Art v 1 IgE epitopes of patients and humanized mice are conformational.

BACKGROUND: Worldwide, pollen of the weed mugwort (Artemisiavulgaris) is a major cause of severe respiratory allergy, with its major allergen, Art v 1, being the key pathogenic molecule for millions of patients. Humanized mice transgenic for a human T-cell receptor specific for the major Art v 1 T-cell epitope and the corresponding HLA have been made. OBJECTIVE: We sought to characterize IgE epitopes of Art v 1-sensitized patients and humanized mice for molecular immunotherapy of mugwort allergy. METHODS: Four overlapping peptides incorporating surface-exposed amino acids representing the full-length Art v 1 sequence were synthesized and used to search for IgE reactivity to sequential epitopes. For indirect mapping, peptide-specific rabbit antibodies were raised to block IgE against surface-exposed epitopes on folded Art v 1. IgE reactivity and basophil activation studies were performed in clinically defined mugwort-allergic patients. Secondary structure of recombinant (r) Art v 1 and peptides was determined by circular dichroism spectroscopy. RESULTS: Mugwort-allergic patients and humanized mice sensitized by allergen inhalation showed IgE reactivity and/or basophil activation mainly to folded, complete Art v 1 but not to unfolded, sequential peptide epitopes. Blocking of allergic patients' IgE with peptide-specific rabbit antisera identified a hitherto unknown major conformational IgE binding site in the C-terminal Art v 1 domain. CONCLUSIONS: Identification of the new major conformational IgE binding site on Art v 1, which can be blocked with IgG raised against non-IgE reactive Art v 1 peptides, is an important basis for the development of a hypoallergenic peptide vaccine for mugwort allergy.

Antibody Conjugates Bispecific for Pollen Allergens and ICAM-1 with Potential to Prevent Epithelial Allergen Transmigration and Rhinovirus Infection.

Allergy and rhinovirus (RV) infections are major triggers for rhinitis and asthma, causing a socioeconomic burden. As RVs and allergens may act synergistically to promote airway inflammation, simultaneous treatment strategies for both causative agents would be innovative. We have previously identified the transmembrane glycoprotein intercellular adhesion molecule 1 (ICAM-1) as an anchor for antibody conjugates bispecific for ICAM-1 and Phleum pratense (Phl p) 2, a major grass pollen allergen, to block allergen transmigration through the epithelial barrier. Since ICAM-1 is a receptor for the major group RVs, we speculated that our bispecific antibody conjugates may protect against RV infection. Therefore, we created antibody conjugates bispecific for ICAM-1 and the major grass pollen allergen Phl p 5 and analyzed their capacity to affect allergen penetration and RV infection. Bispecific antibody conjugates significantly reduced the trans-epithelial migration of Phl p 5 and thus the basolateral Phl p 5 concentration and allergenic activity as determined by humanized rat basophilic leukemia cells and inhibited RV infection of cultured epithelial cells. A reduction in allergenic activity was obtained only through the prevention of allergen transmigration because the Phl p 5-specific IgG antibody did not block the allergen-IgE interaction. Our results indicate the potential of allergen/ICAM-1-specific antibody conjugates as a topical treatment strategy for allergy and RV infections.

Vaccine based on folded receptor binding domain-PreS fusion protein with potential to induce sterilizing immunity to SARS-CoV-2 variants.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing global COVID-19 pandemic. One possibility to control the pandemic is to induce sterilizing immunity through the induction and maintenance of neutralizing antibodies preventing SARS-CoV-2 from entering human cells to replicate in. METHODS: We report the construction and in vitro and in vivo characterization of a SARS-CoV-2 subunit vaccine (PreS-RBD) based on a structurally folded recombinant fusion protein consisting of two SARS-CoV-2 Spike protein receptor-binding domains (RBD) fused to the N- and C-terminus of hepatitis B virus (HBV) surface antigen PreS to enable the two unrelated proteins serving as immunologic carriers for each other. RESULTS: PreS-RBD, but not RBD alone, induced a robust and uniform RBD-specific IgG response in rabbits. Currently available genetic SARS-CoV-2 vaccines induce mainly transient IgG1 responses in vaccinated subjects whereas the PreS-RBD vaccine induced RBD-specific IgG antibodies consisting of an early IgG1 and sustained IgG4 antibody response in a SARS-CoV-2 naive subject. PreS-RBD-specific IgG antibodies were detected in serum and mucosal secretions, reacted with SARS-CoV-2 variants, including the omicron variant of concern and the HBV receptor-binding sites on PreS of currently known HBV genotypes. PreS-RBD-specific antibodies of the immunized subject more potently inhibited the interaction of RBD with its human receptor ACE2 and their virus-neutralizing titers (VNTs) were higher than median VNTs in a random sample of healthy subjects fully immunized with registered SARS-CoV-2 vaccines or in COVID-19 convalescent subjects. CONCLUSION: The PreS-RBD vaccine has the potential to serve as a combination vaccine for inducing sterilizing immunity against SARS-CoV-2 and HBV by stopping viral replication through the inhibition of cellular virus entry.

Neutralization of SARS-CoV-2 requires antibodies against conformational receptor-binding domain epitopes.

BACKGROUND: The determinants of successful humoral immune response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of critical importance for the design of effective vaccines and the evaluation of the degree of protective immunity conferred by exposure to the virus. As novel variants emerge, understanding their likelihood of suppression by population antibody repertoires has become increasingly important. METHODS: In this study, we analyzed the SARS-CoV-2 polyclonal antibody response in a large population of clinically well-characterized patients after mild and severe COVID-19 using a panel of microarrayed structurally folded and unfolded SARS-CoV-2 proteins, as well as sequential peptides, spanning the surface spike protein (S) and the receptor-binding domain (RBD) of the virus. RESULTS: S- and RBD-specific antibody responses were dominated by immunoglobulin G (IgG), mainly IgG1 , and directed against structurally folded S and RBD and three distinct peptide epitopes in S2. The virus neutralization activity of patients´ sera was highly correlated with IgG antibodies specific for conformational but not sequential RBD epitopes and their ability to prevent RBD binding to its human receptor angiotensin-converting enzyme 2 (ACE2). Twenty percent of patients selectively lacked RBD-specific IgG. Only immunization with folded, but not with unfolded RBD, induced antibodies against conformational epitopes with high virus-neutralizing activity. Conformational RBD epitopes required for protection do not seem to be altered in the currently emerging virus variants. CONCLUSION: These results are fundamental for estimating the protective activity of antibody responses after natural infection or vaccination and for the design of vaccines, which can induce high levels of SARS-CoV-2-neutralizing antibodies conferring sterilizing immunity.

Identification of Epitopes on Rhinovirus 89 Capsid Proteins Capable of Inducing Neutralizing Antibodies.

Rhinoviruses (RVs) are major causes of the common cold, but they can also trigger exacerbations of asthma. More than 160 different RV strains exist and can be classified into three genetic species (RV-A, RV-B and RV-C) which bind to different receptors on human cells including intracellular adhesion molecule 1 (ICAM-1), the low-density lipoprotein receptor (LDLR) or the cadherin-related family member 3 (CDHR3). Epitopes located in the RV capsid have mainly been determined for RV2, a minor-group RV-A strain binding to LDLR, and for RV14, a major-group RV-B strain binding to ICAM-1. In order to study epitopes involved in the neutralization of RV89, an ICAM-1-binding RV-A strain which is highly different from RV2 and RV14 in terms of receptor specificity and sequence, respectively, we analyzed the specificity and epitopes of a highly neutralizing antiserum using recombinantly produced RV89 capsid proteins (VP1, VP2, VP3 and VP4), recombinant fragments and synthetic overlapping peptides thereof. We found that the antiserum which neutralized in vitro RV89 infection up to a dilution of 1:24,000 reacted with the capsid proteins VP1 and VP2 but not with VP3 and VP4. The neutralizing antibodies recognized recombinant fragments comprising approximately 100 amino acids of the N- and C-terminus of VP1 and the middle part of VP2, in particular, three peptides which, according to molecular modeling based on the three-dimensional structure of RV16, were surface-exposed on the viral capsid. Two recombinant fusion proteins containing the identified peptides fused to hepatitis B (HBV)-derived preS as a carrier protein induced upon immunization of rabbits antibodies capable of neutralizing in vitro RV89 infections. Interestingly, the virus-neutralizing epitopes determined for RV89 corresponded to those determined for minor-group RV2 binding to LDL and major-group RV14 belonging to the RV-B species, which are highly different from RV89. Our results indicate that highly different RV strains, even when reacting with different receptors, seem to engage similar parts of their capsid in the infection process. These results may be important for the design of active and passive immunization strategies for RV.

Toward personalization of asthma treatment according to trigger factors.

Asthma is a severe and chronic disabling disease affecting more than 300 million people worldwide. Although in the past few drugs for the treatment of asthma were available, new treatment options are currently emerging, which appear to be highly effective in certain subgroups of patients. Accordingly, there is a need for biomarkers that allow selection of patients for refined and personalized treatment strategies. Recently, serological chip tests based on microarrayed allergen molecules and peptides derived from the most common rhinovirus strains have been developed, which may discriminate 2 of the most common forms of asthma, that is, allergen- and virus-triggered asthma. In this perspective, we argue that classification of patients with asthma according to these common trigger factors may open new possibilities for personalized management of asthma.

Rhinovirus Species-Specific Antibodies Differentially Reflect Clinical Outcomes in Health and Asthma.

Rationale: Rhinoviruses (RVs) are major triggers of common cold and acute asthma exacerbations. RV species A, B, and C may have distinct clinical impact; however, little is known regarding RV species-specific antibody responses in health and asthma.Objectives: To describe and compare total and RV species-specific antibody levels in healthy children and children with asthma, away from an acute event.Methods: Serum samples from 163 preschool children with mild to moderate asthma and 72 healthy control subjects from the multinational Predicta cohort were analyzed using the recently developed PreDicta RV antibody chip.Measurements and Main Results: RV antibody levels varied, with RV-C and RV-A being higher than RV-B in both groups. Compared with control subjects, asthma was characterized by significantly higher levels of antibodies to RV-A and RV-C, but not RV-B. RV antibody levels positively correlated with the number of common colds over the previous year in healthy children, and wheeze episodes in children with asthma. Antibody levels also positively correlated with asthma severity but not with current asthma control.Conclusions: The variable humoral response to RV species in both groups suggests a differential infectivity pattern between RV species. In healthy preschoolers, RV antibodies accumulate with colds. In asthma, RV-A and RV-C antibodies are much higher and further increase with disease severity and wheeze episodes. Higher antibody levels in asthma may be caused by a compromised innate immune response, leading to increased exposure of the adaptive immune response to the virus. Importantly, there is no apparent protection with increasing levels of antibodies.

Reduced CDHR3 expression in children wheezing with rhinovirus.

BACKGROUND: Rhinovirus-induced wheezing in young children has been associated with increased asthma risk at school age. Recently, the transmembrane protein cadherin-related family member 3 (CDHR3) was identified as the RV-C receptor and the genetic variant rs6967330 (p.Cys529Tyr) was reported to be associated with enhanced RV-C binding and increased replication in vitro. The aim of this study was to examine rs6967330 genotypes and mRNA expression of CDHR3 in relation to presence of rhinovirus and clinical symptoms in children with acute wheezing and compare to a group of age-matched healthy children. METHODS: rs6967330;G>A was genotyped (n = 216), and CDHR3 mRNA expression was measured in peripheral blood leukocytes (n = 69) from a subgroup of children wheezing with RV infection acute and at a follow-up visit 2-3 months later, and in healthy controls. Standardized TaqMan assays were used. RESULTS: The risk allele rs6967330-A was over-represented in the wheezing group (P < .001). Reduced mRNA levels of CDHR3 were found in children with acute wheezing as compared to the control group (P = .001). Children with the rs6967330 genotypes AA/AG showed the largest differences in CDHR3 expression between acute and follow-up visit (P < .04). CONCLUSIONS: Preschool children with RV-induced wheezing were shown to have reduced CDHR3 mRNA levels, which might result in an increased permeability of the epithelial layers of the airways and thereby an increased vulnerability. Thus, measuring CDHR3 mRNA levels might help to identify a more severe phenotype of wheezing preschool children.

Development and characterization of a recombinant, hypoallergenic, peptide-based vaccine for grass pollen allergy.

BACKGROUND: Grass pollen is one of the most important sources of respiratory allergies worldwide. OBJECTIVE: This study describes the development of a grass pollen allergy vaccine based on recombinant hypoallergenic derivatives of the major timothy grass pollen allergens Phl p 1, Phl p 2, Phl p 5, and Phl p 6 by using a peptide-carrier approach. METHODS: Fusion proteins consisting of nonallergenic peptides from the 4 major timothy grass pollen allergens and the PreS protein from hepatitis B virus as a carrier were expressed in Escherichia coli and purified by means of chromatography. Recombinant PreS fusion proteins were tested for allergenic activity and T-cell activation by means of IgE serology, basophil activation testing, T-cell proliferation assays, and xMAP Luminex technology in patients with grass pollen allergy. Rabbits were immunized with PreS fusion proteins to characterize their immunogenicity. RESULTS: Ten hypoallergenic PreS fusion proteins were constructed, expressed, and purified. According to immunogenicity and induction of allergen-specific blocking IgG antibodies, 4 hypoallergenic fusion proteins (BM321, BM322, BM325, and BM326) representing Phl p 1, Phl p 2, Phl p 5, and Phl p 6 were included as components in the vaccine termed BM32. BM321, BM322, BM325, and BM326 showed almost completely abolished allergenic activity and induced significantly reduced T-cell proliferation and release of proinflammatory cytokines in patients' PBMCs compared with grass pollen allergens. On immunization, they induced allergen-specific IgG antibodies, which inhibited patients' IgE binding to all 4 major allergens of grass pollen, as well as allergen-induced basophil activation. CONCLUSION: A recombinant hypoallergenic grass pollen allergy vaccine (BM32) consisting of 4 recombinant PreS-fused grass pollen allergen peptides was developed for safe immunotherapy of grass pollen allergy.

Infection with Rhinovirus Facilitates Allergen Penetration Across a Respiratory Epithelial Cell Layer.

BACKGROUND: Rhinovirus infections are a major risk factor for asthma exacerbations. We sought to investigate in an in vitro system whether infection with human rhinovirus reduces the integrity and barrier function of a respiratory epithelial cell layer and thus may influence allergen penetration. METHODS: We cultured the human bronchial epithelial cell line 16HBE14o- in a transwell culture system as a surrogate of respiratory epithelium. The cell monolayer was infected with human rhinovirus 14 at 2 different doses. The extent and effects of transepithelial allergen penetration were assessed using transepithelial resistance measurements and a panel of (125)I-labeled purified recombinant respiratory allergens (rBet v 1, rBet v 2, and rPhl p 5). RESULTS: Infection of respiratory cell monolayers with human rhinovirus decreased transepithelial resistance and induced a pronounced increase in allergen penetration. CONCLUSIONS: Our results indicate that infection with rhinovirus damages the respiratory epithelial barrier and allows allergens to penetrate more efficiently into the subepithelial tissues where they may cause increased allergic inflammation.

Entry of human rhinovirus 89 via ICAM-1 into HeLa epithelial cells is inhibited by actin skeleton disruption and by bafilomycin.

HRV89, a major-group rhinovirus, uses intercellular adhesion molecule 1 (ICAM-1) for cell entry, while minor-group HRV2 uses the LDL receptor for clathrin-mediated endocytosis. Entry of HRV89 into HeLa epithelial cells was found to be inefficient, and infectious virus was still detected on the plasma membrane after 3 h of incubation with the cells. Endocytosis, and consequently infection, of HRV89 but not of HRV2, was almost completely blocked by the actin-polymerization inhibitor cytochalasin D, while the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on infection with either virus. Cytochalasin D also inhibited major-group HRV infection of rhabdomyosarcoma cells expressing ICAM-1 when the time available for uncoating was limited to 30 min. Although cholesterol depletion strongly inhibited HRV89 infection of HeLa cells, it only slightly affected HRV89 endocytosis, indicating that a lipid raft environment was not essential for virus uptake. The sodium-proton exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) significantly reduced cell entry and infection by HRV89 only at a concentration that also inhibited HRV2 infection and Alexa 488-transferrin entry. These data rule out classical macropinocytosis as an infectious entry pathway of HRV89 in HeLa cells. Notably, the proton ATPase inhibitor bafilomycin strongly affected cell entry of both viruses, suggesting a role for submembraneous pH in rhinovirus endocytosis.

Determinants of immunoglobulin G responses to respiratory syncytial virus and rhinovirus in children and adults.

Introduction: Exposure to respiratory viruses is a significant cause of morbidity and affects virus-specific antibody levels. Little is known about determinants associated with immune response to these viruses. We aimed to investigate the determinants of respiratory syncytial virus (RSV)- and rhinovirus (RV)- specific IgG responses in both children and adults. Methods: The study is based on the EGEA cohort, composed of 530 samples of children in EGEA1 (1991-95) and 1241 samples of adults in EGEA2 (2003-07). Cumulative RV-specific IgG levels (species A, B and C) and IgG levels to RSV-G protein were measured by using micro-array technoloy. Multiple linear mixed models (random effect to account for familial dependence) were performed to assess associations between age, sex, body mass index (BMI), tobacco smoke exposure and season of blood sampling with RSV-and RV-specific IgG levels. Results: In children (11.1 ± 2.8 years old, 57% boys), higher RV-specific IgG levels were associated with older age (only for RV-B), female sex and lower BMI, while only older age was associated with higher RSV-specific IgG levels. In adults (43.5 ± 16.7 years old, 48% men), younger age, female sex, lower BMI, active smoking and all seasons except summer were associated with higher RV-specific IgG levels. Older age, active smoking and all seasons except summer were associated with higher RSV-specific IgG levels. Conclusion: Personal and seasonal determinants of RSV- and RV-specific IgG levels seem to vary according to the respiratory virus type and between children and adults, suggesting different patterns of responses along the life course.

Misdirected antibody responses against an N-terminal epitope on human rhinovirus VP1 as explanation for recurrent RV infections.

Rhinoviruses (RVs) are the primary cause of upper respiratory tract infections, generally known as the common cold. Moreover, RV infections can trigger severe exacerbations of asthma and chronic obstructive pulmonary disease (COPD). We expressed the 4 major RV capsid proteins, VP1-VP4, in Escherichia coli and used these proteins as well as recombinant and synthetic VP1 fragments to study and map antibody responses in RV-infected humans. VP1, which on infection binds to ICAM 1, was identified as a major target for the memory immune response, residing in the IgG1 subclass and IgA class. Interestingly, this response was mainly directed against an N-terminal 20 mer peptide in VP1, P1a, which becomes exposed on intact RV only when it docks to its receptor ICAM 1. Molecular modeling using the 3-dimensional RV capsid structures revealed that P1a was localized inside the capsid and outside the areas involved in receptor binding or RV neutralization. Our results suggest misdirection of antibody responses against a nonprotective epitope as a mechanism how RV escapes immunity and causes recurrent infections. Based on these findings, it may be possible to design vaccines against RV infections and RV-induced respiratory diseases.

Allergen-specific immunotherapy: towards combination vaccines for allergic and infectious diseases.

IgE-mediated allergies affect more than 25% of the population. Allergen-specific immunotherapy (SIT) is an antigen-specific and disease-modifying form of treatment. It is based on the therapeutic administration of the disease-causing allergens to allergic patients. However, the fact that only allergen extracts of insufficient quality are currently available and the possible occurrence of side effects during treatment limit the broad use of SIT and prophylactic vaccination is has not yet been performed. In the last 20 years the DNA sequences of the most common allergens have been isolated and the corresponding allergens have been produced as recombinant allergens. Based on the progress made in the field of allergen characterization it is possible to improve the quality and safety of allergy vaccines and to develop new, more effective strategies for a broad application of SIT and even for prophylactic treatment. Here we discuss the development of combination vaccines for allergy and infectious diseases. This approach is based on the selection of allergen-derived peptides with reduced IgE- and T cell reactivity in order to minimize IgE- and T cell-mediated side effects as well as the potential of the vaccine to induce allergic sensitization. These peptides are fused by recombinant technology onto a viral carrier protein to obtain a combination vaccine which induces protective immunity against allergy and viral infections. The application of such combination vaccines for therapy and prophylaxis of allergy and infectious diseases is discussed.

Recombinant allergens: what does the future hold?

This year we are celebrating not only the centenary of allergen-specific immunotherapy but also the 10-year anniversary of the first administration of recombinant allergen-based vaccines to allergic patients. By using recombinant DNA technology, defined and safe allergy vaccines can be produced that allow us to overcome many, if not all, of the problems associated with the use of natural allergen extracts, such as insufficient quality, allergenic activity, and poor immunogenicity. Here we provide an update of clinical studies with recombinant allergen-based vaccines, showing that some of these vaccines have undergone successful clinical evaluation up to phase III studies. Furthermore, we introduce a strategy for allergen-specific immunotherapy based on recombinant fusion proteins consisting of viral carrier proteins and allergen-derived peptides without allergenic activity, which holds the promise of being free of side effects and eventually being useful for prophylactic vaccination.

A hypoallergenic cat vaccine based on Fel d 1-derived peptides fused to hepatitis B PreS.

BACKGROUND: Allergen-specific immunotherapy is clinically effective for the treatment of cat allergy but shows a high rate of side effects. OBJECTIVE: We sought to engineer recombinant fusion proteins for cat immunotherapy that allow reducing both IgE-mediated and T cell-mediated side effects. METHODS: Fusion proteins consisting of the hepatitis B virus-derived PreS domain and 2 nonallergenic Fel d 1-derived peptides were expressed in Escherichia coli and purified. IgE reactivity and allergenic activity of Fel d 1 and the fusion proteins were compared by using IgE-binding assays and basophil activation tests in patients with cat allergy. Mice and rabbits were immunized subcutaneously with Fel d 1 and the fusion proteins to investigate the allergenicity of the vaccines and the development of Fel d 1-specific IgG antibodies. RESULTS: The recombinant fusion proteins showed no relevant IgE reactivity and exhibited more than 1000-fold reduced allergenic activity in basophil activation tests. On immunization of mice and rabbits, the fusion proteins induced Fel d 1-specific IgG antibodies that inhibited the binding of allergic patients' IgE to the allergen without allergic sensitization to Fel d 1. CONCLUSION: The described recombinant fusion proteins exhibit strongly reduced IgE-mediated allergenic activity, contain less than 40% of the Fel d 1 sequence, and thus lack many of the specific T-cell epitopes. Therefore they should represent safe vaccines for the treatment of cat allergy.

Cumulative IgE-levels specific for respiratory allergens as biomarker to predict efficacy of anti-IgE-based treatment of severe asthma.

Molecular therapies, including anti-IgE, biologicals and small molecules are increasingly used for treatment of asthma. The effectiveness of these therapies may be increased with biomarkers. Aim of this study was to assess the value of measuring cumulative IgE levels specific for respiratory allergens to increase the efficacy of anti-IgE therapy for severe bronchial asthma. One hundred and thirty seven patients with severe asthma were recruited from 2016 to 2022. Standard empirical allergy diagnosis (i.e., anamnesis, skin testing, allergen-specific IgE measurement), blood eosinophil counting, measurement of total IgE and of cumulative IgE-specific for respiratory allergens by Phadiatop™ were performed. Thirty four patients with severe allergic asthma, for whom all three diagnostic methods were performed, were then used to analyze the efficacy of anti-IgE treatment in patients stratified in two groups according to cumulative IgE levels specific for respiratory allergens determined by Phadiatop™. Group #1 patients (n = 8) had cumulative specific IgE values ≥ 0.35 and < 1.53 PAU/l while in group #2 patients (n = 26) they were ≥ 1.53 PAU/l. Treatment with Omalizumab was performed for at least 12 months. The level of asthma control (ACT questionnaire), the number of asthma exacerbations, the quality of life (AQLQ questionnaire), the need for systemic corticosteroids, and the respiratory function (FEV1) was determined by "before-after" analysis for each group, followed by a comparison of the dynamics between groups. In group 2 patients with an initial allergen-specific IgE level ≥ 1.53 kUA/L, the efficacy of Omalizumab treatment was better regarding asthma control, number of exacerbations, and quality of life than in group 1 patients. Our study provides evidence that measuring cumulative levels of IgE specific for respiratory allergens could be a useful screening method for detecting an allergic phenotype of severe asthma and may serve as biomarker to enhance the success of IgE-targeted therapy.

Microarray-Based Analyses of Rhinovirus Species-Specific Antibody Responses in Exacerbated Pediatric Asthma in a German Pediatric Cohort.

Rhinoviruses (RV) account for a significant number of asthma exacerbations, and RV species C may be associated with a severe course in vulnerable patient groups. Despite important evidence on the role of RV reported by clinicians and life scientists, there are still unanswered questions regarding their influence on asthma exacerbation in young patients. Thus, we measured the RVspecies-specific IgG titers in our German pediatric exacerbation cohort using a microarray-based technology. For this approach, human sera of patients with exacerbated asthma and wheeze, as well as healthy control subjects (n = 136) were included, and correlation analyses were performed. Concordantly with previously published results, we observed significantly higher cumulative levels of RV species A-specific IgG (p = 0.011) and RV-C-specific IgG (p = 0.051) in exacerbated asthma group compared to age-matched controls. Moreover, atopic wheezers had increased RV-specific IgG levels for species A (p = 0.0011) and species C (p = 0.0009) compared to non-atopic wheezers. Hypothesizing that bacterial infection positively correlates with immune memory against RV, we included nasopharyngeal swab results in our analyses and detected limited correlations. Interestingly, the eosinophil blood titer positively correlated with RV-specific IgG levels. With these observations, we add important observations to the existing data regarding exacerbation in pediatric and adolescent medicine. We propose that scientists and clinicians should pay more attention to the relevance of RV species in susceptible pediatric patients.

A combination vaccine for allergy and rhinovirus infections based on rhinovirus-derived surface protein VP1 and a nonallergenic peptide of the major timothy grass pollen allergen Phl p 1.

Allergens and rhinovirus infections are among the most common elicitors of respiratory diseases. We report the construction of a recombinant combination vaccine for allergy and rhinovirus infections based on rhinovirus-derived VP1, the surface protein which is critically involved in infection of respiratory cells, and a nonallergenic peptide of the major grass pollen allergen Phl p 1. Recombinant hybrid molecules consisting of VP1 and a Phl p 1-derived peptide of 31 aa were expressed in Escherichia coli. The hybrid molecules did not react with IgE Abs from grass pollen allergic patients and lacked allergenic activity when exposed to basophils from allergic patients. Upon immunization of mice and rabbits, the hybrids did not sensitize against Phl p 1 but induced protective IgG Abs that cross-reacted with group 1 allergens from different grass species and blocked allergic patients' IgE reactivity to Phl p 1 as well as Phl p 1-induced basophil degranulation. Moreover, hybrid-induced IgG Abs inhibited rhinovirus infection of cultured human epithelial cells. The principle of fusing nonallergenic allergen-derived peptides onto viral carrier proteins may be used for the engineering of safe allergy vaccines which also protect against viral infections.