Nontypeable Haemophilus Influenzae Vaccine Research Articles

Lung mucosal immunity to NTHi vaccine antigens: Antibodies in sputum of chronic obstructive pulmonary disease patients

ABSTRACT Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory illness in older adults. A major cause of COPD-related morbidity and mortality is acute exacerbation of COPD (AECOPD). Bacteria in the lungs play a role in exacerbation development, and the most common pathogen is non-typeable Haemophilus influenzae (NTHi). A vaccine to prevent AECOPD containing NTHi surface antigens was tested in a clinical trial. This study measured IgG and IgA against NTHi vaccine antigens in sputum. Sputum samples from 40 COPD patients vaccinated with the NTHi vaccine were collected at baseline and 30 days after the second dose. IgG and IgA antibodies against the target antigens and albumin were analyzed in the sputum. We compared antibody signals before and after vaccination, analyzed correlation with disease severity and between sputum and serum samples, and assessed transudation. Antigen-specific IgG were absent before vaccination and present with high titers after vaccination. Antigen-specific IgA before and after vaccination were low but significantly different for two antigens. IgG correlated between sputum and serum, and between sputum and disease severity. Sputum albumin was higher in patients with severe COPD than in those with moderate COPD, suggesting changes in transudation played a role. We demonstrated that immunization with the NTHi vaccine induces antigen-specific antibodies in sputum. The correlation between IgG from sputum and serum and the presence of albumin in the sputum of severe COPD patients suggested transudation of antibodies from the serum to the lungs, although local IgG production could not be excluded. Clinical Trial Registration: NCT02075541

Ex-vivo RNA expression analysis of vaccine candidate genes in COPD sputum samples

BackgroundChronic obstructive pulmonary disease (COPD) is a lung disease characterised by airflow-limiting inflammation and mucus production. Acute exacerbations are a major cause of COPD-related morbidity and mortality and are mostly associated with bacterial or viral infections. A vaccine targeting non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat), the main bacteria associated with exacerbations, was tested in a Phase 2 trial. We assessed “ex-vivo” expression of vaccine candidate and housekeeping genes pd, pe, pilA, gapA, ompP6 of NTHi, and uspA2, parE, polA of Mcat in sputum samples of COPD patients and determined whether expression of the vaccine candidate genes pd, pe, pilA (NTHi) and uspA2 (Mcat) differed between stable and exacerbation samples.MethodsA single-centre, prospective, observational cohort study was conducted where 123 COPD patients were seen on enrolment, followed monthly for 2 years, and reviewed after onset of acute exacerbations. We selected 69 patients with sputum samples positive for NTHi or Mcat by PCR during at least one stable and one exacerbation visit. mRNA was isolated from the sputum, and expression of NTHi and Mcat genes was analysed with RT-PCR. Statistical analyses compared mRNA concentrations between stable and exacerbation samples and in relationship to COPD severity and exacerbation frequency.ResultsThe vaccine candidate genes were variably expressed in sputum samples, suggesting they are expressed in the lung. Absolute and relative expression of all NTHi vaccine candidate genes and Mcat uspA2 were similar between exacerbation and stable samples. Expression of pd and pilA was slightly associated with the number of exacerbations in the year before enrolment, and uspA2 with the disease severity status at enrolment.ConclusionsThe NTHi-Mcat vaccine candidate genes were expressed in sputum samples, and each gene had a specific level of expression. No statistically significant differences in gene expression were detectable between stable and exacerbation samples. However, the history of COPD exacerbations was slightly associated with the expression of pd, pilA and uspA2.Trial registration NCT01360398 (https://www.clinicaltrials.gov)Graphical abstract

Invasive Haemophilus influenzae disease among adults in Japan during 2014-2018.

We describe the epidemiology of invasive Haemophilus influenzae disease (IHD) among adults in Japan. Data for 200 adult IHD patients in 2014-2018 were analyzed. The capsular type of H. influenzae was determined by bacterial agglutination and polymerase chain reaction (PCR), and non-typeable Haemophilus influenzae (NTHi) was identified by PCR. The annual incidence of IHD (cases per 100,000 population) was 0.12 for age 15-64years and 0.88 for age ≥ 65years in 2018. The median age was 77years, and 73.5% were aged ≥ 65years. About one-fourth of patients were associated with immunocompromising condition. The major presentations were pneumonia, followed by bacteremia, meningitis and other than pneumonia or meningitis (other diseases). The case fatality rate (CFR) was 21.2% for all cases, and was significantly higher in the ≥ 65-year group (26.1%) than in the 15-64-year group (7.5%) (p = 0.013). The percentage of cases with pneumonia was significantly higher in the ≥ 65-year group than in the 15-64-year group (p < 0.001). The percentage of cases with bacteremia was significantly higher in the 15-64-year group than in the ≥ 65-year group (p = 0.027). Of 200 isolates, 190 (95.0%) were NTHi strains, and the other strains were encapsulated strains. 71 (35.5%) were resistant to ampicillin, but all were susceptible to ceftriaxone. The clinical presentations of adult IHD patients varied widely; about three-fourths of patients were age ≥ 65years and their CFR was high. Our findings support preventing strategies for IHD among older adults, including the development of NTHi vaccine.

Examination of phase-variable haemoglobin-haptoglobin binding proteins in non-typeable Haemophilus influenzae reveals a diverse distribution of multiple variants.

Non-typeable Haemophilus influenzae (NTHi) is a major human pathogen for which there is no globally licensed vaccine. NTHi has a strict growth requirement for iron and encodes several systems to scavenge elemental iron and heme from the host. An effective NTHi vaccine would target conserved, essential surface factors, such as those involved in iron acquisition. Haemoglobin–haptoglobin binding proteins (Hgps) are iron-uptake proteins localized on the outer-membrane of NTHi. If the Hgps are to be included as components of a rationally designed subunit vaccine against NTHi, it is important to understand their prevalence and diversity. Following analysis of all available Hgp sequences, we propose a standardized grouping method for Hgps, and demonstrate increased diversity of these proteins than previously determined. This analysis demonstrated that genes encoding variants HgpB and HgpC are present in all strains examined, and almost 40% of strains had a duplicate, nonidentical hgpB gene. Hgps are also phase-variably expressed; the encoding genes contain a CCAA(n) simple DNA sequence repeat tract, resulting in biphasic ON–OFF switching of expression. Examination of the ON–OFF state of hgpB and hgpC genes in a collection of invasive NTHi isolates demonstrated that 58% of isolates had at least one of hgpB or hgpC expressed (ON). Varying expression of a diverse repertoire of hgp genes would provide strains a method of evading an immune response while maintaining the ability to acquire iron via heme. Structural analysis of Hgps also revealed high sequence variability at the sites predicted to be surface exposed, demonstrating a further mechanism to evade the immune system—through varying the surface, immune-exposed regions of the membrane anchored protein. This information will direct and inform the choice of candidates to include in a vaccine against NTHi.

Non-typeable Haemophilus influenzae protein vaccine in adults with COPD: A phase 2 clinical trial

Loss of airway microbial diversity is associated with non-typeable Haemophilus influenzae (NTHi) infection and increased risk of exacerbation in chronic obstructive pulmonary disease (COPD). We assessed the safety and immunogenicity of an investigational vaccine containing NTHi antigens, recombinant protein D (PD) and combined protein E and Pilin A (PE-PilA), and AS01 adjuvant in adults with moderate/severe COPD and prior exacerbations.In this phase 2, observer-blind, controlled trial (NCT02075541), 145 COPD patients aged 40–80 years randomly (1:1) received two doses of NTHi vaccine or placebo 60 days apart, on top of standard care.Reactogenicity in the 7-day post-vaccination period was higher following NTHi vaccine than placebo. Most solicited adverse events (AEs) were mild/moderate. At least one unsolicited AE was reported during the 30-day post-vaccination period by 54.8% of NTHi vaccine and 51.4% of placebo recipients. One serious AE (placebo group) was assessed by the investigator as vaccine-related. Anti-PD, anti-PE and anti-PilA geometric mean antibody concentrations increased up to 30 days after each NTHi vaccine dose, waned thereafter, but remained higher than baseline (non-overlapping confidence intervals) up to 13 months post-dose 2. The frequency of specific CD4+ T cells increased following two doses of NTHi vaccine and remained higher than baseline. Exploratory analysis showed a statistically non-significant lower yearly rate of moderate/severe exacerbations in the NTHi vaccine group than following placebo (1.49 versus 1.73) in the one-year period post-dose 2, with estimated vaccine efficacy of 13.3% (95% confidence interval −24.2 to 39.5; p = 0.44).The NTHi vaccine had an acceptable safety and reactogenicity profile and good immunogenicity in adults with COPD.

A Protein E-PilA Fusion Protein Shows Vaccine Potential against Nontypeable Haemophilus influenzae in Mice and Chinchillas.

PE-PilA is a fusion protein composed of immunologically relevant parts of protein E (PE) and the majority subunit of the type IV pilus (PilA), two major antigens of nontypeable Haemophilus influenzae (NTHi). Here we report on the preclinical evaluation of PE-PilA as a vaccine antigen. The immunogenic potential of the PE and PilA within the fusion was compared with that of isolated PE and PilA antigens. When injected intramuscularly into mice, the immunogenicity of PE within the fusion was equivalent to that of isolated PE, except when it was formulated with alum. In contrast, in our murine models PilA was consistently found to be more immunogenic as a subentity of the PE-PilA fusion protein than when it was injected as an isolated antigen. Following immunization with PE-PilA, anti-PE antibodies demonstrated the same capacity to inhibit the binding of PE to vitronectin as those induced after PE immunization. Likewise, PE-PilA-induced anti-PilA antibodies inhibited the formation of NTHi biofilms and disrupted established biofilms in vitro These experiments support the immunogenic equivalence between fused PE-PilA and isolated PE and PilA. Further, the potential of PE-PilA immunization against NTHi-induced disease was evaluated. After intranasal NTHi challenge, colonization of the murine nasopharynx significantly dropped in animals formerly immunized with PE-PilA, and in chinchillas, signs of otitis media were significantly reduced in animals that had received anti-PE-PilA antibodies. Taken together, our data support the use of PE-PilA as an NTHi vaccine antigen.

Intranasal coinfection model allows for assessment of protein vaccines against nontypeable Haemophilus influenzae in mice.

Nontypeable Haemophilus influenzae (NTHi) is a commensal in the human nasopharynx and the cause of pneumonia, meningitis, sinusitis, acute exacerbations of chronic obstructive pulmonary disease and acute otitis media (AOM). AOM is the most common ailment for which antibiotics are prescribed in the United States. With the emergence of new strains of antibiotic-resistant bacteria, finding an effective and broad coverage vaccine to protect against AOM-causing pathogens has become a priority. Mouse models are a cost-effective and efficient way to help determine vaccine efficacy. Here, we describe an NTHi AOM model in C57BL/6J mice, which also utilizes a mouse-adapted H1N1 influenza virus to mimic human coinfection. We tested our coinfection model using a protein vaccine formulation containing protein D, a well-studied NTHi vaccine candidate that can be found in the 10-valent Streptococcus pneumoniae conjugate vaccine. We verified the usefulness of our mouse model by comparing bacterial loads in the nose and ear between protein D-vaccinated and control mice. While there was no measurable difference in nasal bacterial loads, we did detect significant differences in the bacterial loads of ear washes and ear bullae between vaccinated and control mice. The results from this study suggest that our NTHi AOM coinfection model is useful for assessing protein vaccines.

Recognition of conserved antigens by Th17 cells provides broad protection against pulmonary Haemophilus influenzae infection

Nontypeable Haemophilus influenzae (NTHi) is a major cause of community acquired pneumonia and exacerbation of chronic obstructive pulmonary disease. A current effort in NTHi vaccine development has focused on generating humoral responses and has been greatly impeded by antigenic variation among the numerous circulating NTHi strains. In this study, we showed that pulmonary immunization of mice with killed NTHi generated broad protection against lung infection by different strains. While passive transfer of immune antibodies protected only against the homologous strain, transfer of immune T cells conferred protection against both homologous and heterologous strains. Further characterization revealed a strong Th17 response that was cross-reactive with different NTHi strains. Responding Th17 cells recognized both cytosolic and membrane-associated antigens, while immune antibodies preferentially responded to surface antigens and were highly strain specific. We further identified several conserved proteins recognized by lung Th17 cells during NTHi infection. Two proteins yielding the strongest responses were tested as vaccine candidates by immunization of mice with purified proteins plus an adjuvant. Immunization induced antigen-specific Th17 cells that recognized different strains and, upon adoptive transfer, conferred protection. Furthermore, immunized mice were protected against challenge with not only NTHi strains but also a fully virulent, encapsulated strain. Together, these results show that the immune mechanism of cross-protection against pneumonia involves Th17 cells, which respond to a broad spectrum of antigens, including those that are highly conserved among NTHi strains. These mechanistic insights suggest that inclusion of Th17 antigens in subunit vaccines offers the advantage of inducing broad protection and complements the current antibody-based approaches.

Haemophilus influenzae oral vaccination for preventing acute exacerbations of chronic bronchitis and chronic obstructive pulmonary disease.

Chronic bronchitis and chronic obstructive pulmonary disease (COPD) are serious conditions in which patients are predisposed to viral and bacterial infections resulting in potentially fatal acute exacerbations. Chronic obstructive pulmonary disease is defined as a lung disease characterised by obstruction to lung airflow that interferes with normal breathing. Antibiotic therapy has not been particularly useful in eradicating bacteria such as non-typeable Haemophilus influenzae (NTHi) because they are naturally occurring flora of the upper respiratory tract in many people. However, they can cause opportunistic infection. An oral NTHi vaccine has been developed to protect against recurrent infective acute exacerbations in chronic bronchitis. To assess the effectiveness of an oral, whole-cell NTHi vaccine in protecting against recurrent episodes of acute exacerbations of chronic bronchitis and COPD in adults. To assess the effectiveness of NTHi vaccine in reducing NTHi colonising the respiratory tract during recurrent episodes of acute exacerbations of COPD. We searched the following databases: the Cochrane Central Register of Controlled Trials (CENTRAL) (2017, Issue 1), MEDLINE (1946 to January 2017), Embase (1974 to January 2017), CINAHL (1981 to January 2017), LILACS (1985 to January 2017), and Web of Science (1955 to January 2017). We also searched trials registries and contacted authors of trials requesting unpublished data. We included randomised controlled trials comparing the effects of an oral monobacterial NTHi vaccine in adults with recurrent acute exacerbations of chronic bronchitis or COPD when there was overt matching of the vaccine and placebo groups on clinical grounds. The selection criteria considered populations aged less than 65 years and those older than 65 years. Two review authors independently assessed trial quality and extracted data from original records and publications for incidence and severity of bronchitis episodes and carriage rate of NTHi measured in the upper respiratory tract, as well as data relevant to other primary and secondary outcomes. We identified six placebo-controlled randomised controlled trials with a total of 557 participants. These trials investigated the efficacy of enteric-coated, killed preparations of H influenzae in populations prone to recurrent acute exacerbations of chronic bronchitis or COPD. The vaccine preparation and immunisation regimen in all trials consisted of at least three courses of formalin-killed H influenzae in enteric-coated tablets taken at intervals (e.g. days 0, 28, and 56). Each course generally consisted of two tablets taken after breakfast over three consecutive days. In all cases the placebo groups took enteric-coated tablets containing glucose. Risk of bias was moderate across the studies, namely due to the lack of information provided about methods and inadequate presentation of results.Meta-analysis of the oral NTHi vaccine showed a small, non-statistically significant reduction in the incidence of acute exacerbations of chronic bronchitis or COPD (risk ratio (RR) 0.79, 95% confidence interval (CI) 0.57 to 1.10; P = 0.16). There was no significant difference in mortality rate between the vaccine and placebo groups (odds ratio (OR) 1.62, 95% CI 0.63 to 4.12; P = 0.31).We were unable to meta-analyse the carriage levels of NTHi in participants as each trial reported this result using different units and tools of measurement. Four trials showed no significant difference in carriage levels, while two trials showed a significant decrease in carriage levels in the vaccinated group compared with the placebo group.Four trials assessed severity of exacerbations measured by requirement for antibiotics. Three of these trials were comparable and when meta-analysed showed a statistically significant 80% increase in antibiotic courses per person in the placebo group (RR 1.81, 95% CI 1.35 to 2.44; P < 0.001). There was no significant difference between the groups with regard to hospital admission rates (OR 0.96, 95% CI 0.13 to 7.04; P = 0.97). Adverse events were reported in five trials but were not necessarily related to the vaccine; a point estimate is suggestive that they occurred more frequently in the vaccine group, however this result was not statistically significant (RR 1.43, 95% CI 0.70 to 2.92; P = 0.87). Quality of life was not meta-analysed but was reported in two trials, with results at six months showing an improvement in quality of life in the vaccinated group (scoring at least two points better than placebo). Analyses demonstrate that NTHi oral vaccination of people with recurrent exacerbations of chronic bronchitis or COPD does not yield a significant reduction in the number and severity of exacerbations. Evidence was mixed, and the individual trials that showed a significant benefit of the vaccine are too small to advocate widespread oral vaccination of people with COPD.

Transcutaneous Immunization with a Band-Aid Prevents Experimental Otitis Media in a Polymicrobial Model.

Otitis media (OM) is a common pediatric disease, and nontypeable Haemophilus influenzae (NTHI) is the predominant pathogen in chronic OM, recurrent OM, and OM associated with treatment failure. OM is also a polymicrobial disease, wherein an upper respiratory tract viral infection predisposes to ascension of NTHI from the nasopharynx, the site of colonization, to the normally sterile middle ear, resulting in disease. Using a clinically relevant viral-bacterial coinfection model of NTHI-induced OM, we performed transcutaneous immunization (TCI) via a band-aid delivery system to administer each of three promising NTHI vaccine candidates derived from bacterial adhesive proteins and biofilm mediators: recombinant soluble PilA (rsPilA), chimV4, and integration host factor. Each immunogen was admixed with the adjuvant LT(R192G/L211A), a double mutant of Escherichia coli heat-labile enterotoxin, and assessed for relative ability to prevent the onset of experimental OM. For each cohort, the presence of circulating immunogen-specific antibody-secreting cells and serum antibody was confirmed prior to intranasal NTHI challenge. After bacterial challenge, blinded video otoscopy and tympanometry revealed a significant reduction in the proportion of animals with signs of OM compared to levels in animals receiving adjuvant only, with an overall vaccine efficacy of 64 to 77%. These data are the first to demonstrate the efficacy afforded by TCI with a band-aid vaccine delivery system in a clinically relevant polymicrobial model of OM. The simplicity of TCI with a band-aid and the significant efficacy observed here hold great promise for reducing the global burden of OM in the pediatric population.

Multi-site placebo-controlled randomised clinical trial to assess protection following oral immunisation with inactivated non-typeable Haemophilus influenzae in chronic obstructive pulmonary disease.

Previous studies identified factors that modify response to an oral non-typeable Haemophilus influenzae (NTHi) vaccine in chronic obstructive pulmonary disease (COPD): severe COPD, moderate-severe exacerbations as end-point and a threshold prevalence of NTHi in the study population. More data are needed to confirm parameters that influence clinical outcomes. The primary aim was to determine the efficacy of an oral NTHi vaccine (HI-164OV) in reducing the rate of exacerbations requiring systemic corticosteroids or hospitalisation in COPD. Secondary aims included effect on the proportion of patients experiencing such exacerbations, severity of infections and quality of life (St George Respiratory Questionnaire for COPD patients (SGRQ-C)). This multi-centre, double-blind, placebo-controlled study was conducted at 21 Australian sites for 9 months in 2011. Three-hundred and twenty subjects with COPD, FEV1 <60% predicted and ≥1 moderate-severe exacerbations in the previous 12 months were recruited. The primary and secondary end-points for the intention-to-treat population aged 40-88 years were not achieved, and only 5% of subjects had an H. influenzae-positive sputum sample. Subsequent exploratory analysis of patients <65 years (91 subjects) indicated protection with respect to the primary and most of the secondary end-points, with SGRQ-C symptom scores lower at 3 and 6 months. Patients aged 40-88 years with moderate to severe COPD and low rates of H. influenzae-positive sputum were not protected against exacerbations by HI-1640V. Further studies are needed to confirm protection in subjects aged <65 years. Older age and low colonisation rates appear to affect adversely response to this vaccine.

Phase I, randomized, observer-blind, placebo-controlled studies to evaluate the safety, reactogenicity and immunogenicity of an investigational non-typeable Haemophilus influenzae (NTHi) protein vaccine in adults

BackgroundNon-typeable Haemophilus influenzae (NTHi) is a major cause of various respiratory diseases. The development of an effective vaccine against NTHi mandates new approaches beyond conjugated vaccines as this opportunistic bacterium is non-encapsulated. Here we report on the safety, reactogenicity and immunogenicity of a multi-component investigational vaccine based on three conserved surface proteins from NTHi (proteins D [PD], E [PE] and Pilin A [PilA]) in two observer-blind phase I studies. MethodsIn the first study (NCT01657526), 48 healthy 18–40 year-olds received two vaccine formulations (10 or 30μg of each antigen [PD and a fusion protein PE-PilA]) or saline placebo at months 0 and 2. In the second study (NCT01678677), 270 50–70 year-olds, current or former smokers, received eight vaccine formulations (10 or 30μg antigen/dose non-adjuvanted or adjuvanted with alum, AS01E or AS04C) or saline placebo at months 0, 2 and 6 (plain and alum-adjuvanted groups) and at months 0 and 2 (AS-adjuvanted groups). Solicited and unsolicited adverse events (AEs) were recorded for 7 and 30 days post-vaccination, respectively; potential immune-mediated diseases (pIMDs) and serious AEs (SAEs) throughout the studies. Humoral and antigen-specific T-cell immunity (in study 2 only) responses were assessed up to 12 months post-vaccination. ResultsObserved reactogenicity was highest in the AS-adjuvanted groups but no safety concerns were identified with any of the NTHi vaccine formulations. One fatal SAE (cardiac arrest) not considered related to vaccination, and one pIMD (non-serious psoriasis) in the Placebo group, were reported post-dose 3 in Study 2. All formulations generated a robust antibody response while the AS01-adjuvanted formulations produced the highest humoral and cellular immune responses. ConclusionThis study confirms that the NTHi vaccine formulations had an acceptable reactogenicity and safety profile and were immunogenic in adults. These results justify further clinical development of this NTHi vaccine candidate.

Serum Concentrations of Antibodies against Outer Membrane Protein P6, Protein D, and T- and B-Cell Combined Antigenic Epitopes of Nontypeable Haemophilus influenzae in Children and Adults of Different Ages.

Nontypeable Haemophilus influenzae (NTHi) is one of the most common etiologies of acute otitis media, rhinosinusitis, and pneumonia. Outer membrane proteins (OMPs) are the main focus in new vaccine development against NTHi, as the H. influenzae type b (Hib) vaccine does not cover noncapsulated NTHi. The OMPs P6 and protein D are the most promising candidate antigens for an NTHi vaccine, and low antibody levels against them in serum may be correlated with infection caused by NTHi. In the current study, we measured the antibody titers against P6, protein D, and their T- and B-cell combined peptide epitopes in healthy individuals of different ages. We found that children <1 month old had the lowest antibody levels against NTHi P6, protein D, and their T- and B-cell combined antigenic epitopes. Antibody titers increased at ages 1 to 6 months, peaked at 7 months to 3 years, and remained high at 4 to 6 years. The antibody titers started to decrease after 6 years and were the lowest in the 21- to 30-year group. The geometric mean titers (GMTs) of T- and B-cell combined antigenic epitopes in P6 and protein D were positively correlated with those of the protein antigens. Among 12 peptides tested, P6-61, P6-123, and protein D-167 epitopes were better recognized than others in human serum. These findings might contribute to the development of an effective serotype-independent vaccine for H. influenzae.

Haemophilus influenzae oral vaccination for preventing acute exacerbations of chronic bronchitis and chronic obstructive pulmonary disease.

Chronic bronchitis and chronic obstructive pulmonary disease (COPD) are serious conditions in which patients are predisposed to viral and bacterial infections resulting in potentially fatal acute exacerbations. COPD is defined as a lung disease characterised by obstruction to lung airflow that interferes with normal breathing. Antibiotic therapy has not been particularly useful in eradicating bacteria such as non-typeable Haemophilus influenzae (NTHi) because they are naturally occurring flora of the upper respiratory tract in many people. However, they can cause opportunistic infection. An oral NTHi vaccine has been developed to protect against recurrent infective acute exacerbations in chronic bronchitis. To assess the effectiveness of an oral, whole-cell, non-typeable H. influenzae (NTHi) vaccine in protecting against recurrent episodes of acute exacerbations of chronic bronchitis and COPD in adults. To assess the effectiveness of NTHi vaccine in reducing NTHi colonising the respiratory tract during recurrent episodes of acute exacerbations of COPD. We searched the following databases: CENTRAL (2014, Issue 6), MEDLINE (1946 to July week 3, 2014), EMBASE (1974 to July 2014), CINAHL (1981 to July 2014), LILACS (1982 to July 2014) and Web of Science (1955 to July 2014). We also searched trials registries and contacted authors of trials requesting unpublished data. We included randomised controlled trials comparing the effects of an oral monobacterial NTHi vaccine in adults with recurrent acute exacerbations of chronic bronchitis or COPD when there was overt matching of the vaccine and placebo groups on clinical grounds. The selection criteria considered populations aged less than 65 years and those older than 65 years. Two authors independently assessed trial quality and extracted data from original records and publications for incidence and severity of bronchitis episodes and carriage rate of NTHi measured in the upper respiratory tract, as well as data relevant to other primary and secondary outcomes. We identified six placebo-controlled randomised controlled trials with a total of 557 participants. They investigated the efficacy of enteric-coated, killed preparations of H. influenzae in populations prone to recurrent acute exacerbations of chronic bronchitis or COPD. The vaccine preparation and immunisation regime in all trials consisted of at least three courses of formalin-killed H. influenzae in enteric-coated tablets taken at intervals (for example, days 0, 28 and 56). Each course generally consisted of two tablets taken after breakfast over three consecutive days. In all cases the placebo groups took enteric-coated tablets containing glucose. Risk of bias was moderate across the studies, namely due to the lack of information provided about methods and inadequate presentation of results.Meta-analysis of the oral NTHi vaccine showed a small, non-statistically significant reduction in the incidence of acute exacerbations of chronic bronchitis or COPD by 2.048% (risk ratio (RR) 0.97, 95% confidence interval (CI) 0.84 to 1.12, P value = 0.68). There was no significant difference in mortality rate between the vaccine and placebo groups (odds ratio (OR) 1.62, 95% CI 0.63 to 4.12, P value = 0.31).We were unable to meta-analyse the carriage levels of NTHi in participants as each trial reported this result using different units and tools of measurement. Four trials showed no significant difference in carriage levels, while two trials showed a significant decrease in carriage levels in the vaccinated group compared with placebo.Four trials assessed severity of exacerbations measured by requirement for antibiotics. Three of these trials were comparable and when meta-analysed showed a statistically significant 80% increase in antibiotic courses per person in the placebo group (RR 1.81, 95% CI 1.35 to 2.44, P value < 0.0001). There was no significant difference between the groups with regards to hospital admission rates (OR 0.96, 95% CI 0.13 to 7.04, P value = 0.97). Adverse events were reported in all six trials with a point estimate suggestive that they occurred more frequently in the vaccine group, however, this result was not statistically significant (RR 1.43, 95% CI 0.70 to 2.92, P value = 0.87). Quality of life was not meta-analysed but was reported in two trials, with results at six months showing an improvement in quality of life in the vaccinated group (scoring at least two points better than placebo). Analyses demonstrate that NTHi oral vaccination of patients with recurrent exacerbations of chronic bronchitis or COPD does not yield a significant reduction in the number and severity of exacerbations. Evidence is mixed and the individual trials that show a significant benefit of the vaccine are too small to advocate widespread oral vaccination of people with COPD.

Impact of immunization with Protein F on pulmonary clearance of nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) is one of the main aetiologies of childhood bacterial infections as well as exacerbations in COPD patients. Currently, no licensed NTHi vaccine exists. In the present study, we evaluated the potential of the conserved and ubiquitous surface protein Haemophilus Protein F (PF) as a vaccine candidate. Our results show that incubation of NTHi with anti-PF antibodies significantly increased the opsonophygocytosis of human promyelocytic leukemia cell line-derived granulocytes, leading to efficient killing of the bacteria (P≤0.05). The presence of anti-PF IgG titers in healthy adults (n=60) was investigated, and we found that 26% of healthy blood donors carried antibodies with the main antigenic epitope being PF23–48. Finally, mice immunized with PF23–48 attained a significantly increased capacity to clear NTHi as compared to a control group immunized with a peptide derived from Moraxella catarrhalis β-lactamase (P≤0.05). Taken together, our results indicate that PF is a potential NTHi-vaccine candidate.

Epitope-specific immune recognition of the nontypeable Haemophilus influenzae outer membrane protein 26

Previous studies using rodent respiratory infection models of nontypeable Haemophilus influenzae (NTHi) infection have established the 26-kDa outer membrane protein of the bacterium, OMP26, as a potential vaccine antigen for NTHi. This study undertook a comprehensive immunological identification of OMP26 T- and B-cell epitopes. A series of OMP26 peptides were constructed and regions of the OMP26 antigen involved in recognition by lymphocyte receptors and induction of acquired immune responses were identified. The dominant T-cell epitopes for OMP26 were located toward the C-terminus between amino acid residues 95 and 197 (T3+T4 region) as mapped using antigen-specific lymphocyte proliferation assays. The newly identified T-cell epitopes exhibited strong capacity for efficient T-cell activation, suggesting that, compared with other OMP26 regions; epitopes within the T3+T4 region have the highest affinity for binding to major histocompatibility complex molecules. In contrast, the predominant B-cell epitopes of OMP26 were located more centrally within the molecule between amino acid residues 45 and 145 (T2+T3 region) as determined using enzyme-linked immunosorbent assay and surface plasmon resonance assays. The T2+T3 region was immunodominant in several species including chinchilla, mice and rats when assessed using both mucosal and parenteral immunization regimes. In addition, the antibodies directed against the T2+T3 region bound to intact NTHi cell surface, according to flow cytometry. Collectively, these results specifically locate the amino acid sequences containing the OMP26 T- and B-cell epitopes, which, as newly mapped antigenic epitopes for lymphocyte recognition, will be useful to improve existing NTHi vaccine strategies. Comprehensive definition of the minimum epitope length required for optimal B- and T-cell responses requires further study.

Bactericidal antibody response against P6, protein D, and OMP26 of nontypeableHaemophilus influenzaeafter acute otitis media in otitis-prone children

The bactericidal antibody response to three nontypeable Haemophilus influenzae (NTHi) outer membrane proteins (D, P6, and OMP26) was studied in 24 otitis-prone children (aged 7-28months) after an acute otitis media (AOM) caused by NTHi. The study was carried out to understand the contribution of antigen-specific bactericidal antibody responses in the class of children who are most vulnerable to recurrent otitis media infections. Levels of protein D (P=0.005) and P6 (P=0.026) but not OMP26 antibodies were higher in bactericidal sera compared with nonbactericidal sera. For five (24%) and 16 (76%) of 21 bactericidal sera tested, removal of anti-protein D and P6 antibody, respectively, resulted in a two- to fourfold drop in bactericidal antibody. Antibodies to OMP26 did not make any contribution to the overall bactericidal activity in any serum samples. Eleven of 21 sera (52%) had bactericidal activity against a heterologous NTHi (86-028 NP) strain but the titers were significantly lower (P<0.05) as compared to the homologous strains. Future studies of protein D, P6, OMP26, and other potential NTHi vaccine antigens should include studies of bactericidal antibody in children who are otitis prone as a possible correlate of protection.

Construction and Immunogenicity of Recombinant Adenovirus Vaccines Expressing the HMW1, HMW2, or Hia Adhesion Protein of Nontypeable Haemophilus influenzae

The objective of the present study was to construct and assess the immunogenicity of recombinant adenovirus vectors expressing the HMW1, HMW2, or Hia protein of nontypeable Haemophilus influenzae (NTHi). These proteins are critical adhesins and potential protective antigens expressed by NTHi. Segments of the hmw1A and hmw2A structural genes that encode the distal one-half of mature HMW1 or HMW2 were cloned into the T7 expression vector pGEMEX-2. These constructs encoded stable HMW1 or HMW2 recombinant fusion protein that expresses B-cell epitopes common to most NTHi strains. A segment of the hia gene that encodes the surface-exposed portion of mature Hia was also cloned into pGEMEX-2. The resulting T7 gene 10 translational fusions were excised from the parent plasmids and cloned into the shuttle plasmid pDC316. Cotransfection of HEK 293 cells with the pDC316 derivatives and pBHGloxΔE1,3Cre resulted in the production of viral plaques from which recombinant adenoviruses expressing fusion proteins were recovered. Chinchillas immunized intraperitoneally with a single 10(8)-PFU dose of either the HMW2 or Hia adenoviral construct developed high anti-HMW2 or anti-Hia serum antibody titers within 4 weeks of immunization. Chinchillas immunized intranasally with a single 10(7)- to 10(9)-PFU dose of the Hia adenoviral construct also developed high anti-Hia serum antibody titers within 8 weeks of immunization. Recombinant adenoviruses represent a promising system to induce mucosal and systemic immunity and protection against mucosal diseases such as otitis media. Recombinant adenoviruses expressing recombinant HMW1, HMW2, or Hia protein will be important new tools in NTHi vaccine development efforts.

Breast-Feeding Is Associated With a Reduced Frequency of Acute Otitis Media and High Serum Antibody Levels Against NTHi and Outer Membrane Protein Vaccine Antigen Candidate P6

Nontypeable Haemophilus influenzae (NTHi) causes acute otitis media (AOM) in infants. Breast-feeding protects against AOM and/or nasopharyngeal (NP) colonization; however, the mechanism of protection is incompletely understood. Children with AOM and healthy children were studied according to feeding status: breastfed,breast/formula fed, or formula fed. Cumulative episodes of AOM, ELISA titers of serum IgG antibodies to whole-cell NTHi and vaccine candidate outer membrane protein P6, bactericidal titers of serum and NP colonization by NTHi were assessed. A lower incidence of AOM was found in breast- versus formula-fed children. Levels of specific serum IgG antibody to NTHi and P6 were highest in breast-fed, intermediate in breast/formula fed, and lowest in formula-fed infants. Serum IgG antibody to P6 correlated with bactericidal activity against NTHi. Among children with AOM, the prevalence of NTHi in the NP was lower in breast- versus nonbreast-fed infants. We conclude that breast-feeding shows an association with higher levels of antibodies to NTHi and P6, suggesting that breast-feeding modulates the serum immune response to NTHi and P6. Higher serum IgG might facilitate protection against AOM and NP colonization in breast-fed children.

Identification of surface-exposed B-cell epitopes on high molecular-weight adhesion proteins of nontypeable Haemophilus influenzae.

We previously reported that two surface-exposed high-molecular-weight proteins, HMW1 and HMW2, expressed by a prototypic strain of nontypeable Haemophilus influenzae (NTHI) mediate attachment to human epithelial cells. These proteins are members of a family of highly immunogenic proteins common to most nontypeable Haemophilus strains. We also reported that immunization with an HMW1-HMW2 mixture modified the course of disease in an animal model of otitis media, suggesting the potential usefulness of these proteins as NTHI vaccine components. Identification of surface-accessible B-cell epitopes could be important to efforts to develop recombinant or synthetic peptide vaccines based upon these high-molecular-weight proteins. Thus, the purpose of the present study was to identify surface-accessible epitopes on the HMW1 and HMW2 proteins by using monoclonal antibodies (MAbs) and to determine the prevalence of these epitopes among the high-molecular-weight proteins expressed by heterologous nontypeable Haemophilus strains. MAbs were generated by immunizing mice with high-molecular-weight proteins purified from prototype strains and were screened by immunoelectron microscopy (IEM) for the ability to recognize surface epitopes. Two MAbs, designated AD6 and 10C5, that recognized surface epitopes by IEM were recovered. In order to map the epitopes recognized by these two MAbs, we constructed a set of HMW1 and HMW2 recombinant fusion proteins using the pGEMEX vectors and examined the reactivity of the MAbs with these fusion proteins. MAb AD6 recognized an epitope in both HMW1 and HMW2 which mapped to the last 75 amino acids at the carboxy termini of the two proteins. When examined for reactivity with heterologous strains, MAb AD6 recognized high-molecular-weight proteins in 75% of 125 unrelated nontypeable Haemophilus strains and, in addition, reacted with three of three such strains when examined by IEM. MAb 10C5 recognized an epitope that mapped to a 155-amino-acid segment near the carboxy terminus of HMW1. This epitope was adjacent to but distinct from the AD6 epitope and was absent from HMW2. The 10C5 epitope was expressed by 40% of the AD6 reactive strains. Identification of shared surface-exposed epitopes on the high-molecular-weight adhesion proteins suggests the possibility of developing recombinant or synthetic peptide-based vaccines protective against disease caused by the majority of NTHI strains.