HLA antigen frequencies and natural history in Alternaria-sensitive and perennial nonallergic asthmatics

The frequency of HLA-A, -B, and -C loci antigens in random populations of Alternaria-sensitive (N = 100) and perennial nonallergic asthmatics (N = 87) were compared with age- (±5 yr) and sex-matched controls from the same geographic region. There was no association between HLA antigens as measured by frequency analyses and Alternaria-sensitive or perennial nonallergic asthma. Moreover there was no association between HLA antigens and the age of onset of asthma, associated allergic disorders, various environmental factors provoking asthma, total serum IgE levels, and Alternaria-specific IgE antibody.     

The Trypanosoma lewisi immunofluorescence test: A new simple technique for simultaneous determination of total antinuclear antibodies and the detection of antibodies to double-stranded DNA

An indirect immunofluorescence technique was developed for the detection of antibodies to dsDNA and the simultaneous assessment of antinuclear antibodies ‘in toto’ (ANA). This assay was based upon the use as substrate of smears of peripheral blood derived from rats infected with Trypanosoma lewisi. T. lewisi possesses a giant kinetoplast posteriorly to the nucleus. Enzyme digestion and absorption experiments provided strong evidence that T. lewisi kinetoplast contains dsDNA uncontaminated by other nuclear antigens. The T. lewisi immunofluorescent test was evaluated on a total of 130 sera (30 from patients with SLE) and compared with radioimmunoassays for antibodies to dsDNA ([¹²⁵I]dsDNA-RIA) and antibodies to ssDNA ([¹²⁵I]ssDNA-RIA). Excellent correlation was found between kinetoplast immunofluorescence and [¹²⁵I]dsDNA-RIA, whereas no non-SLE sera showing significant ssDNA binding activity gave kinetoplast staining. With a single exception, only SLE sera reacted with T. lewisi kinetoplast. Sera containing auto-antibodies other than ANA did not induce fluorescene of any part of the parasite, including the flagellum and its base. These results indicated that the T. lewisi immunofluorescence test is specific and reliablem and combines the advantages of Crithidia luciliae with those of Trypanosoma gambiense. It may be used routinely for evaluating of total ANA and simultaneous detection of antibodies against dsDNA.     

A simple radioactive binding assay for the detection of rheumatoid factor in serum

A simple radioactive binding assay for the detection of rheumatoid factor (RF-RBA) is described. Test sera are complement inactivated by incubation in 0.13 M ethylene diaminetetraacetic acid (EDTA) at 37°C and then incubated with ¹²⁵I-labelled heat-aggregated with 2.5% (w/v) polyethylene glycol 6000. Sera from 78 patients and 24 controls were tested in the RF-RBA assay and the results compared with those obtained by the rheumatoid latex test and the rheumaton test. 37 sera were positive and 59 sera were negative for rheumatoid factor by the 3 methods used. A positive correlation (r = 0.56, P < 0.01) was observed between the rheumaton titre and the RF-RIA result.     

Development and Evaluation of an Immunodiffusion Test for Diagnosis of Systemic Zygomycosis (Mucormycosis): Preliminary Report

An antigen analysis with filtrate and homogenate precipitinogens of single isolates of the zygomycetes Absidia corymbifera, Mucor pusillus, Rhizopus arrhizus, and Rhizopus oryzae demonstrated the presence of common antigens among the three genera as well as antigens which permit their differentiation. Selected homogenate antigens were valuable in developing a diagnostic immunodiffusion (ID) test for systemic zygomycosis. When sera from 43 patients with various proven mycoses other than zygomycosis were tested against each of the antigens, none formed precipitin bands identical to those formed by A. cormybifera, M. pusillus, and the Rhizopus spp. rabbit reference antisera. Sera from 23 normal persons and 25 diabetics did not react with any of the antigens. Homogenate antigens detected antibody in 8 of the 11 sera (73%) from suspected or proven cases of zygomycosis, whereas ID tests with filtrate antigens detected antibody in only 2 of the 11 sera (18%). Of the eight sera that reacted with the homogenate antigens, five only reacted with a specific Rhizopus sp. antigen, two only reacted with a specific M. pusillus antigen, and one only reacted with a specific A. corymbifera antigen. Study results show the ID test with homogenate antigens to be more specific and sensitive than the ID test with filtrate antigens and indicate that the former is a promising technique for diagnosing human zygomycosis.     

Aspergillus fumigatus: Identification of 16, 18, and 45 kd antigens recognized by human IgG and IgE antibodies and murine monoclonal antibodies

The immunochemical properties of antigens produced by Aspergillus fumigatus were investigated with biochemical purification techniques in conjunction with the production of murine monoclonal antibodies (MAbs) and binding studies with human IgG and IgE antibodies. A. fumigatus antigens were partially purified by gel filtration and hydrophobic interaction chromatography on phenyl-Sepharose. Two fractions that eluted with either 2 mol/L or 0.15 mol/L of NaCl demonstrated strong binding to human IgG and IgE antibodies. Immunoprecipitation analysis with IgG antibodies from six patients with different Aspergillus-related diseases demonstrated that the 2M and 0.15M fractions contained major antigens of molecular weight 18 kd (Asp f I) and 45 kd, respectively. The ¹²⁵I-labeled 2M fraction was used to compare IgG antibodies to A. fumigatus in sera from 25 patients with Aspergillus-related diseases. IgG antibodies were significantly higher in patients with allergic bronchopulmonary aspergillosis (geometric mean, 437 U/ml) than in patients with asthma (geometric mean, 14 U/ml; p < 0.001), but undetectable (<5 U/ml) in 43148 control subjects. A good correlation was found between levels of IgG antibodies to the ¹²⁵I-labeled 0.15M fraction and the ¹²⁵I-labeled 2M fraction in sera from 106 patients with cystic fibrosis (r = 0.77; p < 0.001). Five murine IgG MAbs and two IgM MAbs were raised against the 2M fraction, and immunoprecipitation with the IgG MAb demonstrated two distinct antigens within the 2M fraction, Asp f I, and a 16 kd antigen. The results of a solid phase RIA with IgG MAb 4A6 demonstrated that ≈85% of A. fumigatus-allergic patients with allergic bonchopulmonary aspergillosis had IgE antibodies to Asp f 1. The three protein antigens defined in these studies are useful probes for investigating the immunopathogenesis of diseases associated with colonization by A. fumigatus.     

Widespread immunoglobulin E-mediated hypersensitivity in the Sudan to the “green nimitti” midge,Cladotanytarsus lewisi (diptera: Chironomidae): I. Diagnosis by radioallergosorbent test

A radioallergosorbent test (RAST) has been developed for the diagnosis of hypersensitivity to “green nimitti” chironomid midges of the species Cladotanytarsus lewisi. There was a high percentage binding of ¹²⁵I-anti-IgE to the allergen particle complex by serum from subjects who were clinically hypersensitive, and the RAST was inhibited following incubations of allergic sera with an extract of the allergen. In 104 hypersensitive subjects (i.e., those with a positive skin test or clinical history of bronchial asthma, with or without rhinitis) and 21 controls, the RAST appeared to be specific and of diagnostic value: (1) The percentage binding was appreciably higher in 38 symptomatic individuals (group I) with strongly positive skin tests as compared with 36 patients with moderate skin reactivity (group II). (2) Seven symptomatic subjects with negative skin tests (group III) had a positive (>6% binding) green nimitti RAST. (3) Positive RASTs were demonstrable in 16 and of 17 patients with positive skin tests in whom the history was equivocal (group IV). (4) Six asymptomatic individuals with positive skin tests (group V) had low RAST values. (5) Six asymptomatic Sudanese controls with negative skin tests gave similar values to those of the group V subjects. (6) All of the sera from 15 nonatopic United Kingdom controls gave less than 6% binding of ¹²⁵I-anti-IgE. There was no statistical correlation between the concentrations of total IgE and the green nimitti RAST values. These results suggest that the RAST may be useful diagnostic test in green nimitti hypersensitivity and may also be of value in studies on the epidemiology and in the monitoring of treatment of this important and widespread allergy problem in the Sudan.     

A paper radioimmunosorbent test (PRIST) for the detection of larva-specific antibodies to toxocara canis in human sera

A paper radioimmunosorbent test (PRIST) was shown to be sensitive and reproducible when used with excretory/secretory antigen of Toxocara canis second stage larvae. Whatman No. 50 filter paper (5 mm discs) gave the most consistent and clear results with antigen at a concentration of 100 μg/ml, and could be stored for up to 3 weeks in vacuo at ?70°C. Antigen coated discs were incubated with test sera at 1 : 10 dilution for 3 h at room temperature (21°C), reacted with [¹²⁵I]anti-human IgG for 1 h and counts determined in a gamma counter. Sera from patients with fascioliasis, taeniasis, schistosomiasis, oxyuriasis, trichinellosis and ancyclostomiasis gave counts similar to cord serum controls. Sera from patients with ascariasis gave counts of up to twice as great as controls, but sera from patients with toxicariasis produced counts of 7,000–13,000, a 4–6-fold increase.     

Study of procainamide hapten-specific antibodies in rabbits and humans

Procainamide (PA) is the drug most commonly associated with the induction of autoantibodies and drug-related lupus (DRL). While the majority of these patients express autoantibodies, antibodies to the parent drug and metabolites, PA-hydroxylamine (PAHA) or nitroso-PA (NOPA), have not been reported in humans.Hapten-carrier conjugates were prepared using human hemoglobin (HgB) or autologous rabbit erythrocytes with PAHA or NOPA. PA was conjugated to rabbit serum albumin (RSA) or egg albumin (OVA) via diazotization and condensation methods. Rabbits were immunized with hapten conjugates in Freund's adjuvant. These hapten-carrier compounds (5 – 10 μg/ml) were used as test antigens for antibodies in sera from the rabbits and 40 patients on chronic PA treatment, 10 SLE patients, 33 elderly and 20 young normal controls by ELISA. Type I and II collagens were also used as test antigens for human sera.Sera from rabbits immunized with the PA compounds had elevated IgG antibody values to PA, PAHA and NOPA, but no autoantibodies. Absorption of the rabbit sera with the PA compounds reduced the antibody levels; ssDNA and histones failed to inhibit the total binding values. Mean binding to PA - OVA was 0.95 ± 0.41 for PA patients and 1.37 ± 0.26 standard error of means (S.E.M.) in the SLE patients compared to 0.37 ± 0.14 S.E.M. in the normal sera (P⩽0.05); similar binding values to PAHA - HgB and NOPA - HgB were also observed. Sixty-eight percent of the PA patients had antibodies to type II collagen. Elevated binding values to PA compounds were inhibited by absorption of human sera with ssDNA or total histones; absorption with PA or PAHA had no significant effect. These findings suggest that sera from PA patients containing high titers of autoantibodies cross-react in vitro with unrelated antigens.     

Quantitation of IgE antibody specific for ragweed and grass allergens: Binding of radiolabeled allergens by solid-phase bound IgE

IgE antibody specific for multiple allergens extracted from grass and ragweed pollens was measured by radioimmunoassay. The assay depends on the interaction between IgE antibody bound to a polystyrene solid phase, ¹²⁵I-labeled grass allergens (GA), and ragweed allergens (RW). The binding of ¹²⁵I RW by serum IgE antibody from 37 allergic patients ranged from 0.2 ng to 75 ng RW protein (P) bound per ml. This binding of ¹²⁵I RW by patient's IgE was paralleled by their IgE binding of ¹²⁵I antigen E (AgE), a purified allergen from ragweed pollen (r = 0.90, p < 0.001). Inhibition of patient's IgE binding of ¹²⁵I RW by highly purified AgE ranged from 25% to 85% indicating individual differences in patient's IgE response to inhaled ragweed pollen. The binding of ¹²⁵I GA by serum IgE antibody from 7 grass-sensitive patients ranged from 0.6 ng GA P bound per ml to 15 ng. This assay should be useful in the study of IgE responses to environmental agents containing multiple allergens and has the advantage that other antibody classes cannot interfere with the interaction between IgE antibody and labeled allergens.     

Time course of serum inhibitory activity for facilitated allergen–IgE binding during bee venom immunotherapy in children

Background Immunotherapy for bee venom allergy is effective and provides long‐term protection. Venom‐specific IgG4 levels are increased but with no correlation with clinical improvement. Following grass pollen immunotherapy, elevation of antigen‐specific IgG4 is accompanied by increases in IgG‐dependent serum inhibitory activity for IgE‐facilitated binding of allergen–IgE complexes to B cells. As this ‘functional’ assay of inhibitory antibodies may be more predictive of clinical efficacy, we investigated the time course of serum inhibitory activity for IgE‐facilitated antigen binding during venom immunotherapy (VIT) in children and following 2 years of VIT withdrawal. Methods Ten bee venom‐allergic children (mean age: 9.3 years; m/f, 7/3) with moderate to severe allergic reactions to bee stings received VIT. A separate group of seven children (mean age: 14 years; m/f, 5/2) were investigated 2 years after VIT withdrawal. Ten age‐ and gender‐matched children served as non‐allergic controls. Allergen‐specific serum IgG4 and IgE levels were measured by ELISA at baseline, after 2 years of VIT and 2 years after VIT withdrawal. Serum inhibitory activity was assessed using the facilitated‐allergen binding (FAB) assay. Results Sera obtained during VIT significantly inhibited allergen–IgE binding to B‐cells (pre‐treatment=104±23%; 2 years=46±15%; P<0.001) when compared with sera obtained after treatment withdrawal and sera from normal controls. In parallel to FAB inhibition during VIT, significantly higher IgG4 levels were noted after immunotherapy (pre‐treatment=8.6±2.3 AU; 2 years=26.7±3.5 AU; P<0.001) compared with those observed after withdrawal and in the controls. In contrast, progressively lower IgE concentrations were observed compared with pre‐treatment (44±7 AU) in sera obtained after 2 years of VIT (25±5 AU; P<0.01) and 2 years following the withdrawal of VIT (10±3 AU; P<0.05). Conclusions In contrast to grass pollen immunotherapy, the persistent decline in venom‐specific IgE levels, rather than serum inhibitory activity for FAB, may be more relevant for long‐term clinical efficacy of VIT.     

Measurement of IgG antibodies to house dust mite and grass pollen by a solid-phase radioimmunoassay

Polystyrene microtubes, coated with extracts of either Dermatophagoides pteronyssinus (DPT) or grass pollens, were used as the solid-phase in a radioimmunoassay for the measurement of specific IgG antibodies to the corresponding allergen. Radiolabelled protein A from Staphylococcus aureus (SpA) was used to determine the IgG antibodies attached to the tubes. The binding of IgG from either normal or allergic sera to DPT-coated tubes was antigen specific and mediated by the Fab fragment of the immunoglobulin. IgG antibodies purified by affinity chromatography from non-allergic serum competed with IgE antibodies to DPT. IgE antibodies do not significantly interfere with the assay. Indeed, heating a reaginic serum resulted in a striking reduction of the (¹²⁵I) anti-IgE binding to allergen-coated tubes without modifying the (¹²⁵I)-SpA binding. Furthermore, filtration of a reaginic serum through Sephacryl S-200 separated a peak of IgE antibodies, characterized by a high binding of labelled a-IgE and a low binding of (¹²⁵I)-SpA from the peak of IgG antibodies defined by low a-IgE and high SpA binding. The solid phase method is more sensitive than a double-antibody technique employing the same DPT extract as labelled antigen. Non-allergic subjects had less IgG antibodies to DPT or grass pollens than allergic patients. In untreated patients, there was a good correlation between levels of IgG and IgE antibodies to grass pollens but not to DPT. Patients hyposensitized to house dust mite had on the average three times more specific IgG antibodies than untreated cases.     

Reaction of antibody in sera from cystic fibrosis patients with nontoxic forms ofPseudomonas aeruginosa exotoxin A

Sera from 48 cystic fibrosis patients from two hospitals were screened for antibody against rods, non-toxic macromolecular structures which share antigenic determinants withPseudomonas aeruginosa exotoxin A. A solid-phase radioimmunoassay employing (¹²⁵I)-staphylococcal protein A was used to detect anti-rod IgG. Antibodies recognizing rods, exotoxin A, or both antigens, were demonstrated using a competitive radioimmunoassay in cystic fibrosis patient sera, and in sera from animals immunized with exotoxin A, rods, or infected withPseudomonas aeruginosa. Anti-rod titers of cystic fibrosis patients (1.07 to 14 x control serum levels) inversely correlated with aggregate clinical evaluation scores, and in most instances, with X-ray scores. Since rods are non-toxic and cross-reactive with exotoxin A, they may represent therapeutically useful antigens for producing immunity to exotoxin A.     

Allergenicity of α-caseins from cow, sheep, and goat

The allergic potential of α-caseins from bovine, ovine, and goat's milk sharing more than 85% identical amino acids was compared. Caseins were purified by anion-exchange chromatography and used for a specific IgE and IgG ELISA with diluted human sera. Sera were from 17 children with immediate-type allergy to cow's milk, from 59 children with atopy but without food allergy, and from 27 healthy children without atopic disease. The sera of cow's milk-allergic children showed a significantly higher IgE and IgG binding to α-caseins from all three species than the sera of the other groups. All groups showed an increased antibody binding to bovine a-casein compared to the sheep and goat proteins, but the differences were significant only in the groups of atopic children and of healthy controls. Furthermore, inhibition of the IgE binding to bovine α-casein with α-casein from cow, goat, and sheep revealed that the a-caseins from these species are highly cross-reactive, on the basis of the small differences in their primary structure. In conclusion, the milk of goat and sheep harbor an allergic potential and is not suitable for the nutrition of milk-allergic patients.     

A comparison of methods for labelling DNA for use in the radioimmunoassay of DNA-antibodies

The radioimmunoassay for DNA-antibodies in systemic lupus erythematosus is assessed with DNA labelled with ¹²⁵I via chemical iodination on one hand (¹²⁵I-DNA), and with DNA containing [¹²⁵I]iododeoxyuridine via biological incorporation on the other ([¹²⁵IUdR]DNA). The results show that chemical iodination labels protein impurities in the DNA and reduces the difference in binding of normal vs lupus sera. ¹²⁵IUdR-DNA is a superior product since no label is introduced in proteins. The binding of ¹²⁵I-DNA by normal sera ranges from 5 to 20% and from 40 to 70% lupus sera. The same sera yield values of 0–3% for normal and 92–98% for lupus with ¹²⁵IUdR-DNA. The latter allows a sharper discrimination between normal and slightly elevated values, as in patients under treatment.     

Use of a Synthetic Peptide Epitope of Asp f 1, a Major Allergen or Antigen of Aspergillus fumigatus, for Improved Immunodiagnosis of Allergic Bronchopulmonary Aspergillosis

Allergic bronchopulmonary aspergillosis (ABPA) is an immunologically complex allergic disorder caused by the fungal pathogen Aspergillus fumigatus. Elevated levels of total immunoglobulin E (IgE), specific IgE, and IgG antibodies in sera are important immunodiagnostic criteria for ABPA. International reference standards or standardized immunodiagnostic assays are not available due to a lack of well-defined diagnostic antigens. The present study was carried out to identify and evaluate the immunodiagnostic relevance of synthetic epitopic peptides of Asp f 1, a major allergen, antigen, or cytotoxin of A. fumigatus. Five overlapping peptides were synthesized from the N terminus of Asp f 1, one of the potential immunodominant regions predicted by algorithmic programs. The 11-amino-acid synthetic peptide (P1) significantly inhibited both IgG binding (89.10% ± 4.45%) and IgE binding (77.32% ± 3.38%) of the standardized diagnostic antigen (SDA) (a well-defined pool of diagnostically relevant allergens and antigens of A. fumigatus). With a panel of sera of ABPA patients, allergic patients with skin test negativity to A. fumigatus, and healthy individuals, P1 showed a higher diagnostic efficiency than SDA (specific IgG, 100%; specific IgE, 98.3%). The diagnostic efficiency of P1 could be attributed to the presence of homologous epitopes in various immunodominant allergens or antigens of A. fumigatus. The ability of P1 to induce histamine release from sensitized mast cells and a Th2 type of cytokine profile in peripheral blood mononuclear cells of ABPA patients suggests its potential for use in intradermal testing. P1 could be further explored for development of a standardized, specific, and sensitive immunodiagnostic test for aspergillosis.     

Specific IgE antibodies in atopic eczema

Radioallergosorbent tests (RAST'S) with 35 antigens and total serum IgE levels were performed on sera from 25 patients with atopic eczema, ranging from mild to very severe, who had been evaluated clinically and, when possible, skin-tested to inhalant allergens. IgE levels varied from 95 to 112,000 I.U., with a geometric mean of 2,200 I.U. Individual patients' sera gave an average of 8.4 positive RAST's to 14 inhalant allergens with a range of from 1 to 14 positive tests. The correlation of RAST with skin tests averaged 55 per cent with no difference observed with either the scratch or the prick methods. The degree of correlation was not related to severity of eczema. In eczema patients the great majority of noncorrelating tests were RAST positive and skin-test negative, unlike the noncorrelating tests found in children with asthma and allergic rhinitis, where there are more positive skin tests with negative RAST. The 25 sera were tested by RAST with 18 food antigens and the various sera gave from 1 to 18 positive tests, with an average of 9.7. IgE antibodies reacting with at least one of the DPT antigens were found in 10 of the 25 sera. Sera from 4 of the patients studied contained IgE antibodies that combined with all 35 antigens studied. Control RAST's with these sera were negative. This study shows that much of the elevation of serum IgE observed in atopic eczema represents specific IgE antibodies that can combine with common antigens with relatively high affinity.     

Immunologic cross-reactions among thermophilic actinomycetes associated with hypersensitivity pneumonitis

Antibodies produced in rabbits against Micropolyspora faeni, Thermoactinomyces vulgaris, T. sacchari, Thermoactinomyces candidus, and Saccharomonospora viridis were tested against antigens derived from many strains of thermophilic actinomycetes for precipitating antibodies by immunodiffusion test. It was found that immune sera reacted strongly against antigens from strains belonging to the same species and weakly against antigens from different species of thermophilic actinomycetes. However, sera from farmer's lung patients showed cross-reactivity against antigens from different species. This may be because the patient is sensitized to multiple species of thermophilic actinomycetes present in the environment and developed antibodies against most of them.     

Detection of Alternaria allergens by crossed radioimmunoelectrophoresis

Allergens involved in sensitivity to Alternaria have not been well defined. We used crossed radioimmunoelectrophoresis (CRIE) to study antigens in a crude A. alternata extract, which were capable of binding IgE in human serum. Sera from 35 patients sensitive to Alternaria, 10 not sensitive to Alternaria, and five normal controls were examined. CRIE with hyperimmune rabbit sera demonstrated 22 antigens in the Alternaria extract. After exposure of CRIE gels sequentially with patient serum and 125I-labeled anti-human IgE, autoradiography demonstrated that three of the antigens bound IgE in sera of Alternaria-sensitive subjects. Our results suggest that multiple allergens are involved in A. alternata sensitivity.     

A rapid method for determining whether monoclonal antibodies react with the same or different antigens on the cell surface

A quick and simple method for determining whether monoclonal antibodies react with the same or different antigens is described which utilises an indirect radioactive binding assay against cells. Six antibodies were selected, BK19.45, BK20.20, GenOx4.17, GenOx4.21, W6-34 and W6-46, which detected antigens present either only on leukocytes (BK19.45 and BK20.20) or on a wider range of cell types including fibroblasts, liver cells and neuroblastoma cells. The saturation binding levels for each antibody, in terms of the amount of ¹²⁵I-anti-mouse immunoglobulin bound, were determined with respect to a fixed number of cells. The addition of two antibodies with different specities (BK19.45 or BK20.20 to either W6-34 or W6-46) resulted in a higher plateau value of ¹²⁵I-anti-mouse immunoglobulin bound per fixed number of cells than for either antibody singly. The increase in the amount of antibody bound is equivalent to the sum of the two individual saturation binding levels. In contrast, the addition of BK20.20 to BK19.45 or W6-45 produced no detectable rise in the saturation level. From these data it is concluded that BK20.20 and BK19.45, and W6-34 and W6-46 are bound to either identical epitopes which are in very close spatial relationship. On the other hand 19.45 and W6-34, as expected, detect unrelated antigens. Observations from autoradiograph binding studies on the semi-quantitative distribution on bone marrow cells of the antigens recognised by 19.45 and 20.20 supported the conclusion that the antibodies were recognising identical antigens.A study of the antibodies to HLA (2A1) and β₂ microglobulin (M3) showed an increase in the saturation level, when both antibodies were added together, which was less than the sum of the two individual saturation binding levels. The close association of β₂ microglobulin and HLA on the cell surface may to some extent prevent independent antibody binding. These data suggest that the above approach will differentiate between monoclonal antibodies which detect antigenic determinants that are located on closely associated surface molecules.     

Transcriptional Regulation of the nadA Gene in Neisseria meningitidis Impacts the Prediction of Coverage of a Multicomponent Meningococcal Serogroup B Vaccine

The NadA adhesin is a major component of 4CMenB, a novel vaccine to prevent meningococcus serogroup B (MenB) infection. Under in vitro growth conditions, nadA is repressed by the regulator NadR and poorly expressed, resulting in inefficient killing of MenB strains by anti-NadA antibodies. Interestingly, sera from children infected with strains that express low levels of NadA in laboratory growth nevertheless recognize the NadA antigen, suggesting that NadA expression during infection may be different from that observed in vitro. In a strain panel covering a range of NadA levels, repression was relieved through deleting nadR. All nadR knockout strains expressed high levels of NadA and were efficiently killed by sera from subjects immunized with 4CMenB. A selected MenB strain, NGP165, mismatched for other vaccine antigens, is not killed by sera from immunized infants when the strain is grown in vitro. However, in an in vivo passive protection model, the same sera effectively protected infant rats from bacteremia with NGP165. Furthermore, we identify a novel hydroxyphenylacetic acid (HPA) derivative, reported by others to be produced during inflammation, which induces expression of NadA in vitro, leading to efficient antibody-mediated killing. Finally, using bioluminescent reporters, nadA expression in the infant rat model was induced in vivo at 3 h postinfection. Our results suggest that during infectious disease, NadR repression is alleviated due to niche-specific signals, resulting in high levels of NadA expression from any nadA-positive (nadA⁺) strain and therefore efficient killing by anti-NadA antibodies elicited by the 4CMenB vaccine.