Recombinant PreS-fusion protein vaccine for birch pollen and apple allergy.

BACKGROUND: IgE cross-sensitization to major birch pollen allergen Bet v 1 and pathogenesis-related (PR10) plant food allergens is responsible for the pollen-food allergy syndrome. METHODS: We designed a recombinant protein, AB-PreS, consisting of non-allergenic peptides derived from the IgE-binding sites of Bet v 1 and the cross-reactive apple allergen, Mal d 1, fused to the PreS domain of HBV surface protein as immunological carrier. AB-PreS was expressed in E. coli and purified by chromatography. The allergenic and inflammatory activity of AB-PreS was tested using basophils and PBMCs from birch pollen allergic patients. The ability of antibodies induced by immunization of rabbits with AB-PreS and birch pollen extract-based vaccines to inhibit allergic patients IgE binding to Bet v 1 and Mal d 1 was assessed by ELISA. RESULTS: IgE-binding experiments and basophil activation test revealed the hypoallergenic nature of AB-PreS. AB-PreS induced lower T-cell activation and inflammatory cytokine production in cultured PBMCs from allergic patients. IgG antibodies induced by five injections with AB-PreS inhibited allergic patients' IgE binding to Bet v 1 and Mal d 1 better than did IgG induced by up to 30 injections of six licensed birch pollen allergen extract-based vaccines. Additionally, immunization with AB-PreS induced HBV-specific antibodies potentially protecting from infection with HBV. CONCLUSION: The recombinant AB-PreS-based vaccine is hypoallergenic and superior over currently registered allergen extract-based vaccines regarding the induction of blocking antibodies to Bet v 1 and Mal d 1 in animals.

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.

Importance, Applications and Features of Assays Measuring SARS-CoV-2 Neutralizing Antibodies.

More than three years ago, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused the unforeseen COVID-19 pandemic with millions of deaths. In the meantime, SARS-CoV-2 has become endemic and is now part of the repertoire of viruses causing seasonal severe respiratory infections. Due to several factors, among them the development of SARS-CoV-2 immunity through natural infection, vaccination and the current dominance of seemingly less pathogenic strains belonging to the omicron lineage, the COVID-19 situation has stabilized. However, several challenges remain and the possible new occurrence of highly pathogenic variants remains a threat. Here we review the development, features and importance of assays measuring SARS-CoV-2 neutralizing antibodies (NAbs). In particular we focus on in vitro infection assays and molecular interaction assays studying the binding of the receptor binding domain (RBD) with its cognate cellular receptor ACE2. These assays, but not the measurement of SARS-CoV-2-specific antibodies per se, can inform us of whether antibodies produced by convalescent or vaccinated subjects may protect against the infection and thus have the potential to predict the risk of becoming newly infected. This information is extremely important given the fact that a considerable number of subjects, in particular vulnerable persons, respond poorly to the vaccination with the production of neutralizing antibodies. Furthermore, these assays allow to determine and evaluate the virus-neutralizing capacity of antibodies induced by vaccines and administration of plasma-, immunoglobulin preparations, monoclonal antibodies, ACE2 variants or synthetic compounds to be used for therapy of COVID-19 and assist in the preclinical evaluation of vaccines. Both types of assays can be relatively quickly adapted to newly emerging virus variants to inform us about the magnitude of cross-neutralization, which may even allow us to estimate the risk of becoming infected by newly appearing virus variants. Given the paramount importance of the infection and interaction assays we discuss their specific features, possible advantages and disadvantages, technical aspects and not yet fully resolved issues, such as cut-off levels predicting the degree of in vivo protection.

Dissection of Antibody Responses of Gam-COVID-Vac-Vaccinated Subjects Suggests Involvement of Epitopes Outside RBD in SARS-CoV-2 Neutralization.

Millions of people have been vaccinated with Gam-COVID-Vac but fine specificities of induced antibodies have not been fully studied. Plasma from 12 naïve and 10 coronavirus disease 2019 (COVID-19) convalescent subjects was obtained before and after two immunizations with Gam-COVID-Vac. Antibody reactivity in the plasma samples (n = 44) was studied on a panel of micro-arrayed recombinant folded and unfolded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins and 46 peptides spanning the spike protein (S) and by immunoglobulin G (IgG) subclass enzyme-linked immunosorbent assay (ELISA). The ability of Gam-COVID-Vac-induced antibodies to inhibit binding of the receptor-binding domain (RBD) to its receptor angiotensin converting enzyme 2 (ACE2) was investigated in a molecular interaction assay (MIA). The virus-neutralizing capacity of antibodies was studied by the pseudo-typed virus neutralization test (pVNT) for Wuhan-Hu-1 and Omicron. We found that Gam-COVID-Vac vaccination induced significant increases of IgG1 but not of other IgG subclasses against folded S, spike protein subunit 1 (S1), spike protein subunit 2 (S2), and RBD in a comparable manner in naïve and convalescent subjects. Virus neutralization was highly correlated with vaccination-induced antibodies specific for folded RBD and a novel peptide (i.e., peptide 12). Peptide 12 was located close to RBD in the N-terminal part of S1 and may potentially be involved in the transition of the pre- to post-fusion conformation of the spike protein. In summary, Gam-COVID-Vac vaccination induced S-specific IgG1 antibodies in naive and convalescent subjects in a comparable manner. Besides the antibodies specific for RBD, the antibodies induced against a peptide close to the N-terminus of RBD were also associated with virus-neutralization.

Identification of a Synthetic Polyhydroxyphenolic Resveratrol Analogue, 3,3',4,4',5,5'-Hexahydroxy-trans-Stilbene with Anti-SARS-CoV-2 Activity.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus has been causing the COVID-19 pandemic since December 2019, with over 600 million infected persons worldwide and over six million deaths. We investigated the anti-viral effects of polyphenolic green tea ingredients and the synthetic resveratrol analogue 3,3',4,4',5,5'-hexahydroxy-trans-stilbene (HHS), a compound with antioxidant, antitumor and anti-HIV properties. In the TCID50 assay, four out of nine green tea constituents showed minor to modest cell protective effects, whereas HHS demonstrated the highest reduction (1103-fold) of the TCID50, indicating pronounced inhibition of virus replication. HHS was also a highly effective inhibitor of SARS-CoV-2 proliferation in VeroE6 cells with an IC50 value of 31.1 µM. HSS also inhibited the binding of the receptor-binding domain (RBD) of the spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor (RBD-ACE2) binding with 29% at 100 µM and with 9.2% at 50 µM indicating that the SARS-CoV-2 inhibitory effect might at least in part be attributed to the inhibition of virus binding to ACE2. Based on the chemical similarity to other polyphenols, the oral bioavailability of HHS is likely also very low, resulting in blood levels far below the inhibitory concentration of EGCG against SARS-CoV-2 observed in vitro. However, administration of HHS topically as a nose or throat spray would increase concentrations several-fold above the minimal inhibitory concentration (MIC) in the mucosa and might reduce virus load when administered soon after infection. Due to these promising tissue culture results, further preclinical and clinical studies are warranted to develop HHS as an additional treatment option for SARS-CoV-2 infection to complement vaccines, which is and will be the main pillar to combat the COVID-19 pandemic.

Combined assessment of S- and N-specific IL-2 and IL-13 secretion and CD69 neo-expression for discrimination of post-infection and post-vaccination cellular SARS-CoV-2-specific immune response.

BACKGROUND: Antibody-based tests are available for measuring SARS-CoV-2-specific immune responses but fast T-cell assays remain scarce. Robust T cell-based tests are needed to differentiate specific cellular immune responses after infection from those after vaccination. METHODS: One hundred seventeen individuals (COVID-19 convalescent patients: n = 40; SARS-CoV-2 vaccinees: n = 41; healthy controls: n = 36) were evaluated for SARS-CoV-2-specific cellular immune responses (proliferation, Th1, Th2, Th17, and inflammatory cytokines, activation-induced marker [AIM] expression) by incubating purified peripheral blood mononuclear cells (PBMC) or whole blood (WB) with SARS-CoV-2 peptides (S, N, or M), vaccine antigens (tetanus toxoid, tick borne encephalitis virus) or polyclonal stimuli (Staphylococcal enterotoxin, phytohemagglutinin). RESULTS: N-peptide mix stimulation of WB identified the combination of IL-2 and IL-13 secretion as superior to IFN-γ secretion to discriminate between COVID-19-convalescent patients and healthy controls (p < .0001). Comparable results were obtained with M- or S-peptides, the latter almost comparably recalled IL-2, IFN-γ, and IL-13 responses in WB of vaccinees. Analysis 10 months as opposed to 10 weeks after COVID-19, but not allergic disease status, positively correlated with IL-13 recall responses. WB cytokine responses correlated with cytokine and proliferation responses of PBMC. Antigen-induced neo-expression of the C-type lectin CD69 on CD4+ (p < .0001) and CD8+ (p = .0002) T cells informed best about the SARS-CoV-2 exposure status with additional benefit coming from CD25 upregulation. CONCLUSION: Along with N- and S-peptide-induced IL-2 and CD69 neo-expression, we suggest to include the type 2 cytokine IL-13 as T-cellular recall marker for SARS-CoV-2 specific T-cellular immune responses after infection and vaccination.

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.

Characterization of the antibody response to SARS-CoV-2 in a mildly affected pediatric population.

BACKGROUND: While children usually experience a mild course of COVID-19, and a severe disease is more common in adults, the features, specificities, and functionality of the SARS-CoV-2-specific antibody response in the pediatric population are of interest. METHODS: We performed a detailed analysis of IgG antibodies specific for SARS-CoV-2-derived antigens S and RBD by ELISA in 26 SARS-CoV-2 seropositive schoolchildren with mild or asymptomatic disease course, and in an equally sized, age- and gender-matched control group. Furthermore, a detailed mapping of IgG reactivity to a panel of microarrayed SARS-CoV-2 proteins and S-derived peptides was performed by microarray technology. The capacity of the antibody response to block RBD-ACE2 binding and virus neutralization were assessed. Results were compared with those obtained in an adult COVID-19 convalescent population. RESULTS: After mild COVID-19, anti-S and RBD-specific IgG antibodies were developed by 100% and 84.6% of pediatric subjects, respectively. No difference was observed in regards to symptoms and gender. Mounted antibodies recognized conformational epitopes of the spike protein and were capable to neutralize the virus up to a titer of ≥80 and to inhibit the ACE2-RBD interaction by up to 65%. SARS-CoV-2-specific IgG responses in children were comparable to mildly affected adult patients. CONCLUSION: SARS-CoV-2 asymptomatic and mildly affected pediatric patients develop a SARS-CoV-2-specific antibody response, which is comparable regarding antigen, epitope recognition, and the ability to inhibit the RBD-ACE2 interaction to that observed in adult patients after mild COVID-19.

Immunological imprint of COVID-19 on human peripheral blood leukocyte populations.

BACKGROUND: SARS-CoV-2 has triggered a pandemic that is now claiming many lives. Several studies have investigated cellular immune responses in COVID-19-infected patients during disease but little is known regarding a possible protracted impact of COVID-19 on the adaptive and innate immune system in COVID-19 convalescent patients. METHODS: We used multiparametric flow cytometry to analyze whole peripheral blood samples and determined SARS-CoV-2-specific antibody levels against the S-protein, its RBD-subunit, and viral nucleocapsid in a cohort of COVID-19 convalescent patients who had mild disease ~10 weeks after infection (n = 109) and healthy control subjects (n = 98). Furthermore, we correlated immunological changes with clinical and demographic parameters. RESULTS: Even ten weeks after disease COVID-19 convalescent patients had fewer neutrophils, while their cytotoxic CD8+ T cells were activated, reflected as higher HLA-DR and CD38 expression. Multiparametric regression analyses showed that in COVID-19-infected patients both CD3+ CD4+ and CD3+ CD8+ effector memory cells were higher, while CD25+ Foxp3+ T regulatory cells were lower. In addition, both transitional B cell and plasmablast levels were significantly elevated in COVID-19-infected patients. Fever (duration, level) correlated with numbers of central memory CD4+ T cells and anti-S and anti-RBD, but not anti-NC antibody levels. Moreover, a "young immunological age" as determined by numbers of CD3+ CD45RA+ CD62L+ CD31+ recent thymic emigrants was associated with a loss of sense of taste and/or smell. CONCLUSION: Acute SARS-CoV-2 infection leaves protracted beneficial (ie, activation of T cells) and potentially harmful (ie, reduction of neutrophils) imprints in the cellular immune system in addition to induction of specific antibody responses.

Silencing of SARS-CoV-2 with modified siRNA-peptide dendrimer formulation.

BACKGROUND: First vaccines for prevention of Coronavirus disease 2019 (COVID-19) are becoming available but there is a huge and unmet need for specific forms of treatment. In this study we aimed to evaluate the anti-SARS-CoV-2 effect of siRNA both in vitro and in vivo. METHODS: To identify the most effective molecule out of a panel of 15 in silico designed siRNAs, an in vitro screening system based on vectors expressing SARS-CoV-2 genes fused with the firefly luciferase reporter gene and SARS-CoV-2-infected cells was used. The most potent siRNA, siR-7, was modified by Locked nucleic acids (LNAs) to obtain siR-7-EM with increased stability and was formulated with the peptide dendrimer KK-46 for enhancing cellular uptake to allow topical application by inhalation of the final formulation - siR-7-EM/KK-46. Using the Syrian Hamster model for SARS-CoV-2 infection the antiviral capacity of siR-7-EM/KK-46 complex was evaluated. RESULTS: We identified the siRNA, siR-7, targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) as the most efficient siRNA inhibiting viral replication in vitro. Moreover, we showed that LNA-modification and complexation with the designed peptide dendrimer enhanced the antiviral capacity of siR-7 in vitro. We demonstrated significant reduction of virus titer and lung inflammation in animals exposed to inhalation of siR-7-EM/KK-46 in vivo. CONCLUSIONS: Thus, we developed a therapeutic strategy for COVID-19 based on inhalation of a modified siRNA-peptide dendrimer formulation. The developed medication is intended for inhalation treatment of COVID-19 patients.

Molecular IgE sensitization profiles of urban and rural children in South Africa.

BACKGROUND: Allergens can act as disease-triggering factors in atopic dermatitis (AD) patients. The aim of the study was to elucidate the molecular IgE sensitization profile in children with and without AD living in urban and rural areas of South Africa. METHODS: Specific IgE reactivity was assessed in 166 Black South African children aged 9-38 months using a comprehensive panel of microarrayed allergens. According to clinical characterization children fell in four groups, urban AD cases (n = 32), urban controls (non-AD, n = 40), rural cases (n = 49) and rural controls (non-AD, n = 45). RESULTS: IgE reactivity to at least one of the allergens was detected in 94% of urban and 86% of rural AD children. House dust mite (HDM; 81% urban, 74% rural AD) and animal-derived allergens (50% urban, 31% rural AD) were the most frequently recognized respiratory allergens, whereas IgE to pollen allergens was almost absent. Urban AD children showed significantly higher frequency of IgE reactivity (50%) to mouse lipocalin, Mus m 1, than rural AD children (12%). The most frequently recognized food allergens were from egg (63% urban, 43% rural AD), peanut (31% vs 41%), and soybean (22% vs 27%), whereas milk sensitization was rare. α-gal-specific IgE almost exclusively occurred in rural children (AD: 14%, non-AD: 49%). CONCLUSION: Molecular allergy diagnosis detects frequent IgE sensitization to HDM, animal but not pollen allergens and to egg, peanut, and soy, but not milk allergens in African AD children. Urban AD children reacted more often to Mus m 1, whereas α-gal sensitization is more common in rural children likely due to parasite exposure.

Multiprofessional perinatal care in a pregnant patient with acute respiratory distress syndrome due to COVID-19.

BACKGROUND: The coronavirus disease (COVID-19) pandemic has caused ongoing challenges in health services worldwide. Despite the growing body of literature on COVID-19, reports on perinatal care in COVID-19 cases are limited. CASE PRESENTATION: We describe a case of severe acute respiratory distress syndrome (ARDS) in a 36-year-old G5/P2 pregnant woman with morbid obesity, confirmed severe acute respiratory syndrome coronavirus 2 infection, and fulminant respiratory failure. At 28+ 1 gestational weeks, the patient delivered an uninfected newborn. Using ImmunoCAP ISAC® technology, we found no immunoglobulin (Ig) M antibodies, suggesting that no mother-to-child viral transmission occurred during pregnancy or delivery. The maternal respiratory state improved rapidly after delivery; both maternal and neonatal outcomes were encouraging given the early gestational age and fulminant course of respiratory failure in our patient. CONCLUSIONS: The management of ARDS in pregnant women with COVID-19 is complex and requires an individualized, multidisciplinary approach, while considering maternal and fetal outcomes.

Sensitization to grass pollen allergen molecules in a birth cohort-natural Phl p 4 as an early indicator of grass pollen allergy.

BACKGROUND: Grass pollen allergy is one of the most common allergies worldwide. OBJECTIVE: The aim of this study was to evaluate the usefulness of grass pollen allergen molecules for prediction of grass pollen allergy during childhood and up to adolescence. METHOD: Questionnaire data and sera obtained from the study subjects at the ages of 4, 8, and 16 years from the population-based Barn/Children Allergy Milieu Stockholm Epidemiology birth cohort were used. Sera from 763 representative subjects with serum samples available at all 3 ages were analyzed for IgE reactivity to 8 Phleum pratense (Phl p) allergens (MeDALL [Mechanisms for the Development of Allergies] chip) and to timothy grass extract (ImmunoCAP). Allergic rhinitis to grass pollen (ARg) was defined as upper airway symptoms during grass pollen exposure. RESULTS: The prevalence of sensitization to any Phl p molecule was higher compared with that to timothy extract at all 3 ages: at the age of 4 years, 9.7% versus 6.8%; at the age of 8 years, 28.4% versus 15.3%; and at the age of 16 years, 37.1% versus 27.1%. General estimating equations analyses revealed that among children sensitized at the age of 4 years, the overall odds ratio (OR) of later ARg (up to 16 years) was increased only for IgE reactivity to Phl p 1 (OR = 4.9) and natural Phl p 4 (OR = 6.9). The likelihood of later symptoms increased with the number of allergen molecules; at the age of 4 years, 2 or more molecules predicted ARg to 78% and 3 or more molecules predicted ARg to 95%. A positive test result for timothy extract predicted ARg to 70%. CONCLUSIONS: Natural Phl p 4 is a hitherto unrecognized early indicator of grass pollen allergy, in addition to Phl p 1. To identify grass pollen sensitization and predict later ARg, allergen molecules are of added value to timothy extract alone and may help clinicians improve prediction of grass pollen allergy.

Allergen-specific IgE levels and the ability of IgE-allergen complexes to cross-link determine the extent of CD23-mediated T-cell activation.

BACKGROUND: CD23 mediates IgE-facilitated allergen presentation and subsequent allergen-specific T-cell activation in allergic patients. OBJECTIVE: We sought to investigate key factors regulating IgE-facilitated allergen presentation through CD23 and subsequent T-cell activation. METHODS: To study T-cell activation by free allergens and different types of IgE-Bet v 1 complexes, we used a molecular model based on monoclonal human Bet v 1-specific IgE, monomeric and oligomeric Bet v 1 allergen, an MHC-matched CD23-expressing B-cell line, and a T-cell line expressing a human Bet v 1-specific T-cell receptor. The ability to cross-link Fcε receptors of complexes consisting of either IgE and monomeric Bet v 1 or IgE and oligomeric Bet v 1 was studied in human FcεRI-expressing basophils. T-cell proliferation by monomeric or oligomeric Bet v 1, which cross-links Fcε receptors to a different extent, was studied in allergic patients' PBMCs with and without CD23-expressing B cells. RESULTS: In our model non-cross-linking IgE-Bet v 1 monomer complexes, as well as cross-linking IgE-Bet v 1 oligomer complexes, induced T-cell activation, which was dependent on the concentration of specific IgE. However, T-cell activation by cross-linking IgE-Bet v 1 oligomer complexes was approximately 125-fold more efficient. Relevant T-cell proliferation occurred in allergic patients' PBMCs only in the presence of B cells, and its magnitude depended on the ability of IgE-Bet v 1 complexes to cross-link CD23. CONCLUSION: The extent of CD23-mediated T-cell activation depends on the concentration of allergen-specific IgE and the cross-linking ability of IgE-allergen complexes.

Prevention of allergy by virus-like nanoparticles (VNP) delivering shielded versions of major allergens in a humanized murine allergy model.

BACKGROUND: In high-risk populations, allergen-specific prophylaxis could protect from sensitization and subsequent development of allergic disease. However, such treatment might itself induce sensitization and allergies, thus requiring hypoallergenic vaccine formulations. We here characterized the preventive potential of virus-like nanoparticles (VNP) expressing surface-exposed or shielded allergens. METHODS: Full-length major mugwort pollen allergen Art v 1 was selectively targeted either to the surface or to the inner side of the lipid bilayer envelope of VNP. Upon biochemical and immunological analysis, their preventive potential was determined in a humanized mouse model of mugwort pollen allergy. RESULTS: Virus-like nanoparticles expressing shielded version of Art v 1, in contrast to those expressing surface-exposed Art v 1, were hypoallergenic as they hardly induced degranulation of rat basophil leukemia cells sensitized with Art v 1-specific mouse or human IgE. Both VNP versions induced proliferation and cytokine production of allergen-specific T cells in vitro. Upon intranasal application in mice, VNP expressing surface-exposed but not shielded allergen induced allergen-specific antibodies, including IgE. Notably, preventive treatment with VNP expressing shielded allergen-protected mice from subsequent sensitization with mugwort pollen extract. Protection was associated with a Th1/Treg-dominated cytokine response, increased Foxp3+ Treg numbers in lungs, and reduced lung resistance when compared to mice treated with empty particles. CONCLUSION: Virus-like nanoparticles represent a novel and versatile platform for the in vivo delivery of allergens to selectively target T cells and prevent allergies without inducing allergic reactions or allergic sensitization.

Proteolytic and N-glycan processing of human α1-antitrypsin expressed in Nicotiana benthamiana.

Plants are increasingly being used as an expression system for complex recombinant proteins. However, our limited knowledge of the intrinsic factors that act along the secretory pathway, which may compromise product integrity, renders process design difficult in some cases. Here, we pursued the recombinant expression of the human protease inhibitor α1-antitrypsin (A1AT) in Nicotiana benthamiana. This serum protein undergoes intensive posttranslational modifications. Unusually high levels of recombinant A1AT were expressed in leaves (up to 6 mg g(-1) of leaf material) in two forms: full-length A1AT located in the endoplasmic reticulum displaying inhibitory activity, and secreted A1AT processed in the reactive center loop, thus rendering it unable to interact with target proteinases. We found that the terminal protein processing is most likely a consequence of the intrinsic function of A1AT (i.e. its interaction with proteases [most likely serine proteases] along the secretory pathway). Secreted A1AT carried vacuolar-type paucimannosidic N-glycans generated by the activity of hexosaminidases located in the apoplast/plasma membrane. Notwithstanding, an intensive glycoengineering approach led to secreted A1AT carrying sialylated N-glycan structures largely resembling its serum-derived counterpart. In summary, we elucidate unique insights in plant glycosylation processes and show important aspects of postendoplasmic reticulum protein processing in plants.