Jennelle M. Kyd

Nontypeable Haemophilus influenzae as a pathogen in children.

Nontypeable Haemophilus influenzae is a significant pathogen in children, causing otitis media, sinusitis, conjunctivitis, pneumonia, and occasionally invasive infections. H. influenzae type b conjugate vaccines have no effect on infections caused by nontypeable strains because nontypeable strains are nonencapsulated. Approximately, one-third of episodes of otitis media are caused by nontypeable H. influenzae and the bacterium is the most common cause of recurrent otitis media. Recent progress in elucidating molecular mechanisms of pathogenesis, understanding the role of biofilms in otitis media and an increasing understanding of immune responses have potential for development of novel strategies to improve prevention and treatment of otitis media caused by nontypeable H. influenzae. Feasibility of vaccination for prevention of otitis media due to nontypeable H. influenzae was recently demonstrated in a clinical trial with a vaccine that included the surface virulence factor, protein D.

Developing a nontypeable Haemophilus influenzae (NTHi) vaccine

There is a current high demand for nontypable Haemophilus influenzae (NTHi) vaccines. Various options for the composition of such vaccines are possible. Decisions about the vaccine composition have to take into account the antigenic variability of NTHi, so even complex immunogens such as whole bacteria would preferentially have a tailor-made antigenic composition. We will present a summary of NTHi vaccine development, describing research efforts from SmithKline Beecham and other laboratories. Currently, major (P1, P2, P4, P5) and minor (P6, D15, TbpA/B, ellipsis) outer membrane proteins, LPS, adhesins (HMW, Hia, pili, P5) are being studied. Preclinical results with LPD, P5 (LB1) and OMP26 from our laboratories will be described including the use of animal models of otitis and lung infection.

Microbial pattern recognition receptors mediate M-cell uptake of a gram negative bacterium

ABSTRACT The receptors involved in the sampling of particulate microbial antigens by the gut are largely unknown. Here we demonstrate for the first time in an in vitro M-cell model and in situ in isolated murine intestinal segments that the receptors TLR-4, PAF-R, and α5β1 integrin are all involved in mediating bacterial uptake associated with transcytosis. The pattern of expression of TLR-4 and α5β1 integrin differed between M cells and enterocytes. There was increased apical expression of TLR-4 in M-cell cultures, and it was present on the apical surface of murine M cells but not enterocytes in situ. In contrast, PAF-R was expressed equally by both cell types in vitro and was abundantly expressed throughout the intestinal epithelium. Inhibition of TLR-4 and PAF-R, but not TLR-2, reduced gram-negative bacterial uptake by both cell types, whereas inhibition of the apically expressed α5β1 integrin significantly reduced the ability of M cells to translocate bacteria. Hence, the involvement of each receptor was dependent not only on differences in the level of receptor expression but the cellular localization. Using bacteria that had mutations that affected the bacterial lipooligosaccharide structure indicated that the oligosaccharide moiety was important in bacterial uptake. Taken together, the data suggest that pathogen-associated molecular pattern interactions with pattern recognition receptors are key factors in M-cell recognition of intestinal antigens for mucosal immune priming.

Enhancement of Pulmonary Clearance of Moraxella (Branhamella) catarrhalis following Immunization with Outer Membrane Protein CD in a Mouse Model

Moraxella (Branhamella) catarrhalis is an important human respiratory tract pathogen. Outer membrane protein (OMP) CD is highly conserved among strains and has characteristics that indicate it may be an effective vaccine antigen. This study investigated the effect of immunization with OMP CD on pulmonary clearance following intratracheal challenge of mice with M. catarrhalis. Two routes of immunization were studied: mucosal immunization (intra-Peyers patch followed by intratracheal boost) and intramuscular immunization with OMP CD. Both resulted in enhanced pulmonary clearance of M. catarrhalis compared with sham-immunized controls. Immunization with OMP CD induced specific antibodies in serum and bronchoalveolar lavage fluid and induced a specific lymphocyte proliferative response in T cells from mesenteric lymph nodes from mice mucosally immunized with OMP CD. On the basis of these results, OMP CD should undergo continued testing to determine whether it will induce a protective immune response in humans.

Bacterial Ghosts as Adjuvant Particles

The development of more advanced and effective vaccines is of great interest in modern medicine. These new-generation vaccines, based on recombinant proteins or DNA, are often less reactogenic and immunogenic than traditional vaccines. Thus, there is an urgent need for the development of new and improved adjuvants. Besides many other immunostimulatory components, the bacterial ghost (BG) system is currently under investigation as a potent vaccine delivery system with intrinsic adjuvant properties. BGs are nonliving cell envelope preparations from Gram-negative cells, devoid of cytoplasmic contents, while their cellular morphology and native surface antigenic structures remain preserved. Owing to the particulate nature of BGs and the fact that they contain many well known immune-stimulating compounds, BGs have the potential to enhance immune responses against ghost-delivered target antigens.

Functional differences between M cells and enterocytes in sampling luminal antigens

Oral delivery of agents such as vaccines offers a number of significant advantages over parenteral routes, yet only a small number of oral vaccines are routinely available today. The small intestine contains lymphoid aggregates that are overlaid by M cells. These aggregates are part of the gut-associated lymphoid tissues and are important for determining host responses to particulate antigenic material within the small intestine. Differentiating the receptor requirements for M cell uptake and transcytosis of bacterial antigen from the intestine has progressed although the specific signalling mechanisms that initiate antigen uptake and specifically target antigen to these cells is still relatively unknown. Microbial pathogen-associated molecular patterns (PAMPs) are recognised by the innate immune system through pattern recognition receptors (PRRs) either through direct receptor-bacterial ligand or endogenous adaptor-bacterial molecule interactions. PRRs on the surface of M cells that have been identified as important in antigen transcytosis include toll-like receptor-4, platelet-activating factor receptor and alpha5beta1 integrin. A number of these PRRs are also found on neighbouring enterocytes and therefore the pathways signalled by receptor-ligand binding may differentially trigger different transduction pathways. Elucidation of these signalling pathways may assist in the design of effective oral vaccines that target the gut-associated lymphoid tissue.

The effect of exercise on innate mucosal immunity

Methods The authors conducted a prospective observational study comparing salivary lactoferrin and lysozyme concentration over 5 months (chronic changes) in elite rowers (n=17, mean age 24.3±4.0 years) with sedentary individuals (controls) (n=18, mean age=27.2±7.1 years) and a graded exercise test to exhaustion (acute changes) with a cohort of elite rowers (n=11, mean age 24.7±4.1). Results Magnitudes of differences and changes were interpreted as a standardised (Cohens) effect size (ES). Lactoferrin concentration in the observational study was approximately 60% lower in rowers than control subjects at baseline (7.9±1.2 µg/ml mean±SEM, 19.4±5.6 µg/ml, p=0.05, ES=0.68, ‘moderate’) and at the midpoint of the season (6.4±1.4 µg/ml mean ± SEM, 21.5±4.2 µg/ml, p=0.001, ES=0.89, ‘moderate’). The concentration of lactoferrin at the end of the study was not statistically significant (p=0.1) between the groups. There was no significant difference between rowers and control subjects in lysozyme concentration during the study. There was a 50% increase in the concentration of lactoferrin (p=0.05, ES=1.04, ‘moderate’) and a 55% increase in lysozyme (p=0.01, ES=3.0, ‘very large’) from pre-exercise to exhaustion in the graded exercise session. Conclusion Lower concentrations of these proteins may be indicative of an impairment of innate protection of the upper respiratory tract. Increased salivary lactoferrin and lysozyme concentration following exhaustive exercise may be due to a transient activation response that increases protection in the immediate postexercise period.

Efficacy of the 26-kilodalton outer membrane protein and two P5 fimbrin-derived immunogens to induce clearance of nontypeable Haemophilus influenzae from the rat middle ear and lungs as well as from the chinchilla middle ear and nasopharynx.

ABSTRACT The rat middle ear and lung clearance model has been used to show that the nontypeable Haemophilus influenzae 26-kDa outer membrane protein OMP26 is highly efficacious as a mucosal immunogen, inducing significantly enhanced clearance in immunized rats upon direct challenge of these two anatomic sites. Similarly, the chinchilla model of middle ear and nasopharyngeal clearance has been used to show that two P5 fimbrin adhesin-derived immunogens, LB1 and lipoprotein D (LPD)-LB1(f)2,1,3, are highly efficacious as parenteral immunogens. Both induced significantly augmented clearance of nontypeable H. influenzae upon challenge of these sites. Here, these three nontypeable H. influenzae immunogens in addition to six bovine serum albumin and keyhole limpet hemocyanin conjugates of the synthetic peptide LB1(f) were assayed for relative efficacy in the reciprocal rodent model system. OMP26 was assayed in the chinchilla host by a parenteral immunization route, with clearance of the middle ear and nasopharynx used as outcome measures. Both LB1 and LPD-LB1(f)2,1,3 were assayed in the rat host with a mucosal immunization route and clearance of nontypeable H. influenzae from the lungs and middle ears as outcome measures. Both of the immunogens were found to induce a high-titered and specific immune responses in the heterologous host system. Moreover, each was found to be highly efficacious in the reciprocal host system, providing strong support for the continued development and inclusion of both OMP26 and P5 fimbrin-derived peptides as candidate vaccine antigens directed at otitis media caused by nontypeable H. influenzae.

Validation and quantitation of an in vitro M-cell model

This study has evaluated an in vitro model of the follicle-associated epithelia that overlie Peyers patches of the small intestine. The model shares many phenotypic characteristics of M cells in vivo. Co-cultures of the human adenocarcinoma cell line Caco-2 and freshly isolated Peyers patch cells were established. Fluorescence microscopy and quantitative image analysis were used to validate the model against known markers of M-cell phenotype. Apical expression of alkaline phosphatase was down-regulated in co-cultures and villin was re-distributed from the apical membrane to the cytoplasm. alpha5beta1 integrin was found on the apical surfaces of the monolayers and B and T lymphocytes integrated into the monolayers. Particle transport was temperature-dependent in co-cultures, indicating that a transcytotic route was responsible. This model provides opportunities to study factors that influence M-cell development, assess putative Peyers patch targeting in oral vaccine technologies, and study intestinal uptake in vitro.

Mucosal immunity in the lung and upper airway

The mucosal surfaces of the lungs and upper airways are common sites for infection. Extensive studies of the mechanisms associated with immune responses in the respiratory tract have found that understanding the system is challenging and involves many complex interactions to prevent and eliminate infection. Immune protection against diseases transmitted through the respiratory tract requires an understanding of the important aspects associated with beneficial, detrimental or ineffective immune responses. Two critical aspects of an immune response against a pathogen are that of the inductive stage, either induced by vaccination or primary infection, and the effector stage, the ability to recognise, respond to and eliminate the infection without detriment to the host. An immunisation strategy must not only have a measure of the induced antigen specific response, but this response must also be protective.