Juho Vuononvirta

Nasopharyngeal bacterial colonization and gene polymorphisms of mannose-binding lectin and toll-like receptors 2 and 4 in infants.

Background Human nasopharynx is often colonized by potentially pathogenic bacteria. Gene polymorphisms in mannose-binding lectin (MBL), toll-like receptor (TLR) 2 and TLR4 have been reported. The present study aimed to investigate possible association between nasopharyngeal bacterial colonization and gene polymorphisms of MBL, TLR2 and TLR4 in healthy infants. Methodology/Principal Findings From August 2008 to June 2010, 489 nasopharyngeal swabs and 412 blood samples were taken from 3-month-old healthy Finnish infants. Semi-quantitative culture was performed and pyrosequencing was used for detection of polymorphisms in MBL structural gene at codons 52, 54, and 57, TLR2 Arg753Gln and TLR4 Asp299Gly. Fifty-nine percent of subjects were culture positive for at least one of the four species: 11% for Streptococcus pneumoniae, 23% for Moraxella catarrhalis, 1% for Haemophilus influenzae and 25% for Staphylococcus aureus. Thirty-two percent of subjects had variant types in MBL, 5% had polymorphism of TLR2, and 18% had polymorphism of TLR4. Colonization rates of S. pneumoniae and S. aureus were significantly higher in infants with variant types of MBL than those with wild type (p = .011 and p = .024). Colonization rates of S. aureus and M. catarrhalis were significantly higher in infants with polymorphisms of TLR2 and of TLR4 than those without (p = .027 and p = .002). Conclusions Our study suggests that there is an association between nasopharyngeal bacterial colonization and genetic variation of MBL, TLR2 and TLR4 in young infants. This finding supports a role for these genetic variations in susceptibility of children to respiratory infections.

Differences in avidity of IgG antibodies to pertussis toxin after acellular pertussis booster vaccination and natural infection.

BACKGROUND Pertussis toxin (PT) is a specific virulence factor of Bordetella pertussis and it is included in all acellular pertussis vaccines (aP). Although immunity after infection seems to persist longer than that after vaccination, the exact mechanism(s) is not known. Primary aim of this study was to develop an ELISA method for measuring avidity index (AI) of IgG-anti-PT antibodies and to compare antibody responses after booster vaccination and infection. Secondary aim was to evaluate if the AI-ELISA has potential in the serodiagnosis of pertussis. MATERIAL Serum samples from a total of 409 subjects were included in the study. Paired sera were taken from 97 adolescents who received booster vaccine ten years ago (dTpa-004) and from 80 young adults who received a second booster dose ten years after the previous booster vaccine (dTpa-040). Thirty-two paired sera from culture-confirmed pertussis patients, 161 single sera from serologically diagnosed patients and 39 single sera from healthy controls were included. AI of IgG-anti-PT antibodies were determined with newly developed ELISA using diethylamine (DEA) as a bond breaking agent. The IgG-anti-PT antibodies were measured by standardized ELISA. RESULTS A significant increase was found in antibody concentrations and AI between PRE and one month POST vaccination ten years ago [GMC for antibody: 7.9 IU/ml vs. 98.3 IU/ml (p=0.0001); for AI: 40.4% vs. 56.1% (p=0.0001)]. Similar result was observed after the second booster dose [GMC for antibody: 9.2 IU/ml vs. 92.4 IU/ml (p=0.0001); for AI: 36.1% vs. 59.5% (p=0.0001)] and between the first and second sera of culture-confirmed patients [GMC for antibody: 6.9 IU/ml vs. 285.1 IU/ml (p=0.0001); for AI: 40.5% vs. 68.4% (p=0.0001)]. Healthy controls showed lower levels of both antibodies and AI. CONCLUSIONS Our results suggest that there may be difference in quality and quantity of antibodies to PT after vaccination and after infection. Furthermore, AI could be a help for vaccine studies.

Gene Polymorphism in Toll-like Receptor 4: Effect on Antibody Production and Persistence After Acellular Pertussis Vaccination During Adolescence

BACKGROUND Toll-like receptors play an important role in the regulation of adaptive immunity. This study aimed to investigate whether Toll-like receptor 4 (TLR4) polymorphisms influence the production and persistence of antibodies after acellular pertussis booster vaccination during adolescence. METHODS Seventy-five subjects received a single dose of diphtheria and tetanus toxoids and acellular pertussis vaccine 10 years ago, during adolescence. The same cohort was followed up at 3, 5, and 10 years after this booster vaccination. Pyrosequencing was used for detecting polymorphism in TLR4. Concentrations of anti-pertussis vaccine antibodies were measured by standardized enzyme-linked immunosorbant assay and published elsewhere. RESULTS The fold increase in antibodies to pertussis toxin after original vaccination 10 years ago was significantly lower in subjects with TLR4 polymorphism than in those without (55% vs 86%; P = .028). At the 3-year follow-up evaluation, geometric mean concentrations of anti-pertussis vaccine antibodies were significantly lower in subjects with TLR4 polymorphism, compared with those without the polymorphism (for pertussis toxin, P = .028; for filamentous hemagglutinin, P = .047; and for pertactin, P = .046). CONCLUSIONS This study suggests that TLR4 Asp299Gly polymorphism might influence production and persistence of antibodies after pertussis booster vaccination in adolescents. However, the results should be interpreted with caution as the number of subjects included in this study was limited.

Toll-like receptor 3 L412F polymorphisms in infants with bronchiolitis and postbronchiolitis wheezing.

Background: Innate immunity receptors play a critical role in host defense. In addition, the expression of Toll-like receptors (TLRs) has been connected to allergy and asthma. Aims: To evaluate the association between the TLR3 L412F polymorphism and viral findings, clinical characteristics and subsequent wheezing in young infants with bronchiolitis. Methods: In all, 129 full-term infants hospitalized for bronchiolitis at age <6 months have been followed-up until the mean age of 1.5 years. Genotyping of the TLR3 L412F gene mutation was made by pyrosequencing. Results: TLR3 L412F gene polymorphism including the minor allele T was overrepresented (52%) in infants hospitalized with bronchiolitis. The presence of the major allele C as homozygous was associated with repeated postbronchiolitis wheezing (7.06, 95% confidence interval 2.30–21.66). Conclusion: Preliminary evidence was found that TLR3 L412F gene polymorphism may be associated with bronchiolitis leading to hospitalization and postbronchiolitis wheezing.

The association of genetic variants in toll-like receptor 2 subfamily with allergy and asthma after hospitalization for bronchiolitis in infancy.

Background: Toll-like receptors (TLRs) are a pivotal part of the innate immunity system. Variations in TLR genes have been connected to autoimmune conditions, such as allergy and asthma. The TLR2 subfamily comprises TLR1, TLR2, TLR6 and TLR 10. We hypothesized that polymorphism of the TLR2 subfamily may be associated with prevalence of post-bronchiolitic asthma and/or atopy. Methods: TLR1rs5743618, TLR2rs5743708 and TLR6rs5743810 single nucleotide polymorphisms of 133 children who had been hospitalized for bronchiolitis at <6 months of age were analyzed. Doctor-diagnosed asthma and atopy as well as their occurrence during the first 6 years of life were evaluated during a follow-up visit. Results: At the mean age of 6.4 years, asthma was present in 17 (13%) patients, there was asthma diagnosis during the first 6 years of life in 39 (29%) and current doctor-diagnosed allergic rhinitis in 57 (43%) patients. Twenty-four (24%) children with G/G genotype in TLR1 rs5743618 were diagnosed to have asthma between 1 and 6 years of age (vs. 13 (38%) of those with G/T or T/T genotypes; P = 0.04). In addition, 11/60 (18%) children with TLR6 rs5743810 C/T versus 36/73 (49%) children of other genotypes had atopic eczema at follow up. Only 2 children (8%) with wild genotype in all investigated single nucleotide polymorphisms had asthma during the first 6 years of life (vs. 30% in those with variant genotype of TLR1, TLR2 and/or TLR6). Conclusion: In this study, we demonstrated that TLR1 rs5743618 was associated with asthma and atopic eczema during the first 6 years of life after early bronchiolitis. In addition, TLR6 rs5743810 was associated with present atopy at preschool age.

The Gene Polymorphism of IL-17 G-152A is Associated with Increased Colonization of Streptococcus pneumoniae in Young Finnish Children.

Background: Streptococcus pneumoniae is a common respiratory pathogen, and up to 50% of children acquire S. pneumoniae in their nasopharynx during the first 12 months of life. The cytokine interleukin-17A (IL-17A) plays an important role in host defense against extracellular bacterial pathogens. We investigated the effect of IL-17 G-152A polymorphism on pneumococcal colonization in children. Methods: Nasopharyngeal swabs and blood samples were collected from healthy Finnish children at 2.6 (N = 405), 13 (N = 198) and 24 (N = 176) months of age. Of them, 160 had both nasopharyngeal swabs and blood samples at each time point. The semiquantitative culture method was used for bacterial culture, Sequenom iPlex Gold System for IL-17A genotyping and Luminex 200 for serum IL-17A determination. Results: The frequency of IL-17 G-152A genotypes G/G, G/A and A/A was 36%, 45% and 19% in 405 studied subjects, respectively. The colonization rates of S. pneumoniae increased from 10% at 2.6 months to 33% at 24 months of age. Significantly higher pneumococcal colonization was found in subjects with A/A genotype at 13 and 24 months of age compared with those of G/G (RR, 2.30; P = 0.02; RR, 1.91, P = 0.03). This genotype was associated with lower levels of serum IL-17A, and only 6% of subjects with A/A had detectable serum IL-17A compared with 75% and 33% of subjects with G/G and G/A (P < 0.001 and P < 0.01), respectively. Conclusions: Our results indicate that IL-17 G-152A is associated with increased colonization rate of S. pneumoniae in young children, suggesting that IL-17A plays an important role in protection against pneumococcal colonization.

Burden of Recurrent Respiratory Tract Infections in Children: A Prospective Cohort Study.

Background: The burden of recurrent respiratory infections is unclear. We identified young children with recurrent respiratory infections in order to characterize the clinical manifestations, risk factors and short-term consequences. Methods: In this prospective cohort study, 1089 children were followed from birth to 2 years of age for respiratory infections by a daily symptom diary. Nasal swabs taken during respiratory infections were analyzed for viruses from 714 children. Nasopharyngeal swabs collected at 2 months of age were cultured for bacteria. The 10% of children with the highest number of annual respiratory illness days were defined to have recurrent respiratory tract infections. Results: The 90th percentile in the number of annual respiratory illness days was 98. Children above this limit (n = 109) had a median of 9.6 acute respiratory infections per year. Rhinovirus was detected in 58% of their infections. Of the children with recurrent infections, 60% were diagnosed with at least 3 episodes of acute otitis media, 73% received at least 3 antibiotic treatments and 21% were hospitalized for an acute respiratory infection. Tympanostomy was performed for 35% and adenoidectomy for 13% of the children. Asthma was diagnosed in 12% by 24 months of age. Older siblings increased the risk of recurrent respiratory infections. Early nasopharyngeal colonization with Streptococcus pneumoniae was common in children who later developed recurrent infections. Conclusions: Children with recurrent respiratory infections frequently use health care services and antibiotics, undergo surgical procedures and are at risk for asthma in early life. Having older siblings increases the risk of recurrent infections.

Toll-like receptor 2 subfamily gene polymorphisms are associated with Bacillus Calmette-Guerin osteitis following newborn vaccination

Toll‐like receptor (TLR) 1, 2, 6 and 10, the TLR2 subfamily, are known to be associated with immunity against tuberculosis. We evaluated whether polymorphisms in genes encoding TLR1, TLR2 and TLR6 were associated with osteitis in infants who received the Bacillus Calmette‐Guérin (BCG) vaccination soon after birth.

Risk of Repeated Moraxella catarrhalis Colonization Is Increased in Children With Toll-like Receptor 4 Asp299Gly Polymorphism

Background: Moraxella catarrhalis is a common causative agent of acute otitis media and other respiratory infections in children. Toll-like receptor (TLR) 4 is an important protein of human innate immunity. One polymorphic site Asp299Gly of TLR4 is proven to result in an impaired response to lipopolysaccharide from Gram-negative bacteria. We investigated whether Finnish children who carry Asp299Gly had increased risk of M. catarrhalis colonization during their first 2 years of life. Methods: This was a prospective cohort study carried out in Turku, Finland. We studied M. catarrhalis colonization in 161 Finnish children, whose nasopharyngeal specimens were taken at 2, 12 and 24 months of age. The semiquantitative culture method was used for identification of different bacterial species and the pyrosequencing-based method for detection of TLR4 Asp299Gly. Results: Of 161 subjects, 126 (78%) were TLR4 A/A wild type and 35 (22%) were A/G heterozygote variants. The prevalence of M. catarrhalis increased from 24% at 2 months to 48% at 12 months and to 58% at 24 months of age. Of the 35 subjects with TLR4 variant, 15 (43%) were M. catarrhalis positive at all 3 time points, whereas only 11 (9%) subjects with TLR4 wild type were positive at these time points (relative risk 4.91, 95% confidence interval: 2.482–9.711, P = 0.0001). Moreover, subjects with TLR4 variant had significantly higher bacterial load of M. catarrhalis in their nasopharynx than those with TLR4 wild type (P = 0.0032). Conclusions: Our results indicate that children with TLR4 Asp299Gly polymorphism have an increased risk of repeated M. catarrhalis colonization.

Toll-like receptor 2 subfamily genotypes are not associated with severity of bronchiolitis or postbronchiolitis wheezing in infants

Expression of toll‐like receptor (TLR) 2 subfamily genes, including genes encoding TLR1, TLR2, TLR6 and TLR10, have been connected to allergy and asthma. This controlled study investigated the association of TLR1, TLR2 and TLR6 gene polymorphisms with clinical characteristics and subsequent wheezing in young infants with bronchiolitis.