High performance anion exchange chromatographic and colorimetric methods for quality assessment of total and free polysaccharide content in Haemophilus influenzae type b conjugate vaccine containing lactose.

The presence of lactose as a stabilizer in Haemophilus influenzae type b (Hib) conjugate vaccine is a challenge for chromatographic resolution of its total and free poly ribosyl ribitol phosphate (PRP) content. Sample pretreatment using ultrafiltration was performed and had removed ≥95% of lactose in shorter time compared to the conventional dialysis process. Separation of free unconjugated PRP was performed using solid-phase extraction C4 cartridges. Hib conjugate vaccine was then analyzed for determination of total and free PRP, using two validated techniques: high performance anion exchange chromatography with pulsed amperometry (HPAEC-PAD) for ribitol determination and a colorimetric assay for phosphorus determination. Lactose removal had enabled a rapid chromatographic assay via fast depolymerization of PRP using high temperature treatment. Modifying the burning process in the colorimetric assay reduced the analysis time significantly compared to the pharmacopoeial method. Linearity was obtained over the range of 0.10-10.0 µg mL-1 for the HPAEC method and in the range of 1.0-8.0 µg mL-1 for the colorimetric one. Stability of Hib conjugate vaccine was investigated. The HPAEC results revealed about a 35% increase in free PRP content after storage under stressed conditions (moisture and temperature). The proposed methods offered a reliable and economic platform for assessing the immunogenicity, efficacy and stability of Hib conjugate vaccine containing lactose for the biopharmaceutical industry.

Determination of ribose and phosphorus contents in Haemophilus influenzae type b capsular polysaccharide by a quantitative NMR method using a single internal standard.

The ribose and phosphorus contents in Haemophilus influenzae type b (Hib) capsular polysaccharide (CPS) are two important chemical indexes for the development and quality control of Hib conjugate vaccine. A quantitative 1H- and 31P-NMR method using a single internal standard was developed for simultaneous determination of ribose and phosphorus contents in Hib CPS. Hexamethylphosphoramide (HMPA) was successfully utilized as an internal standard in quantitative 1H-NMR method for ribose content determination. The ribose and phosphorus contents were found to be affected by the concentration of polysaccharide solution. Thus, 15-20 mg·L-1 was the optimal concentration range of Hib CPS in D2O solution for determination of ribose and phosphorus contents by this method. The ribose and phosphorus contents obtained by the quantitative NMR were consistent with those obtained by traditional chemical methods. In conclusion, this quantitative 1H- and 31P-NMR method using a single internal standard shows good specificity, accuracy and precision, providing a valuable approach for the quality control of Hib glycoconjugate vaccines.

Immunogenic characterization of vaccines based on Haemophilus parasuis Nagasaki strain, OmpP2, OmpP5 and OmpD15, in colostrum-deprived pigs experimentally challenged with the same strain.

Three recombinant outer membrane proteins (rOmps) from the Haemophilus parasuis Nagasaki strain (serovar 5 reference strain), rOmpP2, rOmpP5 and rOmpD15, which have previously shown protection against H. parasuis infection in mice, were cloned, expressed and evaluated as vaccine antigens in colostrum-deprived pigs. When these animals were immunized with these rOmps and were later challenged intratracheally with 108 CFUs of the Nagasaki strain, no protection was seen in terms of survival, clinical signs, pathological results and recovery of H. parasuis. We hypothesized that a possible explanation for this lack of protection could be the low number of epitopes accessible to the immune system as a consequence of their poor exposure on the bacterial surface so that the immune response would not be able to protect against experimental infection by H. parasuis when a fully susceptible animal model, such as pigs, was used.

Recombinant C-terminal 311 amino acids of HapS adhesin as a vaccine candidate for nontypeable Haemophilus influenzae: A study on immunoreactivity in Balb/C mouse.

Hap, an auto-transporter protein, is an antigenically conserved adhesion protein which is present on both typeable and nontypeable Haemophilus influenzae. This protein has central role in bacterial attachment to respiratory tract epithelial cells. A 1000bp C-terminal fragment of Hap passenger domain (HapS) from nontypeable Haemophilus influenzae was cloned into a prokaryotic expression vector, pET-24a. BALB/c mice were immunized subcutaneously with purified rC-HapS. Serum IgG responses to purified rC-HapS, serum IgG subclasses were determined by ELISA and functional activity of antibodies was examined by Serum Bactericidal Assay. The output of rC-HapS was approximately 62% of the total bacterial proteins. Serum IgG responses were significantly increased in immunized group with rC-HapS mixed with Freund's adjuvant in comparison with control groups. Analysis of the serum IgG subclasses showed that the IgG1 subclass was predominant after subcutaneous immunization in BALB/c mice (IgG2a/IgG1 < 1). The sera from rC-HapS immunized animals were strongly bactericidal against nontypeable Haemophilus influenzae. These results suggest that rC-HapS may be a potential vaccine candidate for nontypeable Haemophilus influenzae.

Immunogenicity and safety of a fully liquid aluminum phosphate adjuvanted Haemophilus influenzae type b PRP-CRM197-conjugate vaccine in healthy Japanese children: A phase III, randomized, observer-blind, multicenter, parallel-group study.

BACKGROUND: Broad use of monovalent Haemophilus influenzae type b (Hib) conjugate vaccines based on the capsular polysaccharide polyribosyl-ribitol phosphate (PRP), has significantly reduced invasive Hib disease burden in children worldwide, particularly in children aged <1year. In Japan, PRP conjugated to tetanus toxoid (PRP-T) vaccine has been widely used since the initiation of public funding programs followed by a routine vaccination designation in 2013. METHODS: We compared the immunogenicity and safety of PRP conjugated to a non-toxic diphtheria toxin mutant (PRP-CRM197) vaccine with the PRP-T vaccine when administered subcutaneously to healthy Japanese children in a phase III study. Additionally, we evaluated the immunogenicity and safety profiles of a diphtheria-tetanus acellular pertussis (DTaP) combination vaccine when concomitantly administered with either PRP-CRM197 or PRP-T vaccines. The primary endpoint was the "long-term seroprotection rate", defined as the group proportion with anti-PRP antibody titers ⩾1.0µg/mL, after the primary series. RESULTS: Long-term seroprotection rates were 99.3% in the PRP-CRM197 group and 95.6% in the PRP-T group. The intergroup difference (PRP-CRM197 group - PRP-T group) was 3.7% (95% confidence interval: 0.099-7.336), demonstrating that PRP-CRM197 vaccine was non-inferior to PRP-T vaccine (p<0.0001). Furthermore, the "short-term seroprotection rate" (anti-PRP antibody titer ⩾0.15µg/mL) before booster vaccination was higher in the PRP-CRM197 group than in PRP-T. Concomitant administration of PRP-CRM197 vaccine with DTaP vaccine showed no differences in terms of immunogenicity compared with concomitant vaccination with PRP-T vaccine and DTaP vaccine. Although CRM197 vaccine had higher local reactogenicity, overall, both Hib vaccines had acceptable safety and tolerability profiles. CONCLUSION: The immunogenicity of PRP-CRM197 vaccine administered subcutaneously as a three-dose primary series in children followed by a booster vaccination 1year after the primary series induced protective levels of Hib antibodies with no safety or tolerability concerns. CLINICAL TRIAL REGISTRY: Registered on ClinicalTrials.gov: NCT01379846.

Introduction of pentavalent vaccine in Indonesia: a policy analysis.

The introduction of pentavalent vaccine containing Haemophilus influenzae type b antigen in Indonesia's National Immunization Program occurred nearly three decades after the vaccine was first available in the United States and 16 years after Indonesia added hepatitis B vaccine into the program. In this study, we analyzed the process that led to the decision to introduce pentavalent vaccine in Indonesia. Using process tracing and case comparison, we used qualitative data gathered through interviews with key informants and data extracted from written sources to identify four distinct but interrelated processes that were involved in the decision making: (a) pentavalent vaccine use policy process, (b) financing process, (c) domestic vaccine development process and (d) political process. We hypothesized that each process is associated with four necessary conditions that are jointly sufficient for the successful introduction of pentavalent vaccine in Indonesia, namely (a) an evidence-based vaccine use recommendation, (b) sufficient domestic financing capacity, (c) sufficient domestic vaccine manufacturing capacity and (d) political support for introduction. This analysis of four processes that led to the decision to introduce a new vaccine in Indonesia may help policy makers and other stakeholders understand and manage activities that can accelerate vaccine introduction in the future.

Neumonía adquirida en la comunidad: tratamiento ambulatorio y prevención / Community acquired pneumonia in children: Outpatient treatment and prevention

La neumonía adquirida en la comunidad (NAC) en la edad pediátrica ha sufrido, en la última década, una serie de cambios epidemiológicos, clínicos, etiológicos y de resistencias a antibióticos, que obligan a replantear su abordaje terapéutico. En este documento, dos de las principales sociedades de especialidades pediátricas involucradas en el diagnóstico y tratamiento de esta entidad, como son la Sociedad Española de Infectología Pediátrica y la Sociedad Española de Neumología Pediátrica, así como el Comité Asesor de Vacunas de la AEP, proponen unas pautas consensuadas de tratamiento y prevención, con el fin de proporcionar a todos los pediatras una guía actualizada. En esta primera parte del consenso, se aborda el tratamiento de los pacientes sin enfermedades de base relevantes con NAC que no precisan ingreso hospitalario, así como la prevención global de esta patología con vacunas. En un siguiente documento se expondrá el abordaje terapéutico tanto de aquellos pacientes en situaciones especiales como de las formas complicadas de la enfermedad

There have been significant changes in community acquired pneumonia (CAP) in children in the last decade. These changes relate to epidemiology and clinical presentation. Resistance to antibiotics is also a changing issue. These all have to be considered when treating CAP. In this document, two of the main Spanish pediatric societies involved in the treatment of CAP in children, propose a consensus concerning therapeutic approach. These societies are the Spanish Society of Paediatric Infectious Diseases and the Spanish Society of Paediatric Chest Diseases. The Advisory Committee on Vaccines of the Spanish Association of Paediatrics (CAV-AEP) has also been involved in the prevention of CAP. An attempt is made to provide up-to-date guidelines to all paediatricians. The first part of the statement presents the approach to ambulatory, previously healthy children. We also review the prevention with currently available vaccines. In a next second part, special situations and complicated forms will be addressed

Evaluation of Haemophilus influenzae type b carrier status among children 10 years after the introduction of Hib vaccine in Brazil.

The aim of the present study was to assess the prevalence of Haemophilus influenzae type b (Hib) nasopharyngeal (NP) colonisation among healthy children where Hib vaccination using a 3p+0 dosing schedule has been routinely administered for 10 years with sustained coverage (> 90%). NP swabs were collected from 2,558 children who had received the Hib vaccine, of whom 1,379 were 12-< 24 months (m) old and 1,179 were 48-< 60 m old. Hi strains were identified by molecular methods. Hi carriage prevalence was 45.1% (1,153/2,558) and the prevalence in the 12-< 24 m and 48-< 60 m age groups were 37.5% (517/1,379) and 53.9% (636/1,179), respectively. Hib was identified in 0.6% (16/2,558) of all children in the study, being 0.8% (11/1,379) and 0.4% (5/1,179) among the 12-< 24 m and 48-< 60 m age groups, respectively. The nonencapsulate Hi colonisation was 43% (n = 1,099) and was significantly more frequent at 48-< 60 m of age (51.6%, n = 608) compared with that at 12-< 24 m of age (35.6%, n = 491). The overall resistance rates to ampicillin and chloramphenicol were 16.5% and 3.7%, respectively; the co-resistance was detected in 2.6%. Our findings showed that the Hib carrier rate in healthy children under five years was very low after 10 years of the introduction of the Hib vaccine.

Evaluation of Haemophilus influenzae type b carrier status among children 10 years after the introduction of Hib vaccine in Brazil

The aim of the present study was to assess the prevalence of Haemophilus influenzaetype b (Hib) nasopharyngeal (NP) colonisation among healthy children where Hib vaccination using a 3p+0 dosing schedule has been routinely administered for 10 years with sustained coverage (> 90%). NP swabs were collected from 2,558 children who had received the Hib vaccine, of whom 1,379 were 12-< 24 months (m) old and 1,179 were 48-< 60 m old. Hi strains were identified by molecular methods. Hi carriage prevalence was 45.1% (1,153/2,558) and the prevalence in the 12-< 24 m and 48-< 60 m age groups were 37.5% (517/1,379) and 53.9% (636/1,179), respectively. Hib was identified in 0.6% (16/2,558) of all children in the study, being 0.8% (11/1,379) and 0.4% (5/1,179) among the 12-< 24 m and 48-< 60 m age groups, respectively. The nonencapsulate Hi colonisation was 43% (n = 1,099) and was significantly more frequent at 48-< 60 m of age (51.6%, n = 608) compared with that at 12-< 24 m of age (35.6%, n = 491). The overall resistance rates to ampicillin and chloramphenicol were 16.5% and 3.7%, respectively; the co-resistance was detected in 2.6%. Our findings showed that the Hib carrier rate in healthy children under five years was very low after 10 years of the introduction of the Hib vaccine.

International collaborative study for establishment of the 2nd WHO International Standard for Haemophilus influenzae type b polysaccharide.

In this report we present the results of a collaborative study for the preparation and calibration of a replacement International Standard (IS) for Haemophilus influenzae type b polysaccharide (polyribosyl ribitol phosphate; 5-d-ribitol-(1 â†’ 1)-ß-d-ribose-3-phosphate; PRP). Two candidate preparations were evaluated. Thirteen laboratories from 9 different countries participated in the collaborative study to assess the suitability and determine the PRP content of two candidate standards. On the basis of the results from this study, Candidate 2 (NIBSC code 12/306) has been established as the 2nd WHO IS for PRP by the Expert Committee of Biological Standards of the World Health Organisation with a content of 4.904 ± 0.185mg/ampoule, as determined by the ribose assays carried out by 11 of the participating laboratories.

Preclinical evaluation of a Haemophilus influenzae type b conjugate vaccine process intended for technology transfer.

Introduction of Haemophilus influenzae type b (Hib) vaccine in low- and middle-income countries has been limited by cost and availability of Hib conjugate vaccines for a long time. It was previously recognized by the Institute for Translational Vaccinology (Intravacc, originating from the former Vaccinology Unit of the National Institute of Public Health [RIVM] and the Netherlands Vaccine Institute [NVI]) that local production of a Hib conjugate vaccine would increase the affordability and sustainability of the vaccine and thereby help to speed up Hib introduction in these countries. A new affordable and a non-infringing production process for a Hib conjugate vaccine was developed, including relevant quality control tests, and the technology was transferred to a number of vaccine manufacturers in India, Indonesia, and China. As part of the Hib technology transfer project managed by Intravacc, a preclinical toxicity study was conducted in the Netherlands to test the safety and immunogenicity of this new Hib conjugate vaccine. The data generated by this study were used by the technology transfer partners to accelerate the clinical development of the new Hib conjugate vaccine. A repeated dose toxicity and local tolerance study in rats was performed to assess the reactogenicity and immunogenicity of a new Hib conjugate vaccine compared to a licensed vaccine. The results showed that the vaccine was well tolerated and immunogenic in rats, no major differences in both safety and immunogenicity in rats were found between the vaccine produced according to the production process developed by Intravacc and the licensed one. Rats may be useful to verify the immunogenicity of Hib conjugate vaccines and for preclinical evaluation. In general, nonclinical evaluation of the new Hib conjugate vaccine, including this proof of concept (safety and immunogenicity study in rats), made it possible for technology transfer partners, having implemented the original process with no changes in the manufacturing process and vaccine formulation, to start directly with phase 1 clinical trials.

Safety, immunogenicity, and antibody persistence following an investigational Streptococcus pneumoniae and Haemophilus influenzae triple-protein vaccine in a phase 1 randomized controlled study in healthy adults.

We investigated a protein-based nontypeable Haemophilus influenzae (NTHi) and pneumococcal (HiP) vaccine containing pneumococcal histidine triad D (PhtD), detoxified pneumolysin (dPly), and NTHi protein D (PD) in adults. In a phase I study, 40 healthy 18- to 40-year-old subjects were randomized (2:2:1) to receive two HiP doses administered 60 days apart, with or without AS03 adjuvant (HiP-AS and HiP groups, respectively), or Engerix B (GlaxoSmithKline, Belgium) as a control. Safety, antibodies, and antigen-specific CD4(+) T-cell immune responses were assessed before and until 480 days after vaccination. No serious adverse events were reported, and no subject withdrew due to an adverse event. Local and systemic symptoms were reported more frequently in the HiP-AS group than in the other two groups. The frequency and intensity of local and systemic symptoms appeared to increase after the second dose of HiP-AS or HiP but not Engerix B. Antibody geometric mean concentrations (GMCs) for PhtD, dPly, and PD increased after each dose of HiP-AS or HiP, with higher GMCs being observed in the HiP-AS group (statistically significant for anti-PD after dose 1 and anti-Ply after dose 2). GMCs remained higher at day 420 than prior to vaccination in both the HiP-AS and HiP groups. Antigen-specific CD4(+) T cells increased after each dose but were unmeasurable by day 480. Two doses of an investigational PhtD-dPly-PD protein vaccine induced humoral immunity and antigen-specific CD4(+) T-cell responses after each dose, with generally higher responses when the vaccine was administered with AS03. HiP combined with AS03 appeared to be more reactogenic than the antigens alone. (This study has been registered at ClinicalTrials.gov under registration no. NCT00814489.).

Neutral and acidic oligosaccharides supplementation does not increase the vaccine antibody response in preterm infants in a randomized clinical trial.

BACKGROUND: In preterm infants, a decreased immunological response and lower serological effectiveness are observed after immunizations due to ineffectiveness of both humoral and cellular immune mechanisms. OBJECTIVE: To determine the effect of 80% neutral oligosaccharides [small-chain galacto-oligosaccharides/long-chain fructo-oligosaccharides (scGOS/lcFOS)] in combination with 20% pectin-derived acidic oligosaccharides (pAOS) on antibody concentrations after DTaP-IPV-Hib immunization in preterm infants. DESIGN: In this randomized clinical trial, preterm infants with gestational age <32 weeks and/or birth weight <1500 g received enteral supplementation with scGOS/lcFOS/pAOS or placebo (maltodextrin) between days 3 and 30 of life. Blood samples were collected at 5 and 12 months of age. RESULTS: In total, 113 infants were included. Baseline and nutritional characteristics were not different in both groups. Geometric mean titers were not different after prebiotic supplementation at 5 months, Ptx (37/44 EU/ml), FHA (78/96 EU/ml), Prn (78/80 EU/ml), Diphtheria (0.40/0.57 IU/ml), Tetanus (0.74/0.99 IU/ml) and Hib (0.35/0.63 µg/ml), and at 12 months Ptx (55/66 EU/ml), FHA (122/119 EU/ml), Prn (116/106 Eu/ml), Diphtheria (0.88/1.11 IU/ml), Tetanus (1.64/1.79 IU/ml) and Hib (2.91/2.55 µg/ml). CONCLUSIONS: Enteral supplementation of neutral (scGOS/lcFOS) and acidic oligosaccharides (pAOS) does not improve the immunization response in preterm infants. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN16211826 ISRCTN16211826.

New insights in cellular immune response in colostrum-deprived pigs after immunization with subunit and commercial vaccines against Glässer's disease.

Four groups of colostrum-deprived pigs were immunized with Porcilis Glässer® (PG) or with subunit vaccines developed by us (rTbpA, NPAPT(M) or NPAPT(Cp)) against Glässer's disease, and they were challenged with 3×10(8)CFU of Haemophilus parasuis. A strong reduction in CD3(+)γδTCR(+) cells was seen in non-immunized control and scarcely protected (rTbpA) groups, suggesting that these cells could represent a target of H. parasuis infection. A significant increase in CD172α(+)CD163(+) cells was detected in all groups but PG, while a reduction in SLAIIDR(+) molecules expression was observed after challenge in control animals. Significant increases in CD3ε(+)CD8α(+)CD8ß(+) and B cells were detected respectively in control and NPAPT groups, and in scarcely (rTbpA) and well-protected (NPAPT(M) and NPAPT(Cp)) groups. Finally, a greater response in CD4(+)CD8α(-) cells was observed in NPAPT(Cp) compared to NPAPT(M) and PG groups. These results state the potential of NPAPT antigen for developing effective vaccines against Glässer's disease.

Acute phase protein concentrations in colostrum-deprived pigs immunized with subunit and commercial vaccines against Glässer's disease.

Haemophilus parasuis is the etiological agent of Glässer's disease, which is characterized by fibrinous polyserositis, polyarthritis and meningitis in pigs. This study was focused on the characterization of the acute-phase response after immunization and infection of colostrum-deprived pigs with H. parasuis serovar 5, by measuring serum concentrations of three positive acute-phase proteins (APPs) (pig major acute-phase protein pig, MAP; haptoglobin, HPG; C-reactive protein, CRP) and one negative APP (apolipoprotein A-I, ApoA-I). Six experimental groups were established: a non-immunized but infected control group (CTL); two groups immunized with either a recombinant transferrin-binding protein (Tbp) A or TbpB fragment from H. parasuis Nagasaki strain (rTbpA and rTbpB, respectively); two groups immunized with native outer membrane proteins with affinity to porcine transferrin (NPAPT), one of them inoculated intramuscularly (NPAPTim) and the other intratracheally (NPAPTit), and the last group receiving a commercially available bacterin (PG). The greatest concentrations of the three positive APPs and the lowest concentration of the negative APP were detected in CTL group, as well as in those animals belonging to rTbpA or rTbpB groups that died in response to challenge. Significant differences (P<0.005) were found in these groups when comparing challenge with the following days after it. However, no significant differences were seen for the remaining vaccinated groups (NPAPTim, NPAPTit and PG), which were effectively protected against Glässer's disease. Therefore, APPs could be used as useful biomarkers for both evaluating disease progression and determining vaccination effectiveness.

Seroprotection associated with infant vitamin A supplementation given with vaccines is not related to antibody affinity to Hepatitis B and Haemophilus influenzae type b vaccines.

Vaccines are usually assessed by analyses of their safety and immunogenicity to determine the effectiveness of eliciting antibody responses against target organisms. However, it is equally important to establish antibody affinity because of its specific role in protection from infection. Antibody affinity can be determined by comparisons of various antibody concentrations in dose-response curves. During a study on the immunogenicity of a pentavalent vaccine in 888 infants, antibody affinity analyses of the hepatitis B and Haemophilus influenzae type b components were investigated in infants given 15mg RE vitamin A with their vaccination and those who were not given vitamin A. In this paper we present the results of 222 infants; a 25% sub-sample of the original study. Analyses were carried out using dilutions of serum samples from fitted values corresponding to optical densities from antibody detection assays. These were obtained from the ligand binding equation and mid point titres in dose-response curves were then calculated. Vitamin A supplementation had no effect on the midpoint titres of Hepatitis B and H. influenzae type b vaccine derived antibodies. The significant effect of vitamin A supplementation on the Hepatitis B vaccine component observed in a previous seroprotection analysis is probably due to the amount of antibodies since affinity was unaffected.

Invasive Haemophilus influenzae Disease, Europe, 1996-2006.

An international collaboration was established in 1996 to monitor the impact of routine Haemophilus influenzae type b (Hib) vaccination on invasive H. influenzae disease; 14 countries routinely serotype all clinical isolates. Of the 10,081 invasive H. influenzae infections reported during 1996-2006, 4,466 (44%, incidence 0.28 infections/100,000 population) were due to noncapsulated H. influenzae (ncHi); 2,836 (28%, 0.15/100,000), to Hib; and 690 (7%, 0.036/100,000), to non-b encapsulated H. influenzae. Invasive ncHi infections occurred in older persons more often than Hib (median age 58 years vs. 5 years, p<0.0001) and were associated with higher case-fatality ratios (12% vs. 4%, p<0.0001), particularly in infants (17% vs. 3%, p<0.0001). Among non-b encapsulated H. influenzae, types f (72%) and e (21%) were responsible for almost all cases; the overall case-fatality rate was 9%. Thus, the incidence of invasive non-type b H. influenzae is now higher than that of Hib and is associated with higher case fatality.

A single nasal dose of fms-like tyrosine kinase receptor-3 ligand, but not peritoneal application, enhances nontypeable Haemophilus influenzae-specific long-term mucosal immune responses in the nasopharynx.

Nasal vaccination is an effective therapeutic regimen for preventing otitis media. In the development of nasal vaccine, an appropriate adjuvant is required. In the present study, we examined the efficacy of fms-like tyrosine kinase receptor-3 ligand (Flt3L) as a mucosal adjuvant. Flt3L was administered intranasally or peritoneally to mice, which were then immunized intranasally with P6 protein of nontypeable Haemophilus influenzae (NTHi), and P6-specific immune responses were examined. In addition, NTHi challenges were performed and the level of NTHi was quantified in nasal washes. Nasal application of Flt3L induced an increase in the number of dendritic cells in nasal-associated lymphoid tissue. P6-specific nasal wash immunoglobulin (Ig)A and serum IgG titers were elevated significantly after nasal immunization. Enhanced NTHi clearance from the nasopharynx was also observed. The effect of nasal vaccination with P6 combined with nasal Flt3L application was prolonged. These results indicate the potential of Flt3L as an effective mucosal adjuvant and suggest that nasal vaccination with P6 in combination with nasal Flt3L might be an effective regimen for the induction of NTHi-specific protective immunity.

Acute-phase protein response in pigs experimentally infected with Haemophilus parasuis.

The acute-phase protein (APP) response to an infection caused by Haemophilus parasuis, the etiological agent of Glässer's disease in pigs, was characterized measuring serum concentrations of pig major acute-phase protein (pig MAP), haptoglobin (HPT), C-reactive protein (CRP) and apolipoprotein A-I (ApoA-I) in colostrum-deprived pigs. They were divided into six experimental groups: non-immunized control group (I); immunized with a non-commercial bacterin (II); with an OMP-vaccine (III); with a sublethal dose (IV); and with two commercial bacterins (V and VI). All groups were challenged intratracheally with 5 × 10(9)CFU of H. parasuis 37 days after immunisation. The highest levels of the positive APPs (pig MAP, HPT and CRP) and the lowest levels of the negative APPs (ApoA-I) were observed in the animals that died as a consequence of the infection, both those in the non-immunized and in the immunized groups. However, the surviving animals (all of them in groups II, V and VI, two pigs in group III, and three in group IV) showed a minor variation in APP response, mainly on day 1 post-challenge (p.c.), and then tended to recover the initial values. APP response was still less pronounced in the groups of pigs previously immunized with bacterins. In conclusion, APP response can reflect Glässer-disease ongoing, showing a correlation between the severity and duration of the clinical signs and lesions and the magnitude of changes in the APP levels.

Haemophilus influenzae Type B conjugate vaccine introduction in Mali: impact on disease burden and serologic correlate of protection.

In Bamako, Mali, where surveillance revealed a high incidence of Haemophilus influenzae type b (Hib) invasive disease, Hib conjugate vaccine was introduced into the Expanded Program on Immunization and the impact assessed. Annual confirmed Hib hospitalizations for infants 0-11 months of age fell from 175/10(5) to 44/10(5) (P < 0.001); among infants 6-7 months of age Hib hospitalizations fell from 377/10(5) to 69/10(5), (82% decrease, P < 0.001). Invasive Streptococcus pneumoniae hospitalizations remained unchanged. In a baseline serosurvey, only 3/200 infants 6-7 months of age (1.5%) had protective anti-polyribosylribitol phosphate (PRP) titers > or = 0.15 microg/mL and 1(0.5%) had >or = 1.0 microg/mL. In serosurveys 18 and 30 months after vaccine introduction, 168/201 (84%) and 184/200 (92%) infants, respectively, had titers > or = 0.15 microg/mL and 141/201 (70%) and 163/200 (82%) had titers > or = 1.0 microg/mL. Introduction of Hib vaccine led to rises in anti-PRP seroprevalence, significant reductions in Hib disease, and all-cause hospitalizations, whereas S. pneumoniae disease remained unchanged.