Safety evaluation of a glutaraldehyde modified tyrosine adsorbed housedust mite extract containing monophosphoryl lipid A (MPL) adjuvant: a new allergy vaccine for dust mite allergy.

A new allergy vaccine is currently under clinical evaluation for the prevention or relief of symptoms caused by specific housedust mites. It consists of a 50:50 mixture of the mite Dermatophagoides pteronyssinus and D. farinae protein derived from aqueous extracts of the mites which is chemically modified by glutaraldehyde and adsorbed onto L-tyrosine with addition of the immunostimulatory adjuvant, monophosphoryl lipid A (MPL) "Polymite". A specific preclinical safety testing strategy was developed to support clinical use and comprised single and repeat dose toxicity, reproduction toxicity and local tolerance studies. Dose levels of up to 0.5ml for the mouse and up to 1ml for both the rat and the rabbit were used. Overall, the product was shown to produce no toxicological findings of significance at levels greatly in excess to those proposed for clinical use. A not unexpected, but relatively minor, immunostimulatory effect was seen following repeated dosing (once weekly for 13 weeks) at 1ml per rat; the Polymite formulation also resulted in injection site reaction which can largely be attributed to the presence of tyrosine. No reproduction toxicity was found.

A Th1-inducing adjuvant, MPL, enhances antibody profiles in experimental animals suggesting it has the potential to improve the efficacy of allergy vaccines.

BACKGROUND: Monophosphoryl lipid A (MPL) is a detoxified derivative of the lipopolysaccharide (LPS) moiety of Salmonella minnesota R595, which has retained immunostimulatory activities. MPL has been administered to many subjects in clinical trials as an adjuvant component of infectious disease vaccines and is currently a component of a licensed cancer vaccine, Melacine (Corixa Inc., Schering Plough). MPL has, in particular, been shown to promote Th1-type antigen specific responses. L-tyrosine is a depot adjuvant which is fully metabolisable and has been successfully employed in allergy vaccines for a number of years. METHODS: Mice were immunised with MPL adjuvant in conjunction with separate preparations of either ovalbumin or glutaraldehyde-modified ragweed pollen extract both coprecipitated with L-tyrosine. The specific antibody isotypes IgG1, IgG2a, IgG2b and also IgE were measured. Rats received booster injections of keyhole limpet haemocyanin (KLH) in conjunction with MPL adjuvant following priming with KLH in alum alone. KLH-specific antibody responses were measured. RESULTS: It was shown that a combination of L-tyrosine and MPL were synergistic in enhancing murine antigen specific IgG antibody responses without enhancing antigen specific IgE responses. Furthermore, this adjuvant combination promoted strong IgG2 antigen specific responses indicative of a Th1 directed response. In KLH sensitised rats, treatment with MPL was shown to prevent a secondary IgE antibody response when injected with booster injections of antigen. CONCLUSIONS: Immunisation of mice with two different antigens adsorbed to L-tyrosine induced a Th1-skewed primary response when in conjunction with MPL adjuvant which also generally enhances a specific IgG response. Incorporation of MPL adjuvant in the immunisation of rats prevented a secondary specific IgE response. These results suggest that the employment of this new adjuvant in clinical allergy vaccination formulations may result in an improved efficacy which could be utilised in various ways to improve compliance.

A well-tolerated grass pollen-specific allergy vaccine containing a novel adjuvant, monophosphoryl lipid A, reduces allergic symptoms after only four preseasonal injections.

BACKGROUND: We present data showing that a Th1-inducing adjuvant can reduce the number of injections required for allergy vaccination. Allergy vaccination is the only treatment for type 1 hypersensitivity that can alter the underlying disease process. A switch of specific T-cell activity from Th2 >Th1 to Th1 >Th2 is believed to be an important change seen after long-term vaccination therapy. An immunologic adjuvant that enhances such a switch could be used to reduce the number of injections required. This would improve compliance with the treatment and provide pharmacoeconomic advantages. Such an adjuvant is 3-deacylated monophosphoryl lipid A (MPL adjuvant, Corixa). METHODS: A multicentre, placebo-controlled, randomized, double-blind clinical study was performed with a new standardized allergy vaccine comprising a tyrosine-adsorbed glutaraldehyde-modified grass pollen extract containing MPL adjuvant. Four subcutaneous injections of the active product were given preseasonally to 81 grass pollen-sensitive subjects, and 60 received placebo injections (tyrosine alone). Diary cards were used to record symptoms and medication taken during approximately 30 days of the grass pollen season. RESULTS: There was a statistical advantage in favour of the active treatment for nasal (P = 0.016) and ocular (P = 0.003) symptoms and combined symptom and medication scores (P=0.013). Titrated skin prick testing revealed a significant reduction of skin sensitivity in the active group compared to placebo (P = 0.04). Grass-pollen-specific IgG antibody was raised by active treatment (P < 0.01). A rise in IgE antibody was seen in the placebo group during the season (P < 0.01). The first year's treatment rise of IgE was not seen in the active group, and no rise occurred during the pollen season. More local adverse events were seen in the active group. There was no difference in generalized adverse events. CONCLUSION: A new, well-tolerated allergy vaccine, incorporating a Th1-inducing adjuvant, MPL, was efficacious and after only four preseasonal injections produced antibody changes normally associated with long injection schedules. This may encourage wider application of allergy vaccination. The vaccine is now available in a number of countries as Pollinex Quattro.

Standardisation of glutaraldehyde-modified tyrosine-adsorbed tree pollen vaccines containing the Th1-inducing adjuvant, monophosphoryl lipid A (MPL).

BACKGROUND: a new range of allergy vaccines has been developed by the introduction of a relatively new Th1-inducing adjuvant known as 3-deacylated monophosphoryl lipid A (MPL). MPL adjuvant is of natural origin, derived from the lipopolysaccharide of Salmonella minnesota R595. This adjuvant is incorporated in a glutaraldehyde-modified pollen extract adsorbed to L-tyrosine (Pollinex Quattro). A major potential benefit provided by MPL adjuvant is the promotion of a Th1 response which enhances the efficacy of allergy vaccination and can consequently allow a reduction in the number of injections required for treatment. The standardisation of Pollinex Quattro tree pollen allergy vaccine is described and we include details of some innovative analytical procedures. METHODS AND RESULTS: an essential feature of the analytical strategy is the assay of the MPL adjuvant using a recently developed HPLC technique. The adjuvant has a complex chemical structure and the analysis is illustrated in detail. We give a full picture of the vaccine standardisation by describing biochemical and immunological characterisation of the allergen extract, together with some brief manufacturing details. CONCLUSIONS: a high overall level of standardisation is illustrated by a number of different tests applied to all stages of vaccine manufacture. Tree pollen allergen potency is measured following the pollen extraction, chemical modification and formulation as a tyrosine adsorbate. Good batch-to-batch reproducibility is shown. The HPLC assay for MPL adjuvant showed high quality resolution which did not vary when measuring raw material or when incorporated in the vaccine and the technically complex assay is shown to be reliable.

Comparison of the efficacy and safety of two preseasonal regimens of glutaraldehyde modified, tyrosine-adsorbed parietaria pollen extract over a period of three years in monosensitive patients.

The purpose of this study was to evaluate the clinical efficacy over a period of three years (1988-90) of two preseasonal dosage regimens of a Parietaria allergoid (Bencard Tyrosine Parietaria) in patients who were only sensitive to this pollen. Fifty patients were included (14 men and 36 women, age: mean, 28 years; range, 14-47 years). Twenty five patients (group A) were treated each january with the basic course of Bencard Tyrosine Parietaria. This consisted of injecting subcutaneously 0.5 ml from each of three vials, with one week between each injection. A further injection using the vial with the highest dose was given one week later. Each january and february, twenty five patients (group B) were treated with the basic course of Bencard Tyrosine Parietaria, repeating the last dose five times, with one week between each injection. Immunotherapy with a tyrosine-adsorbed Parietaria judaica allergoid is an effective method for mitigating nasal (p < 0.0001), bronchial (p < 0.005), conjunctival (p < 0.001) and palatal itching symptoms (p < 0.0001) in patients who are sensitive to this pollen. Sensitivity to Parietaria pollen, as verified by skin test and nasal challenge, decreased during immunotherapy (p < 0.001). Histamine release by peripheral blood basophils decreased during the course of the study, falling from 43.5 ng/ml to 12.3 ng/ml in group A and from 42.9 ng/ml to 10.0 ng/ml in group B; during the second and third years, IgG levels were increased one and four months after starting treatment with the extract, while this was not the case after ten months; IgE levels were also increased. Finally, overall tolerance to this immunotherapy product was good in almost all patients.

Modulation of Basophil Histamine Release and T Cell Reactivity by Chemically Modified Allergen.

Modification of a model allergen ovalbumin (OA) with succinylation (Suc-OA) led to inhibition of allergen histamine releasing activity as it was tested on basophils of OA-sensitive patients. A whole blood leukocyte histamine release was performed by glass fibre based histamine assay. IgE-binding activity of Suc-OA was significantly reduced as it was shown by RAST inhibition technique. Suc-OA stimulated OA-specific T cell hybrid 3DO-548 and ACP:LK35 to produce cytokine release at the same level as in the case of non-modified OA. Succinylation of OA was concluded to result in selective blockade of B cell and preservation of T cell epitopes of the allergen suggesting a new approach for allergen specific immunotherapy.

Design of an optimally-diagnostic skin test solution for diagnosis of sensitivity to timothy grass (Phleum pratense) pollen.

BACKGROUND: Although most of the common allergen extracts that are used for diagnosis of type 1 hypersensitivity are now well standardized, this gives no assurance that they are within the concentration range that gives the best chance of a true diagnosis. OBJECTIVE: The objective of this study was to identify the most appropriate concentration range of timothy grass pollen Phleum pratense extract to diagnose sensitivity to this pollen correctly through skin-testing. METHODS: Dilutions of a well-standardized extract were made and used to skin test "true' positive and "true' negative populations of subjects as identified by case history, challenge tests and radioallergosorbent test (RAST). Weal diameters were measured and the data were submitted to receiver operating characteristics (ROC) analysis. For any particular weal size cut-off, the optimal diagnostic concentration (ODC) range was thus calculated. RESULTS: A 3 mm weal diameter cut-off was chosen as an appropriate size for routine diagnosis. Therefore the ODC range at this diameter was used to establish a product target concentration and specification for formulation of the diagnostic reagent. This method of allergen extract standardization can lead to a true-biological unitage that can be used for labelling purposes. CONCLUSION: The optimum concentration range at which to formulate an allergen extract, in terms of an in vitro immunologically based assay, can be determined by carrying out ROC analysis of the results of clinical studies as described in this communication. Diagnostic units (DU), are now used by us for labelling of such final formulations which conveys the information that the product is at the most appropriate concentration for diagnosis.

Immunological properties of allergen chemically modified with synthetic copolymer of N-vinylpyrrolidone and maleic anhydride.

Several conjugates of model allergen ovalbumin (OA) and the copolymer of N-vinyl pyrrolidone and maleic anhydride (VMA) modified with epsilon-aminocaproic acid (Acp) were prepared in different OA/Acp-VMA ratios. All conjugates were separated by ultrafiltration and analyzed by HPLC. Their compositions were determined by amino acid analysis and UV spectrometry. To detect immunogenicity, all conjugates were injected intraperitoneally into (CBAxC57BL/6)F1 mice three times in 3-week intervals in OA doses equivalent to 0.5, 10, and 100 micrograms/mouse. Only the conjugate containing 20%OA (OA(20%)-Acp-VMA) did not induce significant quantities of anti-OA IgE, but did induce anti-OA IgG antibodies in dose-dependent manner comparable to that of unmodified OA. Mixtures of OA and Acp-VMA or OA modified only with VMA without Acp activation with Acp induced dose-dependent anti-OA IgE and IgG antibody formation comparable to that of OA. Using passive cutaneous anaphylaxis, RAST inhibition and leukocyte histamine release, a significant reduction of allergenicity was noted using OA(20%)-Acp-VMA. This conjugate stimulated activation of the OA-specific T-cell hybrid 3DO-548 comparable to that of unconjugated OA. During experimental allergen-specific hyposensitization with OA(20%)-Acp-VMA, suppression of anti-OA IgE response and elevation of anti-OA IgG responses were noted when compared with unmodified OA. Selective blockade of B-cell epitopes of allergen may occur using the carrier Acp-VMA to reduce allergenicity while not affecting T-cell epitopes, thereby preserving immunogenicity. This approach of chemical modification of allergen suggests new opportunities in the creation of preparations for allergen-specific immunotherapy.

Grass pollen specific antibody in the plasma of normal dogs.

An investigation carried out with healthy beagle dogs showed that their plasmas contained detectable levels of grass pollen specific IgG antibody, as measured by a radiometric assay involving the binding of IgG to radio-iodinated staphylococcal protein A. These samples did not, however, contain detectable homocytotropic-type antibodies specific for grass pollen extract.

Pollinex Parietaria (Bencard Parietaria), a new allergoid for treatment of patients sensitive to Parietaria pollen.

Two matching groups each of eleven patients suffering from allergy to Parietaria pollen were treated either with tyrosine-adsorbed glutaraldehyde-modified extract of Parietaria judaica pollen (Bencard Parietaria/Pollinex Parietaria) or with alum-adsorbed pyridine-extract (Alavac). The side effects of therapy were similar in both groups and were mostly local in nature. Nasal symptoms were significantly less at the end of treatment in the group of patients treated with Pollinex. P. judaica-specific IgG levels were significantly higher in patients following treatment with Pollinex. The majority of patients in both groups showed reduced nasal and/or skin sensitivity following therapy as measured by provocation testing. The results indicate that Pollinex Parietaria is an effective vaccine for the treatment of immediate hypersensitivity to Parietaria pollen.

Hyposensitization therapy of Parietaria-sensitive patients with a tyrosine adsorbed allergoid, Pollinex Parietaria (Bencard Parietaria).

Thirty patients suffering from allergy to Parietaria pollen were treated with either a new tyrosine-adsorbed allergoid of Parietaria judaica pollen (Pollinex Parietaria) or a commercially available alum-adsorbed extract (Alavac) as control. A reduced response to nasal provocation was seen in 7 out of 11 patients following treatment with Pollinex and 1 out of 10 after control treatment. 9 out of 11 and 3 out of 10, respectively, showed reduced skin test activity. Patients who received Pollinex tended to have fewer nasal symptoms during the pollen season. Pollinex induced larger increases in P. judaica-specific IgG antibody than did the control product. Side effects of therapy were similar between the two groups of patients. Pollinex Parietaria thus shows good potential for the control of allergy to Parietaria pollen.

IgE-mediated sensitization to English plantain pollen in seasonal respiratory allergy: identification and partial characterisation of its allergenic components.

Characterisation by SDS-PAGE immunoblotting of plantain pollen extract showed that components of 16,000-20,000 M(r) were frequently reactive with IgE antibody in the sera of subjects with seasonal respiratory allergy. Other, more weakly IgE-binding allergens were seen in the range of 40,000-60,000 M(r). HPLC followed by RAST inhibition demonstrated that components of approximately 17,000 M(r) were also responsible for much of the IgE-binding activity of the extract. These components appeared to have pI values between 4.5 and 5.2. RAST inhibition showed that there were no common IgE-binding epitopes in grass pollen and plantain pollen extracts, indicating that skin test responses should not necessarily be interpreted in terms of cross-reaction. 82 subjects with a clinical history of seasonal, respiratory allergy were screened in a skin prick test survey. 28% were skin test positive to plantain pollen extract. The frequency of positive skin test reactions to plantain pollen extract was greater than that to Betula (23%) and Artemisa (16%), both which are considered to be important allergens. In a larger survey positive RAST scores to plantain pollen were given by 34% of sera from subjects with respiratory allergy. Plantain pollen sensitivity should therefore be considered during diagnosis of seasonal allergy.