Comparison of rhinovirus antibody titers in children with asthma exacerbations and species-specific rhinovirus infection.

BACKGROUND: Asthma exacerbations are associated with human rhinovirus (HRV) infections, and more severe exacerbations are associated with HRV-C. We have previously shown that the HRV-C-specific antibody response is low in healthy adult sera and that most of the antibody to HRV-C is cross-reactive with HRV-A. OBJECTIVES: To compare the antibody response to each HRV species in asthmatic and nonasthmatic children in whom the type of HRV infection was known. METHODS: Total and specific IgG1 binding to HRV viral capsid protein antigens of HRV-A, -B, and -C were tested in the plasma from nonasthmatic children (n = 47) and children presenting to the emergency department with asthma exacerbations (n = 96). HRV, found in most of the children at the time of their exacerbation (72%), was analyzed using molecular typing. RESULTS: Asthmatic children had higher antibody responses to HRV. The titers specific to HRV-A, and to a lesser extent HRV-B, were higher than in nonasthmatic controls. The species-specific responses to HRV-C were markedly lower than titers to HRV-A and HRV-B in both asthmatic and nonasthmatic children (P < .001). The titers both at presentation and after convalescence were not associated with the HRV genotype detected during the exacerbation. CONCLUSIONS: The higher total anti-HRV antibody titers of asthmatic children and their higher anti-HRV-A and -B titers show their development of a heightened antiviral immune response. The low species-specific HRV-C titers found in all groups, even when the virus was found, point to a different and possibly less efficacious immune response to this species.

Quantitation of IgE binding to the chitinase and chitinase-like house dust mite allergens Der p 15 and Der p 18 compared to the major and mid-range allergens.

BACKGROUND: The prevalence of IgE binding to the group 15 and 18 house dust mite (HDM) allergens of the Dermatophagoides species is reported to be >50% and they are the major allergens of HDM-sensitised dogs. The objective was to quantitate the IgE titres to Der p 15 and Der p 18 and evaluate their importance in human HDM sensitisation compared to the known major and mid-tier allergens. METHODS: Der p 15 and Der p 18 were produced in Pichia pastoris, and their structure validated by circular dichroism. IgE binding was measured in 37 Australian HDM-allergic adults using a quantitative DELFIA™ assay. RESULTS: The prevalence of IgE titres to Der p 15 and Der p 18 >0.1 ng/ml was low (38%) and only one subject had a titre >10 ng/ml to either allergen. The mean anti-Der p 15 and Der p 18 titres were 1.2 and 2.6 ng/ml, respectively, i.e. approximately 10- to 20-fold lower than the response to the major Der p 1 and Der p 2 allergens (p < 0.001). The IgE responses to Der p 15 and Der p 18 were lower than the mid-tier allergens Der p 5 and Der p 7 and although they correlated with each other, they did not correlate with titres to either the major or mid-tier allergens. CONCLUSIONS: Sensitisation to Der p 15 and Der p 18 makes a minor contribution to anti-HDM IgE titres, and the titres do not correlate with the size of the response to the major allergens.

Antibacterial antibody responses associated with the development of asthma in house dust mite-sensitised and non-sensitised children.

BACKGROUND: Infants who develop house dust mite (HDM) allergy and HDM-sensitised children with severe persistent asthma have low antibody responses to the P6 antigen of Haemophilus influenzae. OBJECTIVE: To measure the development of antibody to two ubiquitous bacteria of the respiratory mucosa in a prospective birth cohort at high risk of allergic disease and to assess which responses are associated with asthma and atopy. METHODS: IgG1 and IgG4 antibody to H influenzae (P4 and P6) and Streptoccocus pneumoniae (PspA and PspC) surface antigens was measured in yearly blood samples of children aged 1-5 years. IgE to the P6 antigen was examined for the 5-year group. The children were stratified based on HDM sensitisation and asthma at 5 years of age. RESULTS: HDM-sensitised children had lower IgG1 antibody titres to the bacterial antigens, and early responses (<3 years and before the development of HDM sensitisation and asthma) corrected for multiple antigens were significantly reduced for P4, P6 and PspC (p=0.008, p=0.004 and p=0.028, respectively). Similar associations with asthma were also found (p=0.008, p=0.004 and p=0.032 for P4, P6 and PspC, respectively). The IgG4 antibody titre and prevalence were similar in both HDM-sensitised and non-sensitised groups, but sensitised children had a slower downregulation of the IgG4 response. Children with asthma (27/145 at 5 years) had lower anti-P6 IgE responses (p<0.05). CONCLUSIONS: HDM-sensitised children have early defective antibody responses to bacteria that are associated with asthma. Surprisingly, antibacterial IgE was associated with a reduced risk for asthma.

Pyroglyphid house dust mite allergens.

Mites of the family Pyroglyphidae are the most important source of house dust mite allergens. A small number of allergens, namely those of groups 1, 2, 4, 5 and 7 constitute the known major and mid-potency specificities, with possible important contributions of the groups 11, 14 and 15 requiring further definition. Most of the allergens can be identified by sequence homologies and the structures of the major allergens have been solved. There are however challenges in determining the nature of the group 5 and 7 allergens and in obtaining detailed structures of the significant allergens to be used for genetic engineering.

Species-specific and cross-reactive IgG1 antibody binding to viral capsid protein 1 (VP1) antigens of human rhinovirus species A, B and C.

BACKGROUND: Human rhinoviruses (HRV) are associated with upper and lower respiratory illnesses, including severe infections causing hospitalization in both children and adults. Although the clinical significance of HRV infections is now well established, no detailed investigation of the immune response against HRV has been performed. The purpose of this study was to assess the IgG1 antibody response to the three known HRV species, HRV-A, -B and -C in healthy subjects. METHODS: Recombinant polypeptides of viral capsid protein 1 (VP1) from two genotypes of HRV-A, -B and -C were expressed as glutathione S-transferase (GST) fusion proteins and purified by affinity and then size exclusion chromatography. The presence of secondary structures similar to the natural antigens was verified by circular dichroism analysis. Total and species-specific IgG1 measurements were quantitated by immunoassays and immunoabsorption using sera from 63 healthy adults. RESULTS: Most adult sera reacted with the HRV VP1 antigens, at high titres. As expected, strong cross-reactivity between HRV genotypes of the same species was found. A high degree of cross-reactivity between different HRV species was also evident, particularly between HRV-A and HRV-C. Immunoabsorption studies revealed HRV-C specific titres were markedly and significantly lower than the HRV-A and HRV-B specific titres (P<0.0001). A truncated construct of HRV-C VP1 showed greater specificity in detecting anti-HRV-C antibodies. CONCLUSIONS: High titres of IgG1 antibody were bound by the VP1 capsid proteins of HRV-A, -B and -C, but for the majority of people, a large proportion of the antibody to HRV-C was cross-reactive, especially to HRV-A. The improved specificity found for the truncated HRV-C VP1 indicates species-specific and cross-reactive regions could be defined.

IgE and IgG binding patterns and T-cell recognition of Fel d 1 and non-Fel d 1 cat allergens.

BACKGROUND: Cat allergy affects approximately 15% of the population and is a major risk factor for asthma. The relative importance of cat allergens other than Fel d 1 is not known. OBJECTIVE: To compare IgE and IgG antibody binding and T-cell recognition of the major cat allergen Fel d 1 with other cat proteins with known IgE binding properties. METHODS: IgE, IgG1, and IgG4 antibody to Fel d 1, 2, 3, 4, 7, 8, and the undesignated IgE binding proteins haptoglobin and S100A12 were measured in the plasma of 96 individuals with cat allergy and 78 individuals without cat allergy. Cytokines were measured from T cells stimulated with the cat allergens. RESULTS: An allergen other than Fel d 1 had the highest IgE binding specificity for 35% of individuals with cat allergy, and it bound more than 50% of their IgE antibody in 70% of these sera. Fel d 4, 7, and 8 were identified as the main contributors to the non-Fel d 1 IgE binding response and elicited inflammatory Th2 cytokines to a similar degree as Fel d 1. As expected, the average percentage of IgE binding to Fel d 1 for individuals was 55%. IgG4 binding to Fel d 1 was detected in both subjects with allergy (30%) and subjects without allergy (19%). IgG4 binding to the other allergens was less prevalent but was found for both groups. IgG1 antibody was not detected to any of the newly described cat proteins. CONCLUSION: Fel d 4, 7, and 8 are allergens that should be included in the diagnosis and investigation of cat allergy.

Effect of early carriage of Streptococcus pneumoniae on the development of pneumococcal protein-specific cellular immune responses in infancy.

BACKGROUND: The aim of this study was to examine the relationship between nasopharyngeal pneumococcal colonization in early life and the subsequent development of pneumococcal-specific T cell responses. METHODS: Pernasal swabs were collected from Papua New Guinean infants at the ages of 1 and 2 weeks (n = 279). At 9 months, in vitro cellular immune responses to choline-binding protein A (n = 132), pneumococcal surface protein A (n = 132), pneumolysin (n = 99), and the pneumococcal conjugate vaccine carrier CRM197 were determined. Responses were compared based on the children's carriage status within the first 2 weeks of life. RESULTS: Within the first 2 weeks of life, 40% of the study children carried Streptococcus pneumoniae. Early carriage was associated with lower interferon-γ and interleukin 10 responses to pneumococcal proteins at age 9 months when children had not received pneumococcal conjugate vaccines during the study period. CONCLUSIONS: Early pneumococcal carriage may result in enhanced disease susceptibility and suboptimal vaccine responses by modulating the development of pneumococcal immune responses.

House dust mite allergens in asthma and allergy.

IgE antibodies in house dust mite (HDM) allergy follow a predictable pattern. Half are directed against two dominant allergens and the remainder largely against four midpotency allergens. This hierarchical pattern is not changed by the titre of the IgE response or severity of disease. The structures of these allergens are known and they can be produced as authentic recombinant allergens. There is also evidence that the allergenicity is augmented by the biological activity of the key allergens, validating them as targets for vaccination. Collectively, these developments should facilitate the development of new diagnostics, improve immunotherapy and allow vaccination with defined reagents. Highly purified recombinant polypeptides representing the important mite allergens are now available so that informative and reproducible experiments can be performed with mite allergens in place of poorly defined and variable extracts.

Maternal antibodies to pneumolysin but not to pneumococcal surface protein A delay early pneumococcal carriage in high-risk Papua New Guinean infants.

Immunization of pregnant women can be an efficient strategy to induce early protection in infants in developing countries. Pneumococcal protein-based vaccines may have the capacity to induce pneumococcal serotype-independent protection. To understand the potential of maternal pneumococcal protein-specific antibodies in infants in high-risk areas, we studied the placental transfer of naturally acquired antibodies to pneumolysin (Ply) and pneumococcal surface protein A family 1 and 2 (PspA1 and PspA2) in relation to onset of pneumococcal nasopharyngeal carriage in infants in Papua New Guinea (PNG). In this study, 76% of the infants carried Streptococcus pneumoniae in the upper respiratory tract within the first month of life, at a median age of 19 days. Maternal and cord blood antibody titers to Ply (rho = 0.824, P < 0.001), PspA1 (rho = 0.746, P < 0.001), and PspA2 (rho = 0.631, P < 0.001) were strongly correlated. Maternal pneumococcal carriage (hazard ratio [HR], 2.60; 95% confidence interval [CI], 1.25 to 5.39) and younger maternal age (HR, 0.74; 95% CI, 0.54 to 1.00) were independent risk factors for early carriage, while higher cord Ply-specific antibody titers predicted a significantly delayed onset (HR, 0.71; 95% CI, 0.52 to 1.00) and cord PspA1-specific antibodies a significantly younger onset of carriage in PNG infants (HR, 1.57; 95% CI, 1.03 to 2.40). Maternal vaccination with a pneumococcal protein-based vaccine should be considered as a strategy to protect high-risk infants against pneumococcal disease by reducing carriage risks in both mothers and infants.

Structural biology of allergens.

Major allergens may have special aerobiological properties and allergenic structures. It would also be instructive to consider the properties of nonallergens and nonallergenic responses. In some cases, nonallergenic responses appear to result from a lack of antigenicity and in others from regulation. Proteolytic activity has been proposed as an adjuvant for allergenicity, but lipid binding is far more common and is found for more than 50% of the major allergens. Such structures can enhance allergenicity via Toll-like receptor (TLR) or CD1 pathways. TLR signaling can enhance both Th1 and Th2 responses and be induced by peptides as well as nonproteinaceous ligands.

Genetically engineered vaccines.

The application of recombinant DNA technology to allergen research has provided the sequence information and genetic material to produce new types of allergy vaccines. One general strategy has been to use the knowledge to produce synthetic peptides that represent selected T-cell or B-cell epitopes. The production of genetically engineered allergens provides an alternative strategy to construct hypoallergenic vaccines, which can provide a better and less selected representation of the epitopes. Many strategies have been used to produce such hypoallergens, and their ability to reduce allergenicity has been amply demonstrated by skin and nasal provocation tests. The retention of T cell-stimulating activity has also been demonstrated, and a consistent feature of the vaccines has been, despite the reduced immunoglobulin E (IgE)-binding reactivity, the ability to induce anti-allergen IgG antibody. The lead hypoallergens have been polypeptide fragments and trimeric constructs of the birch allergen Bet v 1. A clinical trial with these medicaments has shown the ability to modify IgE and IgG antibody production, skin test reactivity, and symptom scores. This is the first trial of a recombinant allergy vaccine, and it has set a benchmark for further studies. A new generation of hypoallergens is now being produced based on the detailed knowledge of the tertiary structures of the allergens and of the T-cell and B-cell epitopes. The modifications have been made to change the topography of the allergens while retaining a stable, folding structure. In the case of Bet v 1, tertiary structures of hypoallergens have been determined. Structurally modeled hypoallergens have been produced for pollen, venom, food, and latex allergens, with promising characteristics from preclinical studies.

The allergenic specificities of the house dust mite.

The most important house dust mites are Dermatophagoides pteronyssinus and in drier areas D. farinae. In subtropical and tropical regions the glycyphagid mite Blomia tropicalis is a major source of allergen, which co-exists with D. pteronyssinus. The group 1 and 2 allergens of Dermatophagoides mites are clearly major specificities and it is likely that these allergens could be the basis of new strategies of immunotherapy for many mite-allergic subjects. About 20% of patients, however, do not have IgE antibody to the group 1 and 2 allergens, and even though this is a minority, it constitutes a large population. There are also many other house dust mite allergens which have high IgE binding activity but these are present in low and variable concentrations in mite extracts, usually at less than 1% of the group 1 and 2 allergens. It must be appreciated that mite extracts are arbitrary preparations that do not accurately represent the relative concentrations of allergens in inhaled air. There is now the opportunity to produce more representative and more balanced formulations of allergens, possibly by mixtures of recombinant allergens. It is likely that the group 3, 5, 7 and 9 allergens will be important along with the high molecular weight group 11, 14, 15 and 18. The tropomyosin group 10 may be an important cross-reacting allergen. B. tropicalis is, because of its distribution in highly populated regions with increasing affluence, a very important allergen. It has low-grade cross-reactivity with Dermatophagoides but most allergens only have 30-40% sequence identity between the different families so they require different allergens for immunotherapy and new diagnostic measures are required to distinguish the sensitivity between the mite families. Studies on B. tropicalis allergens are required to identify the major allergens that do not appear to be the group land 2 specificities. Component resolved diagnosis is a newly developing procedure that uses allergen arrays to provide a diagnostic format to differentiate between cross-reacting allergens and to identify the optimal formulation of allergens for different patients.

Recombinant allergens for analysing T-cell responses.

T-cell responses constitute a central element of allergic disease and a model for studying Th1 and Th2 cytokine pathways. Most studies to date have used extracts of allergens which contain variable quantities of different allergens and non-allergenic antigens. Recombinant allergens provide the tools for studying the responses to allergens in a reproducible and dose-dependent manner and the different T-cell responses of allergic and non-allergic subjects provide a method for verifying the responses and their relationship to allergic sensitisation. Most allergies show dominant responses to one or a few major allergens. These allergens have been described for the common allergies and have been produced as recombinant allergens. A particular problem for allergens is that many are mixtures of proteins from multi-gene families or are highly polymorphic. Information now exists so the sequence variation can be represented. Purified recombinant allergens produced by standard expression systems stimulate the expected T-cell responses from the peripheral blood of allergic and non-allergics to allergen extracts. Although stimulation with recombinant allergens which are not produced with a natural IgE binding activity can provide a measure of allergenicity, the altered tertiary structure can reduce Th2 responses. The sequence information now available provides the means to use PCR to produce cDNA for the production of recombinant allergens from readily available sources. The production of the highly reactive recombinant Der p 2 allergen of house dust mite from natural sources is described.

Characterization and immunobiology of house dust mite allergens.

The examination of house dust mite extracts has indicated that over 30 different proteins can induce IgE antibody in patients allergic to the house dust mite. There are however dominant specificities especially the group 1 and 2 allergens which can account for much of the allergenicity of extracts. Of the 19 denominated allergens, the major IgE binding has been reported for the group 1, 2, 3, 9, 11, 14 and 15 allergens. The high-molecular-weight group 11, 14 and 15 allergens have only recently been described and although high IgE binding has been anticipated from immunoblotting, there is a need for considerable corroboration. Similarly, the study of the group 3 and 9 serine protease allergens has been incomplete. The group 4, 5, 7 and 8 allergens have shown intermediate IgE binding and the group 10 tropomyosins are of interest because of their potential cross-reactivity with allergen from disparate species. Although the progress with the production of recombinant group 1 allergens has been recent, many of the allergens can be produced as high IgE-binding polypeptides. The tertiary structure of the group 2 allergens has been determined from recombinant proteins and they are an excellent model for the investigation of modified allergens. An unexpected property of the group 1, 2 and 3 allergens has been the high degree of polymorphism found by cDNA analysis. It has however been possible to identify sequences to represent the variation in the natural allergens. The group 7 and 14 allergens show secondary modifications which vary in different extracts creating batch variation. While some estimate of the importance of allergens can be obtained from IgE binding, few analyses of T-cell responses have been made and these regulate both the development of, and the protection from sensitization.