infection prone vs. non-prone children. We conclude that lower expression of TNFRs, lower proliferation of B-cells in response to BAFF and lower expression of B7-1 and B7-2 by B-cells may contribute to reduced antibody responses to Spn and consequent infection proneness in young children.

Functional Immune Cell Differences Associated With Low Vaccine Responses in Infants.

BACKGROUND: We sought to understand why some children respond poorly to vaccinations in the first year of life. METHODS: A total of 499 children (6-36 months old) provided serum and peripheral blood mononuclear cell samples after their primary and booster vaccination. Vaccine antigen-specific antibody levels were analyzed with enzyme-linked immunosorbent assay, and frequency of memory B cells, functional T-cell responses, and antigen-presenting cell responses were assessed in peripheral blood mononuclear cell samples with flow cytometric analysis. RESULTS: Eleven percent of children were low vaccine responders, defined a priori as those with subprotective immunoglobulin G antibody levels to ≥66% of vaccines tested. Low vaccine responders generated fewer memory B cells, had reduced activation by CD4(+) and CD8(+) T cells on polyclonal stimulation, and displayed lower major histocompatibility complex II expression by antigen-presenting cells. CONCLUSIONS: We conclude that subprotective vaccine responses in infants are associated with a distinct immunologic profile.

Dual orientation of the outer membrane lipoprotein Pal in Escherichia coli.

Peptidoglycan associated lipoprotein (Pal) of Escherichia coli (E. coli) is a characteristic bacterial lipoprotein, with an N-terminal lipid moiety anchoring it to the outer membrane. Since its discovery over three decades ago, Pal has been well studied for its participation in the Tol-Pal complex which spans the periplasm and has been proposed to play important roles in bacterial survival, pathogenesis and virulence. Previous studies of Pal place the lipoprotein in the periplasm of E. coli, allowing it to interact with Tol proteins and the peptidoglycan layer. Here, we describe for the first time, a subpopulation of Pal which is present on the cell surface of E. coli. Flow cytometry and confocal microscopy detect anti-Pal antibodies on the surface of intact E. coli cells. Interestingly, Pal is surface exposed in an 'all or nothing' manner, such that most of the cells contain only internal Pal, with fewer cells ( < 20  %) exhibiting surface Pal.

Human antibodies to PhtD, PcpA, and Ply reduce adherence to human lung epithelial cells and murine nasopharyngeal colonization by Streptococcus pneumoniae.

Streptococcus pneumoniae adherence to human epithelial cells (HECs) is the first step in pathogenesis leading to infections. We sought to determine the role of human antibodies against S. pneumoniae protein vaccine candidates PhtD, PcpA, and Ply in preventing adherence to lung HECs in vitro and mouse nasopharyngeal (NP) colonization in vivo. Human anti-PhtD, -PcpA, and -Ply antibodies were purified and Fab fragments generated. Fabs were used to test inhibition of adherence of TIGR4 and nonencapsulated strain RX1 to A549 lung HECs. The roles of individual proteins in adherence were tested using isogenic mutants of strain TIGR4. Anti-PhtD, -PcpA, and -Ply human antibodies were assessed for their ability to inhibit NP colonization in vivo by passive transfer of human antibody in a murine model. Human antibodies generated against PhtD and PcpA caused a decrease in adherence to A549 cells (P < 0.05). Anti-PhtD but not anti-PcpA antibodies showed a protective role against mouse NP colonization. To our surprise, anti-Ply antibodies also caused a significant (P < 0.05) reduction in S. pneumoniae colonization. Our results support the potential of PhtD, PcpA, and Ply protein vaccine candidates as alternatives to conjugate vaccines to prevent non-serotype-specific S. pneumoniae colonization and invasive infection.

Differential impact of respiratory syncytial virus and parainfluenza virus on the frequency of acute otitis media is explained by lower adaptive and innate immune responses in otitis-prone children.

BACKGROUND: Acute otitis media (AOM) is a leading cause of bacterial pediatric infections associated with viral upper respiratory infections (URIs). We examined the differential impact of respiratory syncytial virus (RSV) and parainfluenza virus URIs on the frequency of AOM caused by Streptococcus pneumoniae (Spn) and nontypeable Haemophilus influenzae (NTHi) in stringently defined otitis-prone (sOP) and non-otitis-prone (NOP) children as a potential mechanism to explain increased susceptibility to AOM. METHODS: Peripheral blood and nasal washes were obtained from sOP and NOP children (n = 309). Colonization events and antiviral responses consisting of total specific immunoglobulin G (IgG) responses, neutralizing antibody responses, and T-cell responses were determined. Isolated neutrophils were infected with varying multiplicities of infection of both viruses, and opsonophagocytosis potential was measured. RESULTS: A significant increase was found in frequency of AOM events caused by Spn and NTHi, with a concurrent RSV infection in sOP children. These results correlated with diminished total RSV-specific IgG, higher viral nasal burdens, and lower IgG neutralizing capacity. The sOP children had diminished T-cell responses to RSV that correlated with lower Toll-like receptor 3/7 transcript and decreased expression of HLA-DR on antigen-presenting cells. RSV interfered with the Spn phagocytic capacity of neutrophils in a dose-dependent manner. Parainfluenza virus infections did not differentially affect AOM events in sOP and NOP children. CONCLUSIONS: Lower innate and adaptive immune responses to RSV in sOP children may slow the kinetics of viral clearance from the nasopharynx and allow for viral interference with antibacterial immune responses, thus contributing to increased frequency of AOMs.

Transcriptome signature in young children with acute otitis media due to non-typeable Haemophilus influenzae.

Non-typeable Haemophilus influenzae (NTHi) causes acute otitis media (AOM) in young children. In our recent paper in Microbes and Infection we described the transcriptome signature elicited from PBMCs at onset of AOM caused by Streptococcus pneumoniae. In the current study we found very different results with NTHi AOM infections; 5.1% of 29 187 genes were differentially regulated by more than 2-fold at the onset of AOM compared with the pre-infection healthy state in the same children. Among the 1487 transcripts, 100 genes associated with the immune defense response were specifically analyzed. About half of the differentially regulated genes associated with antibacterial activity and the cell-mediated immune response were activated and half were suppressed. The important signatures for NTHi in children suggested that the balance of the immune response was toward suppression. Moreover, 90% of the genes associated with a pro-inflammatory cytokine response were down-regulated. The genes associated with the classic complement pathway were down-regulated, although the alternative complement pathway genes were up-regulated. These results provide the first human transcriptome data identifying gene expression in the immune response to be predominantly down-regulated at the onset of AOM due to NTHi.

Correlation of nasopharyngeal cultures prior to and at onset of acute otitis media with middle ear fluid cultures.

BACKGROUND: We sought to determine if nasopharyngeal (NP) cultures taken at times of healthy visits or at onset of acute otitis media (AOM) could predict the otopathogen mix and antibiotic-susceptibility of middle ear isolates as determined by middle ear fluid (MEF) cultures obtained by tympanocentesis. METHODS: During a 7-year-prospective study of 619 children from Jun 2006-Aug 2013, NP cultures were obtained from 6-30 month olds at healthy visits and NP and MEF (by tympanocentesis) at onset of AOM episodes. RESULTS: 2601 NP and 530 MEF samples were collected. During healthy visits, S. pneumoniae (Spn) was isolated from 656 (31.7%) NP cultures compared to 253 (12.2%) for Nontypeable Haemophilus influenzae (NTHi) and 723 (34.9%) for Moraxella catarrhalis (Mcat). At onset of AOM 256 (48.3%) of 530 NP samples were culture positive for Spn, 223 (42%) for NTHi and 251 (47.4%) for Mcat, alone or in combinations. At 530 AOM visits, Spn was isolated from 152 (28.7%) of MEF compared to 196 (37.0%) for NTHi and 104 (19.6%) for Mcat. NP cultures collected at onset of AOM but not when children were healthy had predictive value for epidemiologic antibiotic susceptibility pattern assessments. CONCLUSIONS: NP cultures at onset of AOM more closely correlate with otopathogen mix than NP cultures at healthy visits using MEF culture as the gold standard, but the correlation was too low to allow NP cultures to be recommended as a substitute for MEF culture. For epidemiology purposes, antibiotic susceptibility of MEF isolates can be predicted by NP culture results when samples are collected at onset of AOM.