Immunogenicity of H1N1 influenza virus-like particles produced in Nicotiana benthamiana.

The H1N1 influenza pandemic of 2009 stimulated interest in developing safe and effective subunit influenza vaccines using rapid and cost-effective recombinant technologies that can avoid dependence on hens' eggs supply and live viruses for production. Among alternative approaches to subunit vaccine development, virus-like particles (VLPs) represent an attractive strategy due to their safety and immunogenicity. Previously, we have produced a recombinant monomeric hemagglutinin (HA) protein derived from the A/California/04/09 (H1N1) strain of influenza virus in a plant-based transient expression system and demonstrated immunogenicity and safety of this monomeric HA in animal models and human volunteers. In an effort to produce higher potency influenza vaccine in plants, we have designed and generated enveloped VLPs using the ectodomain of HA from the A/California/04/09 strain and heterologous sequences. The resulting H1 HA VLPs (HAC-VLPs) elicited robust hemagglutination inhibition antibody responses in mice at doses lower than 1 µg in the presence or absence of Alhydrogel adjuvant. These results suggest enhanced immunogenicity of recombinant HA in the form of an enveloped VLP over soluble antigen.

Changes in cat specific IgE and IgG antibodies with decreased cat exposure.

BACKGROUND: Current understanding of the effects of reducing exposure to cat allergens is limited. It has also become clear that there are different forms of immune response to cat allergens. OBJECTIVE: To investigate changes in skin tests and cat specific IgG and IgE antibodies when students from a home with a cat move to a college dormitory. METHODS: Ninety-seven college students participated in a prospective study that consisted of allergy skin prick testing and serum measurement of IgE and IgG antibodies to cat at the beginning and end of one academic year in college. A subgroup returned for follow-up at the end of 2 years. RESULTS: Among 97 students, 33% had IgG antibodies to Fel d 1 but no evidence of sensitization, 25% had positive skin test results and/or serum IgE antibodies, and 42% had negative skin test results and no detectable serum antibodies. Among the non-cat sensitized students with IgG antibodies, the titers decreased during 8 months (P = .002). Titers of IgG4 to Fel d 1 also decreased (P < .001). Among the sensitized students, no change in IgE antibodies to cat occurred in 8 months (P = .20), whereas Fel d 1 specific IgG antibodies decreased (P < .001). Thus, ratios of IgG to IgE decreased highly significantly (P = .007). Among the students with negative skin test results who returned for follow-up (n = 56), none developed positive skin test results or serum IgE antibodies. CONCLUSION: Under conditions of marked decrease in exposure, no participants developed new-onset sensitization. Among the individuals sensitized at study entry, there were major decreases in the ratio of IgG to IgE.

Delayed anaphylaxis, angioedema, or urticaria after consumption of red meat in patients with IgE antibodies specific for galactose-alpha-1,3-galactose.

BACKGROUND: Carbohydrate moieties are frequently encountered in food and can elicit IgE responses, the clinical significance of which has been unclear. Recent work, however, has shown that IgE antibodies to galactose-alpha-1,3-galactose (alpha-gal), a carbohydrate commonly expressed on nonprimate mammalian proteins, are capable of eliciting serious, even fatal, reactions. OBJECTIVE: We sought to determine whether IgE antibodies to alpha-gal are present in sera from patients who report anaphylaxis or urticaria after eating beef, pork, or lamb. METHODS: Detailed histories were taken from patients presenting to the University of Virginia Allergy Clinic. Skin prick tests (SPTs), intradermal skin tests, and serum IgE antibody analysis were performed for common indoor, outdoor, and food allergens. RESULTS: Twenty-four patients with IgE antibodies to alpha-gal were identified. These patients described a similar history of anaphylaxis or urticaria 3 to 6 hours after the ingestion of meat and reported fewer or no episodes when following an avoidance diet. SPTs to mammalian meat produced wheals of usually less than 4 mm, whereas intradermal or fresh-food SPTs provided larger and more consistent wheal responses. CAP-RAST testing revealed specific IgE antibodies to beef, pork, lamb, cow's milk, cat, and dog but not turkey, chicken, or fish. Absorption experiments indicated that this pattern of sensitivity was explained by an IgE antibody specific for alpha-gal. CONCLUSION: We report a novel and severe food allergy related to IgE antibodies to the carbohydrate epitope alpha-gal. These patients experience delayed symptoms of anaphylaxis, angioedema, or urticaria associated with eating beef, pork, or lamb.

Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose.

BACKGROUND: Cetuximab, a chimeric mouse-human IgG1 monoclonal antibody against the epidermal growth factor receptor, is approved for use in colorectal cancer and squamous-cell carcinoma of the head and neck. A high prevalence of hypersensitivity reactions to cetuximab has been reported in some areas of the United States. METHODS: We analyzed serum samples from four groups of subjects for IgE antibodies against cetuximab: pretreatment samples from 76 case subjects who had been treated with cetuximab at multiple centers, predominantly in Tennessee, Arkansas, and North Carolina; samples from 72 control subjects in Tennessee; samples from 49 control subjects with cancer in northern California; and samples from 341 female control subjects in Boston. RESULTS: Among 76 cetuximab-treated subjects, 25 had a hypersensitivity reaction to the drug. IgE antibodies against cetuximab were found in pretreatment samples from 17 of these subjects; only 1 of 51 subjects who did not have a hypersensitivity reaction had such antibodies (P<0.001). IgE antibodies against cetuximab were found in 15 of 72 samples (20.8%) from control subjects in Tennessee, in 3 of 49 samples (6.1%) from northern California, and in 2 of 341 samples (0.6%) from Boston. The IgE antibodies were shown to be specific for an oligosaccharide, galactose-alpha-1,3-galactose, which is present on the Fab portion of the cetuximab heavy chain. CONCLUSIONS: In most subjects who had a hypersensitivity reaction to cetuximab, IgE antibodies against cetuximab were present in serum before therapy. The antibodies were specific for galactose-alpha-1,3-galactose.

Analysis of CD25hiCD4+ "regulatory" T-cell subtypes in atopic dermatitis reveals a novel T(H)2-like population.

BACKGROUND: It is unresolved whether circulating CD25hiCD4+ T cells in patients with atopic dermatitis who have elevated IgE (IgE(high)) are regulatory or effector in nature. OBJECTIVE: To analyze the properties of CD25hi T-cell subtypes in IgE(high) atopic dermatitis. METHODS: The phenotype of circulating CD25hi T cells was analyzed by flow cytometry using PBMCs from patients with atopic dermatitis (total IgE > 250 IU/mL). Cytokines induced in CD25hi subtypes were analyzed after activation with anti-CD3 mAb (+/-IL-2) and in the presence of activated autologous effector T cells (CD25negCD4+). Reactivity to bacterial superantigen derived from the skin-colonizing organism Staphylococcus aureus was also evaluated. RESULTS: CD25(hi) T cells expressing regulatory T-cell markers (Foxp3, CCR4, cutaneous lymphocyte-associated antigen) were increased in atopic dermatitis compared with IgE(low) controls. This phenomenon was linked to disease severity. Two subtypes of CD25hi T cells were identified on the basis of differential expression of the chemokine receptor CCR6. Although the ratio of CCR6+ and CCR6neg subtypes within the CD25hi subset was unaltered in atopic dermatitis, each subtype proliferated spontaneously ex vivo, suggesting in vivo activation. Activated CCR6neg cells secreted T(H)2 cytokines, and coculture with effector T cells selectively enhanced IL-5 production. Moreover, induction of a T(H)2-dominated cytokine profile on activation with bacterial superantigen was restricted to the CCR6neg subtype. CONCLUSION: Despite a regulatory phenotype, activated CD25hi T cells that lack expression of CCR6 promote T(H)2 responses.

Targeting Fel d 1 to FcgammaRI induces a novel variation of the T(H)2 response in subjects with cat allergy.

BACKGROUND: Induction of CD4+ T cells that produce IL-10 or IFN-gamma is central to the protective effects of conventional allergen immunotherapy. OBJECTIVE: We examined the T-cell modulatory capacity of a fusion protein (H22-Fel d 1) that targets Fel d 1 to the high-affinity IgG receptor (FcgammaRI) on antigen-presenting cells. METHODS: Monocyte-derived dendritic cells pulsed with H22-Fel d 1 were analyzed for surface phenotype and cytokine secretion by flow cytometry and cytometric bead assay, respectively. CD4+ T cells generated after coculture with H22-Fel d 1-pulsed dendritic cells were analyzed at the single-cell level by flow cytometry after intracellular cytokine staining. The T-cell repertoire was compared for subjects with (IgE+) and without cat allergy (IgE(neg)IgG(neg)), including subjects with a modified T(H)2 response (IgE(neg)IgG+). RESULTS: H22-Fel d 1 induced a semimature phenotype in dendritic cells in conjunction with a selective increase in IL-5+ and IL-10+ CD4+ T cells compared with nonreceptor-targeted Fel d 1. Amplified T cells included diverse subtypes characteristic of T(H)0 (IL-5+IFN-gamma+), regulatory T(H)1 (IL-10+IFN-gamma+) and regulatory T(H)2 (IL-10+IL-5+ cells. T-cell qualitative changes were restricted to subjects with allergy and were distinct from a modified T(H)2 response. Blocking IL-10 induced by H22-Fel d 1 selectively increased IL-5+ CD4+ T cells, suggesting that T(H)2 responses were controlled. CONCLUSION: Targeting Fel d 1 to FcgammaRI induces a novel variation of the T(H)2 response that incorporates major elements of a protective T-cell response.

Prevalence and titer of IgE antibodies to mouse allergens.

BACKGROUND: Positive skin tests to allergens derived from mouse urine have been reported among patients with asthma. Very few data are available detailing the titer of IgE Ab to mouse allergen and how it varies by location and population. OBJECTIVE: To evaluate further the prevalence and titer of IgE Ab to mouse-derived allergens and their relevance to total IgE and asthma. METHODS: IgE Ab to mouse allergens was measured in 1165 sera from diverse populations including children and adults. The results were compared with IgE Ab to other allergens and total serum IgE. RESULTS: Positive results were found in 79 sera, but only 15 had an IgE Ab titer >or=10 IU/mL. Results for IgE Ab to Mus m 1 showed a close quantitative correlation with IgE Ab to mouse allergen (r = 0.93; P < .001). Cohorts in neither Atlanta nor Virginia contained sera in which IgE Ab to mouse was dominant over other allergens or contributed significantly to total IgE. By contrast, among 319 mothers from minority groups in Boston, 11 sera had >or=10 IU/mL. In these sera, specific IgE Ab to mouse made a significant contribution to the total. CONCLUSION: Mouse allergen sensitization may contribute significantly to total IgE and allergy in African American and Hispanic populations from some northern cities. Analysis of the significance of an IgE Ab response should include quantitative comparison with other responses and total IgE. CLINICAL IMPLICATIONS: Significance of rodent infestation and IgE Ab varies dramatically in different populations and areas of the United States.

Simultaneous detection of total and allergen-specific IgE by using purified allergens in a fluorescent multiplex array.

BACKGROUND: Testing serum samples for total and allergen-specific IgE requires separate testing for each antibody and allergen specificity. OBJECTIVE: To apply fluorescent suspension array technology to allow simultaneous detection of total and allergen-specific IgE in serum in a single quantitative test. METHODS: A 7-plex suspension array for the simultaneous detection of total IgE and IgE specific to Der p 1, Der p 2, Fel d 1, Can f 1, Bet v 1, and Phl p 5 was developed, using mAb or purified allergens covalently coupled to fluorescent microspheres. The multiplex array was validated by comparing total and allergen-specific IgE levels in serum from patients with allergy with results obtained by enzyme immunoassays. RESULTS: There was a highly significant correlation between total IgE levels measured by multiplex array and fluorescent enzyme immunoassay (r = 0.97; P < .001; n = 63). Total and allergen-specific IgE levels also correlated with enzyme-linked and fluorescent enzyme immunoassay results (r = 0.44-0.94; n = 95 or 106). The multiplex array was reproducible (r = 0.86-0.99; mean coefficient of variance percentage, 12% to 25%). The sample volume required for a 7-plex assay was <20 microL per sample, compared with >400 microL in current immunoassays. CONCLUSION: The multiplex array is a high-throughput system that allows simultaneous quantification of allergen-specific and total IgE. CLINICAL IMPLICATIONS: Our results suggest that fluorescent multiplex technology will facilitate large-scale epidemiologic studies of allergic sensitization. The reduced serum volume is an advantage for pediatric studies.

The relevance of microbial allergens to the IgE antibody repertoire in atopic and nonatopic eczema.

BACKGROUND: A propensity to microbial skin infections has been reported in atopic ("high IgE") and nonatopic ("low IgE") forms of eczema. However, the relationship between antimicrobial IgE antibodies and nonatopic disease is unclear. OBJECTIVE: We examined the relevance of microbial allergens to the allergen-specific IgE antibody repertoire in patients with atopic dermatitis. METHODS: Patients with IgE levels of less than 150 IU/mL were stratified according to sensitivity (n = 22) or no sensitivity (n = 27) to 11 common food allergens and aeroallergens. The prevalence and titers of antimicrobial IgE antibodies were compared with those of patients (n = 36) with increased total IgE levels (>150 IU/mL). Skin-derived serum chemokines were also analyzed. RESULTS: Patients with low IgE levels showed decreased disease severity, increased age of onset, a striking female predominance, and a distinct distribution of skin lesions. High titer IgE antibodies (sum of 8 bacterial and fungal allergens = 29.8 +/- 32.6 IU/mL) and multisensitization specific for microbial allergens was characteristic of patients with high IgE levels, with an overall 84% positivity; however, antimicrobial IgE antibodies comprised 3% or less of allergen-specific IgE antibodies. By contrast, antimicrobial IgE antibodies were detected in only 20% of patients with low IgE, and titers were negligible, irrespective of sensitization to common allergens. These patients were monosensitized, and exclusive microbial sensitivity was uncommon (10%). Patients with low IgE with no sensitivity to common allergens had lower levels of serum macrophage inflammatory protein 3alpha compared with their sensitized counterparts. CONCLUSION: Antimicrobial IgE antibodies are uncommon in patients with atopic dermatitis with low IgE levels. CLINICAL IMPLICATIONS: Hypersensitivity to microbial allergens is an unlikely trigger for eczematous eruptions in patients with low IgE levels.

Asian ladybugs (Harmonia axyridis): a new seasonal indoor allergen.

BACKGROUND: Harmonia axyridis, the Asian ladybug (ALB), was repeatedly introduced between 1916 and 1990. These beetles are intolerant to cold and move indoors during the winter. OBJECTIVE: To investigate sensitization to ALB. METHODS: Proteins in ALB extracts were purified by gel filtration and ion exchange chromatography. Purified fractions were screened for IgE antibody using the streptavidin CAP technique in sera from 20 patients with allergy living in ALB-infested houses. Two proteins were fully purified. Serum antibodies were also assessed in sera from 68 adult patients with asthma. RESULTS: Fifteen of the 20 sera had measurable IgE antibody, 7 with high titers, > 10 IU/mL, to ALB extract. The 2 proteins, Har a 1, 10 kd, and Har a 2, 55 kd, bound IgE antibody in 65% and 75% of the sera, respectively. Sequencing revealed a novel N-terminal sequence for Har a 1. Sequencing of Har a 2 demonstrated homology to a dehydrogenase from the red flour beetle. Although sera from 18 of the patients with asthma were positive for IgE antibody to ALB, they were also positive to Blatella germanica. These subjects did not report exposure to H axyridis, and inhibition studies with B germanica blocked > or = 95% of ALB IgE antibody binding. CONCLUSION: Two proteins of ALB have been purified and used to demonstrate that patients exposed to this beetle can develop high-titer IgE antibody. Cross-reactivity with B germanica was found but was significant only among patients primarily exposed to cockroaches. CLINICAL IMPLICATIONS: Asian ladybug has become an important seasonal indoor allergen in the United States.

Bla g 6: a troponin C allergen from Blattella germanica with IgE binding calcium dependence.

BACKGROUND: The known cockroach allergens do not appear to account for the full repertoire of IgE responses. OBJECTIVE: To identify and investigate the importance of other Blattella germanica allergens contributing to cockroach allergy. METHODS: A B germanica cDNA library was screened with pooled sera from patients with cockroach allergy. Three isoallergens of troponin C (Bla g 6) were cloned and expressed in Pichia pastoris. Homology modeling was performed by using Swiss-Model. IgE responses to purified allergens were simultaneously measured in 104 sera by using a fluorescent multiplex array system. The effect of calcium on IgE binding was investigated by ELISA. RESULTS: Three isoallergens, Bla g 6.0101, Bla g 6.0201, and Bla g 6.0301, were identified which share homology with insect troponin Cs and vertebrate calmodulins (61% to 78% and 42% to 44% amino acid identity, respectively) and have 2 EF-hand calcium binding domains. Molecular models of Bla g 6 showed 2 structurally homologous lobes connected by a linker that confers flexibility to the allergen. The prevalence of IgE binding to recombinant Bla g 6 was 14%. Calcium depletion by 10 mmol/L ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraacetic acid did not significantly affect IgE binding in most cases. Interestingly, addition of 10 mmol/L CaCl2 after calcium depletion increased IgE binding by approximately 2-fold, a finding not previously reported for calcium binding allergens. CONCLUSION: Bla g 6 is a troponin allergen with a calcium dependent IgE reactivity that may be involved in muscle contraction. CLINICAL IMPLICATIONS: Bla g 6 homologous allergens may occur among other insects and cause cosensitization or allergenic cross-reactivity.

Quantitative measurement of IgE antibodies to purified allergens using streptavidin linked to a high-capacity solid phase.

BACKGROUND: Commercially available assays for IgE antibody provide results in international units per milliliter for many allergen extracts, but this is not easily achieved with purified or novel allergens. OBJECTIVE: To develop assays for IgE antibody suitable for purified or novel allergens by using a commercially available immunosorbent. METHODS: Streptavidin coupled to a high-capacity immunosorbent (CAP) was used to bind biotinylated purified allergens from mite (Der p 1 and Der p 2), cat (Fel d 1), and dog (Can f 1). Assays for IgE antibody to these allergens were performed on sera from children (asthma and control) as well as adults with atopic dermatitis. RESULTS: The results were validated by serial dilution of sera with high and low levels of IgE antibody and were quantitated in international units per milliliter by using a standard curve. Values for IgE antibody to Der p 1, Der p 2, and Fel d 1 correlated with values obtained with the allergen extracts (r2 = 0.80, 0.84, and 0.95, respectively; P < .001 in each case). Furthermore, the values for IgE antibody in sera from children with high exposure to mite and cat allergens demonstrated 10-fold higher levels of IgE antibody to Der p 1 and Der p 2 than to Fel d 1 (P < .001). CONCLUSION: The streptavidin immunosorbent technique provides a new method for quantifying IgE antibody to purified proteins. The results provide evidence about the high quantities of IgE antibody to purified inhalant allergens in patients with atopic dermatitis. In addition, the results demonstrate major differences in IgE antibodies specific for mite and cat allergens among children with high exposure to both allergens.

Specific IgE and IgG antibody-binding patterns to recombinant cockroach allergens.

BACKGROUND: The specificity of serum antibody responses to different cockroach allergens has not been studied. OBJECTIVE: We sought to quantitate serum IgE and IgG antibodies to a panel of purified cockroach allergens among cockroach-sensitized subjects. METHODS: IgE antibodies to recombinant cockroach allergens (rBla g 1, rBla g 2, rBla g 4, rBla g 5, and rPer a 7) were measured in sera containing IgE antibodies to Blattella germanica extract (n = 118) by using a streptavidin CAP assay and a multiplex flow cytometric assay. Specific IgG antibodies were determined by using radioimmunoprecipitation techniques. RESULTS: Specific IgE antibodies measured by means of CAP assay and multiplex assay were strongly correlated ( r = 0.8, P < .001). The sum of IgE antibodies (in international units per milliliter) against all 5 allergens equated to IgE antibodies to cockroach extract. Although the prevalence of IgE antibodies was highest for rBla g 2 (54.4%) and rBla g 5 (37.4%), patterns of IgE antibody binding were unique to each subject. Surprisingly, only 16% of cockroach-sensitized subjects with IgE antibodies to house dust mite exhibited IgE antibody binding to cockroach tropomyosin (rPer a 7). Specific IgE antibodies were associated with increased IgG antibody levels, although detection of IgG in the absence of IgE was not uncommon. CONCLUSION: The techniques described offer a new approach for defining the hierarchy of purified allergens. IgE antibodies directed against 5 allergens constitute the majority of the IgE antibody repertoire for cockroach. Such distinct patterns of IgE-IgG responsiveness to different cockroach allergens highlight the complexity of B-cell responses to environmental allergens.

Strain-specific variants of the mouse Cftr promoter region reveal transcriptional regulatory elements.

Regulation of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels is not well understood. Mouse Cftr mRNA shows strain-dependent expression differences that cannot be fully explained by variation at non-Cftr loci. Differences in tracheal and colonic expression appear to be due predominantly to elements linked to Cftr. Fifteen single nucleotide sequence variations were found within 1.4 kb 5' to the translation start site between the inbred lines A/J, C57BL/6J and 129/SvJ. In addition, 129/SvJ carries a 100 bp deletion relative to the other two strains. These variants were investigated by sequentially deleting 5' regions and measuring luciferase reporter activity from transfected, mouse epithelial cell lines derived from pancreatic duct, renal collecting duct, salivary gland and trachea. These assays identified a region between -524 and -834 in the C57BL/6J promoter, but not in A/J or 129/SvJ, capable of repressing expression. Sequence analysis and gel mobility shift assays suggest that the transcription factor MZF is involved in the strain-dependent effect. It was also apparent that several reporter constructs displayed expression differences between cell lines, possibly indicating the presence of tissue-specific elements.