There are several characteristics, which define an ideal protein vaccine candidate. Proteins should be conserved among all/most clinically relevant strains in order to elicit broad protection. To determine if the targets are conserved, it is important to have access to a large collection of bacterial strains. 18 The conservation of the target protein can be determined at several levels: first, whether or not the gene is present in all strains; second, how conserved the protein amino acid sequence is among strains; third, how conserved is the global structure of the protein among strains (i.e., can monoclonal antibodies bind to the protein in all bacterial strains). Even though a protein is identified as conserved across strains, it is also critical to predict the toxicity of the protein. For instance, after introduction of the protein into the system, the protein should not stimulate an unwanted autoimmune response, since the point of the protein is to stimulate production of specific antibodies and help induce memory immunity. If the protein is a toxin, then a genetic modification of the protein or a chemical treatment may be required, such as what was done for the pertussis toxin. 18 Lastly, based on the previous two characteristics, the protein vaccine candidate must be immunogenic, meaning that the components must elicit a protective immune response. This requires the protein to also be surface exposed and able to interact with human cells without bacterial lysis. Proteins must be able to induce the generation of antibodies in the host, and those antibodies must be functional, that is bactericidal, opsonic, or confer blocking of adherence/infection. 18 Based on the criteria above, the research groups under
Experts in the field of otitismedia vaccine research have strongly advocated the development of mucosal immunization strategies for the prevention of otitismedia (26). Recombinant adenoviral vectors are particularly attractive as vaccine candi- dates for immunization of the upper respiratory tract (61). The middle ear cavity is lined by a very thin mucosa that is contin- uous with the mucosa that lines the nasopharynx and the Eu- stachian tube. Components of both the innate and adaptive immune systems are thought to mediate protection of the middle ear space against infection (36, 38, 59). The nasal mu- cosa-associated lymphoid tissue (NALT) has been proposed as a primary inductive site for upper respiratory tract mucosal immune responses (36, 56), and recent studies have begun to investigate the trafficking of lymphocytes to the middle ear mucosa (35, 48, 59). In response to antigenic stimulation, den- dritic cells in the chinchilla nasal mucosa have been shown to traffic to the NALT, where they presumably initiate an antigen- specific immune response (48). Recombinant adenoviral vec- tors have been shown to be potent activators of dendritic cell maturation in several experimental systems (42, 52). Presenta- tion of a vaccine antigen to nasal dendritic cells by a recombi- nant adenoviral vector could be a particularly effective way to induce a protective middle ear immune response.
ABSTRACT Indigenous populations experience high rates of otitismedia (OM), with increased chronicity and severity, compared to those experienced by their nonindig- enous counterparts. Data on immune responses to otopathogenic bacteria in these high-risk populations are lacking. NontypeableHaemophilus inﬂuenzae (NTHi) is the predominant otopathogen in Australia. No vaccines are currently licensed to target NTHi; however, protein D (PD) from NTHi is included as a carrier protein in the 10- valent pneumococcal polysaccharide conjugate vaccine (PHiD10-CV), and other promising protein vaccine candidates exist, including outer membrane protein 4 (P4) and protein 6 (P6). We measured the levels of serum and salivary IgA and IgG against PD, P4, and P6 in Aboriginal and non-Aboriginal children with chronic OM who were undergoing surgery and compared the levels with those in healthy non- Aboriginal children (controls). We found that Aboriginal cases had lower serum IgG titers to all NTHi proteins assessed, particularly PD. In contrast, serum IgA and sali- vary IgA and IgG titers to each of these 3 proteins were equivalent to or higher than those in both non-Aboriginal cases and healthy controls. While serum antibody lev- els increased with age in healthy controls, no changes in titers were observed with age in non-Aboriginal cases, and a trend toward decreasing titers with age was ob- served in Aboriginal cases. This suggests that decreased serum IgG responses to NTHi outer membrane proteins may contribute to the development of chronic and severe OM in Australian Aboriginal children and other indigenous populations. These data are important for understanding the potential beneﬁts of PHiD10-CV implemen- tation and the development of NTHi protein-based vaccines for indigenous popula- tions.
Clinical trials are under way to assess the effect of antibiotics and the 10-valent pneumococcal H. influenzaeprotein D- conjugate vaccine (PHiD-CV; Synflorix) on NTHi nasopharyn- geal carriage and infection (for example, NCT01735084 and NCT01174849). An 11-valent precursor to PHiD-CV reduced NTHi-associated acuteotitismedia by 35% (12). Although a re- duction in H. influenzae nasopharyngeal carriage was observed following primary and booster vaccination with the 11-valent vac- cine (17), no substantial effect has been observed on NTHi car- riage in subsequent studies with PHiD-CV, which has 8 of 10 serotypes conjugated to protein D (20, 21). Whether or not PHiD-CV will afford protection against OM or other NTHi infec- tions, particularly in high-risk populations, is yet to be deter- mined.
space of E. coli, indicating the role of acylation in hydrophobicity of PD as an outer membrane-anchored lipoprotein  . PD also increases bacterial adhesion and internalization into human monocytes. In vivo and in vitro studies have demonstrated that the NTHi virulence in the upper respiratory tract is related to PD expression. For instance, a 100 fold decrease in virulence was found in a PD-deficient strain compared to the PD-expressing strain in a rat model of otitismedia. In addition, culture specimens, inoculated with the PD-deficient NTHi compared with isogenic wild type NTHi, indicated a reduction in the onset of ciliary activity after 12 hours. Ciliary dysfunction was significantly higher in the PD-expressing strain, as compared to the PD-negative mutant (P<0.01). In addition, a significant loss of cilia was observed in the PD-expressing strain after 48-hour incubation, indicating that PD is responsible for etiology of upper respiratory tract infection caused by NTHi, probably due to increased functional and morphological damages to ciliated epithelial cells. Based on a study on animal models, the protective antibodies against NTHi otitismedia in rat and chinchilla were provoked by PD [42,69-72] .
ABSTRACT Vaccinations with the 10-valent pneumococcal conjugated vaccine (PHiD-CV) started in Iceland in 2011. Protein D (PD) from H. inﬂuenzae, which is coded for by the hpd gene, is used as a conjugate in the vaccine and may provide protection against PD-positive H. inﬂuenzae. We aimed to evaluate the effect of PHiD-CV vaccination on H. inﬂuenzae in children, both in carriage and in acuteotitismedia (AOM). H. inﬂuenzae was isolated from nasopharyngeal swabs collected from healthy children attending 15 day care centers in 2009 and from 2012 to 2017 and from middle ear (ME) samples from children with AOM collected from 2012 to 2017. All isolates were identiﬁed using PCR for the hpd and fucK genes. Of the 3,600 sam- ples collected from healthy children, 2,465 were culture positive for H. inﬂuenzae (68.5% carriage rate); of these, 151 (6.1%) contained hpd-negative isolates. Of the 2,847 ME samples collected, 889 (31.2%) were culture positive for H. inﬂuenzae; of these, 71 (8.0%) were hpd negative. Despite the same practice throughout the study, the annual number of ME samples reduced from 660 in 2012 to 330 in 2017. The proportions of hpd-negative isolates in unvaccinated versus vaccinated children were 5.6% and 7.0%, respectively, in healthy carriers, and 5.4% and 7.8%, respectively, in ME samples. The proportion of hpd-negative isolates increased with time in ME sam- ples but not in healthy carriers. The number of ME samples from children with AOM decreased. The PHiD-CV had no effect on the proportion of the hpd gene in H. inﬂu- enzae from carriage, but there was an increase in hpd-negative H. inﬂuenzae in otitismedia. The proportions of hpd-negative isolates remained similar in vaccinated and unvaccinated children.
NontypeableHaemophilusinfluenzae (NTHi) is an impor- tant cause of otitismedia (OM) in children and respiratory tract diseases in adults (12, 16, 17). NTHi accounts for 25 to 30% of acute OM and for a larger percentage of cases of chronic OM with effusion (4, 23). These numbers may under- estimate the level because a recent study indicated that live NTHi could be found in a large percentage of culture-negative fluid from OM (20). Since NTHi lacks a capsular polysaccha- ride, lipooligosaccharide (LOS) is believed to be a major sur- face-exposed saccharide antigen and a possible virulence factor of NTHi OM (3, 11). The LOS is also a potentially protective antigen for NTHi infection because human antibodies showed bactericidal activity in vitro (1), and a mouse monoclonal an- tibody (MAb) enhanced opsonization and bacterial clearance in a murine pulmonary challenge model (15). We showed that NTHi LOS-protein conjugates elicited bactericidal antibodies in animals and conferred protection againstotitismedia in chinchillas (5, 9). The LOS epitopes which elicit these biolog- ically active antibodies in the host have not been identified.
of protein and lipooligosaccharide antigens have been the subject of detailed investi- gation as potential vaccine candidates (7, 8). In our early work, we demonstrated that development of bactericidal antibody in the sera of children recovered from acute NTHi otitismedia was associated with the appearance of serum antibodies directed against highly immunogenic high molecular weight (HMW) proteins (9). This work led subse- quently to the identiﬁcation and characterization of the HMW1/HMW2 family of proteins (10). The HMW1/HMW2 proteins were later shown to be major adhesins of NTHi (11), as well as targets of human opsonophagocytic antibodies (12, 13) and protective antibodies in the chinchilla otitismodel (14). The HMW1/HMW2-like proteins are expressed by approximately 75% of NTHi strains (10, 15). The 25% of NTHi strains that do not express HMW1/HMW2 proteins express other immunogenic high molecular weight proteins that are recognized by human convalescent-phase serum antibodies (16). Almost all HMW1/HMW2-negative strains express a second distinct class of adhe- sin known as Hia (16). The Hia proteins are members of a large family of bacterial proteins known as autotransporters that are found in many Gram-negative bacteria (17). The Hia proteins have also been demonstrated to serve as targets for antibodies mediating opsonophagocytosis of NTHi (18). Nearly all NTHi strains that lack HMW1/ HMW2 proteins contain an hia gene and express an Hia protein, and conversely, strains that express HMW1/HMW2 proteins lack an hia gene (15, 16, 19). Both the HMW1/ HMW2 and Hia proteins demonstrate strain heterogeneity (15, 20). However, HMW1/ HMW2- and Hia-speciﬁc antisera mediate relatively broad-based killing of heterologous NTHi (21), suggesting that the strain heterogeneity present among these proteins would not preclude their potential use as vaccine components.
Infections due to nontypeableHaemophilusinfluenzae result in enormous global morbidity in two clinical settings: otitismedia in children and respiratory tract infections in adults with chronic obstructive pulmonary disease (COPD). Recurrent otitis me- dia affects up to 20% of children and results in hearing loss, delays in speech and language development and, in developing coun- tries, chronic suppurative otitismedia. Infections in people with COPD result in clinic and emergency room visits, hospital ad- missions, and respiratory failure. An effective vaccine would prevent morbidity, help control health care costs, and reduce antibiotic use, a major contributor to the global crisis in bacterial antibiotic resistance. The widespread use of the pneumococcal conjugate vaccines is causing a relative increase in H. influenzaeotitismedia. The partial protection against H. influenzaeotitismedia induced by the pneumococcal H. influenzaeprotein D conjugate vaccine represents a proof of principle of the feasibility of a vaccine for nontypeable H. influenzae. An ideal vaccine antigen should be conserved among strains, have abundant epitopes on the bacterial surface, be immunogenic, and induce protective immune responses. Several surface proteins of H. influenzae have been identified as potential vaccine candidates and are in various stages of development. With continued research, progress toward a broadly effective vaccine to prevent infections caused by nontypeable H. influenzae is expected over the next several years.
and the antibody levels were held at a low and stable level. The lowest antibody levels were found in 21- to 40-year-old groups, a child-bearing age for women, which explained the low antibody levels to P6 and protein D in infants. In addition, a small increase in the levels of serum antibodies was observed in older groups. Our finding is somewhat contradictory with what was reported by Pichichero et al. (16), who suggested that adults age 18 to 60 years had anti-PD or anti-P6 levels that were higher than or similar to those in children. The discrepancy between these two studies might be due to different sample sizes or different NTHi exposure rates in these two study settings. Nonetheless, we also found that the anti-PD antibody concentration increased in the group ⬎ 50 years old, while the anti-P6 antibody concentration was not in- creased, which is consistent with the finding by Pichichero et al. (16). Why anti-P6 antibody was not increased to the same extent as the anti-PD antibody in the same cohort may due to the differ- ent immunogenicities of the two proteins (29). Khan et al. (29) compared the serum IgG levels of protein D and P6 in paired acute- and convalescent-phase sera from children with acuteotitismedia. They found that the increased IgG titers to protein D were significantly higher than those to P6, indicating that the immuno- genicity of P6, with a low molecular weight, was inferior to that of protein D (PD). It was reported that H. influenzae was one of the five most frequently occurring pathogens in community-acquired pneumonia, and the weakened immune systems in older people may increase their susceptibility to infections (36).
Most cases of OM are caused by three major pathogens: S. pneumoniae (30–40%), nontypeableHaemophilus influen- zae (NTHi, 30%) and M. catarrhalis (20%) [1,2]. These organisms are also pathogens for sinusitis and chronic obstructive pulmonary disease (COPD) . In the past three decades, there has been a dramatic worldwide increase in antibiotic resistance in respiratory pathogens [4,5]. This has resulted in a reduction of the number of effective antibiotics for OM and has begun to pose a major public health threat. There is, thus, an urgent need to develop new and innovative non-antibiotic approaches to prevent and manage this disease. To this end, it is imper- ative to understand the mechanisms that defend the tubotympanum (the middle ear and eustachian tube) against invading pathogens and determine if the defense mechanisms can be augmented to prevent or treat OM. The pathogenesis of OM is multi-factorial with risk factors such as delayed development of the adaptive immune sys- tem, complement system abnormality, as well as other environmental and genetic factors playing an important role [6-10]. Although anatomic immaturity of the eus- tachian tube and delayed development of systemic immu- nity are considered to be the most important factors underlying OM susceptibility among young children, other factors may be of greater importance before the adaptive immune system is fully matured [1,8]. For exam- ple, in a study of children with recurrent acuteotitismedia (rAOM), Prellner and colleagues showed that only 1/3 of two-year-old children with rAOM had significantly lower
N ontypeableHaemophilus influenzae (NTHI) are small nonen- capsulated Gram-negative coccobacilli that lack a polysac- charide capsule and commonly colonize the human pharynx. They also cause respiratory tract infections and are now responsi- ble for more than half of the cases of otitismedia (OM) (1), a very common bacterial infection of the middle ear space in young chil- dren. OM may result in substantial sequelae including deafness and suppurative complications (2), significant antibiotic use (2), and OM with effusion (OME) (3), an important cause of tran- sient, moderately severe hearing loss during a critical period for language development (4).
The hypothesis addressed in the present study is that per- sistent and to some extent cryptic colonization with NTHI is not uncommon in children with recurrent otitismedia and may lead to AOM through endogenous reinfection. The reason for examining nasopharyngeal carriage of NTHI instead of con- centrating on the middle ear is as follows. Using a fluorescein isothiocyanate-conjugated oligonucleotide probe which specif- ically hybridizes with the 16S rRNA of H. influenzae, we have demonstrated in a recent study of children free of clinical infection and with adenoid hypertrophy that the bacterium, even if it is absent from the nasopharyngeal mucosa, could be found to be sequestered in macrophage-like cells located sub- epithelially near the crypts in sections of adenoid tissue (8). Therefore, we have speculated that the adenoid could serve as a reservoir for NTHI, causing endogenous reinfections of the nasopharyngeal mucosa (24). This environment would nearly always be the source of the middle ear infection since it is generally accepted that the bacteria responsible for AOM mi- grate from the nasopharyngeal cavity through the eustachian tube to the middle ear (3, 18).
A 16,600-D outer membrane protein is present in all strains of Haemophilusinfluenzae and antibodies to this protein are present in human serum. This study was designed to assess the role of this outer membrane protein (P6) in nontypeable H. influenzae as a target for human serum bactericidal antibody. P6 was isolated and coupled to an affinity column. Depleting normal human serum of antibodies to P6 by affinity chromatography resulted in reduced bactericidal activity of that serum for nontypeable H. influenzae. Immunopurified antibodies to P6 from human serum were bactericidal. Finally, preincubation of bacteria with a monoclonal antibody that recognizes a surface epitope on P6, inhibited human serum bactericidal killing. Taken together, these experiments establish that P6 is a target for human bactericidal antibodies. This observation provides evidence that P6 plays a potentially important role in human immunity to infection by nontypeable H. influenzae.
The dramatic decline in invasive disease due to Hib since the introduction of Hib conjugate vaccines has been well docu- mented (3, 5, 17, 19, 23, 31, 39). Indeed, during the 8 years of our investigation, there was only one episode of invasive Hib infection at our institution. The available literature provides no evidence for a concomitant increase in invasive disease due to nontypeable H. influenzae (8, 38, 41). Nevertheless, serious NTHi disease is now more important in relative terms, and in areas where the Hib vaccine is universally available, nontype- able strains are now more likely to be the causative agents of invasive Haemophilus disease. Accordingly, it would be bene- ficial to identify the patient populations at high risk for these infections and to characterize the bacterial factors that con- tribute to invasive disease.
Haemophilus haemolyticus has been recently discovered to have the potential to cause invasive disease. It is closely related to nontypeableHaemophilusinfluenzae (NT H. influenzae). NT H. influenzae and H. haemolyticus are often misidentified because none of the existing tests targeting the known phenotypes of H. haemolyticus are able to specifically identify H. haemolyticus. Through comparative genomic analysis of H. haemolyticus and NT H. influenzae, we identified genes unique to H. haemolyticus that can be used as targets for the identification of H. haemolyticus. A real-time PCR targeting purT (encoding phosphoribosylg- lycinamide formyltransferase 2 in the purine synthesis pathway) was developed and evaluated. The lower limit of detection was 40 genomes/PCR; the sensitivity and specificity in detecting H. haemolyticus were 98.9% and 97%, respectively. To improve the discrimination of H. haemolyticus and NT H. influenzae, a testing scheme combining two targets (H. haemolyticus purT and H. influenzae hpd, encoding protein D lipoprotein) was also evaluated and showed 96.7% sensitivity and 98.2% specificity for the identification of H. haemolyticus and 92.8% sensitivity and 100% specificity for the identification of H. influenzae, respectively. The dual-target testing scheme can be used for the diagnosis and surveillance of infection and disease caused by H. haemolyticus and NT H. influenzae.
As we observed that both M. catarrhalis and H. influenzae can deplete AI-2/DPD, yet only one of these species produces the sig- nal, there could be competition between M. catarrhalis and H. in- fluenzae for available AI-2 during coinfection. Based on the AI-2 requirement for H. influenzae to establish a chronic infection, any competition with M. catarrhalis would most likely have a negative impact on H. influenzae persistence. However, the numbers of viable bacteria recovered from coinfected animals were similar to the numbers recovered from those which received H. influenzae alone, indicating that AI-2 uptake by M. catarrhalis did not inter- fere with H. influenzae biofilm formation or persistence. Addi- tionally, the presence of M. catarrhalis during stationary biofilm formation was not observed to negatively impact H. influenzae biofilm formation or antibiotic resistance. Taken together, these observations support a model wherein H. influenzae requires only a certain threshold level of AI-2 but may produce AI-2 in excess of the threshold concentration needed to promote biofilm develop- ment. In this model, AI-2 depleted from the biofilm environment by M. catarrhalis would not have a detrimental impact on H. in- fluenzae biofilm development. Another possible explanation is that M. catarrhalis may require only a minimal concentration of AI-2 to alter biofilm development. This would be advantageous, as M. catarrhalis could utilize any AI-2-producing species as an “in- fection partner” to promote its own persistence without directly competing for the AI-2 signal. Further research will be necessary to determine the minimal AI-2 concentration required by both M. catarrhalis and H. influenzae to promote biofilm development. The data presented in this study and others provide substantial evidence for the influence of polymicrobial infection on severity of disease and the outcome of antibiotic treatment, particularly for chronic infections involving persistence of bacteria within bio- films. Notably, the results of this study imply that vaccination against upper airway pathogens, such as the unencapsulated strains of H. influenzae, may have a greater impact than expected. For instance, successful vaccination against H. influenzae may also disrupt the establishment of disease by M. catarrhalis. Further research is necessary to elucidate the interactions between all three of the leading causative agents of OM and the impact of other polymicrobial upper airway infections on resistance to relevant antibiotics. Knowledge of the bacterial species present during highly recalcitrant infections may provide insight into which course of antibiotic treatment would be most effective. Addition- ally, AI-2 may represent an ideal target for disruption of numer- ous chronic and/or recurrent infections.