WO2009070122A1

WO2009070122A1 - A novel method for assessing if symptoms in a patient are related to a hypersensitivity to a specific allergen

Info

Publication number: WO2009070122A1
Application number: PCT/SE2008/051385
Authority: WO
Inventors: Lars Söderström; Staffan Ahlstedt; Per Matsson; Adnan Custovic
Original assignee: Phadia Ab
Priority date: 2007-11-30
Filing date: 2008-12-01
Publication date: 2009-06-04
Also published as: EP2238457A1; EP2238457A4; JP2011505557A

Abstract

The present invention relates to the field of allergic diseases, and especially to methods for assessing if symptoms in a patient are related to a hypersensitivity to a specific allergen. Furthermore the invention relates to a computer program product comprising means for controlling the steps of said methods for use with an apparatus for analysis of binding assays. The invention also relates to such an apparatus.

Description

A NOVEL METHOD FOR ASSESSING IF SYMPTOMS IN A PATIENT ARE RELATED TO A HYPERSENSITIVITY TO A SPECIFIC ALLERGEN

Field of the invention

The present invention relates to the field of allergic diseases, and especially to methods for assessing if symptoms in a patient are related to a hypersensitivity to a specific allergen.

Background

It is well accepted that allergen specific IgE antibodies are associated to allergic disease when the individual is exposed to the allergen. We have previously demonstrated that the absolute specific IgE antibody levels offer more information about the relationship between IgE-mediated sensitization and respiratory symptoms than just the presence of specific IgE. We found total IgE to be a poorer predictor of wheeze than the sum of specific IgEs. These data suggested that labelling subjects as sensitized or not based on an arbitrary cutoff is an oversimplification of a trait that is not dichotomous in its relationship to wheeze. In certain situations the association is less, particularly after strong stimulation of the immune system such as after immunotherapy. Furthermore, after strong exposure to domestic animals, particularly cats, the association is less pronounced. The explanation for this has been that heavy exposure may overload the IgE stimulation and turn it to an alternative Th2 response with production of more IgG₄ antibodies and less IgE antibodies. This further accentuates the view that dichotomous use of IgE antibodies as an all or nothing phenomenon is an oversimplification.

Summary of the invention

In a first aspect, the present invention relates to a method for obtaining a parameter for assessing if symptoms in a subject are related to a hypersensitivity to an allergen or allergen component, comprising the steps - obtaining from said patient a body fluid sample containing immunoglobulins; - determining the amount in the sample of a first type of immunoglobulin binding specifically to said allergen or allergen component;

- determining the amount in the sample of a second type of immunoglobulin binding specifically to said allergen or allergen component; and

- relating the amount of said first type of immunoglobulin to the amount of said second type of immunoglobulin in said sample.

In a preferred embodiment of the invention, said first and second types of immunoglobulins are selected from the group consisting of IgA, IgD, IgE, IgG and IgM, such as IgG or IgE or subtypes thereof, such as IgG4.

In a preferred embodiment of the invention, the step of relating the amount of said first type of immunoglobulin to the amount of said second type of immunoglobulin in said sample comprises the formation of a ratio between the amounts of said first and second immunoglobulin, respectively.

In one embodiment of the invention, the amount of said first and/or second type of immunoglobulins, respectively, is a sum of serum immunoglobulins.

The allergen is preferably a respiratory allergen and may come from a number of allergen sources such as animals and plants, e.g. cat, cockroach, dog, mouse, grass pollen, tree pollen, horse, house dust mites, moulds and/ or olive pollen.

The symptoms may be any allergy like or allergy related symptoms , such as persistent wheeze, asthma, rhinitis, rhino-conjunctivitis, eczema etc.

In a second aspect the invention relates to a method for assessing if symptoms in a subject are related to a hypersensitivity to an allergen, comprising the steps

- obtaining a parameter according to the method according to the first aspect; - assigning a probability for hypersensitivity to said allergen for said patient by relating the obtained value of said parameter to a predetermined correlation between said parameter and hypersensitivity to said allergen.

In a third aspect, the invention relates to a method for assessing if symptoms in a subject are related to a hypersensitivity to an allergen, comprising the steps

- obtaining a parameter according to the method according to the first aspect - comparing the value of the obtained parameter to at least one previously determined value of said parameter, which previously determined value is in turn coupled to a probability score for the symptoms to be related to a hypersensitivity to the allergen; and

- assessing the probability for the symptoms to be related to a hypersensitivity as higher or lower than the probability score coupled to said previously determined value, based on the comparison between the obtained value and the previously determined value for said parameter.

The subject in the above mentioned aspects may be either a human or an animal.

The determination of amounts of immunoglobulins in the sample from the subject may be done by conventional methods used in the art, e.g. by ImmunoCAP (Phadia AB, Uppsala, Sweden), immunoassays such as ELISA, microarrays etc.

In a fourth aspect the invention relates to computer program products directly loadable into the internal memory of a processing means associated with an apparatus for analysis of binding assays, comprising the software code means adapted for controlling the steps of the method according to the first or second aspect. In a fifth aspect the invention relates to an apparatus for analysis of binding assays, said apparatus being adapted to perform the method according to the first or second aspect and comprising

- means ( 10) for receiving a value representing an amount of a first type of immunoglobulin binding specifically to an allergen;

- means (1 1) for receiving a value representing an amount of a second type of immunoglobulin binding specifically to said allergen; and

- means (20) for relating the amount of said first type of immunoglobulin to the amount of said second type of immunoglobulin in said sample; and

- means (30) for communicating the relation between said first and second type of immunoglobulin to a user.

A schematic drawing of such an apparatus is given in Figure 5. The above described means should typically be regarded as software defined functional means in the digital processing parts of the apparatus, i.e. not necessarily physical entities. The apparatus may be incorporated into one physical apparatus, such as a desk top computer, but may also be distributed over a number of physical apparatuses, such as servers, located at different locations.

Brief description of the figures

Figure 1. Fitted predicted probability curve for persistent wheeze at a given cat-specific IgE value and FeI d 1 -specific IgG value derived from the multivariate logistic regression analysis. Higher IgE antibody level to cat is associated with higher probability of allergic disease, wheeze or asthma, in the presence of low IgG antibody level. Higher IgG antibody level decrease this association.

Figure 2. Fitted predicted probability curve for persistent wheeze at a given cat-specific IgE value and FeI d 1 -specific IgG4 value derived from the multivariate logistic regression analysis. Higher IgE antibody level to cat is associated with higher probability of allergic disease, wheeze or asthma. Higher IgG4 antibody level does not change this association.

Figure 3. Fitted predicted probability curve for the association between cumulative FeI d 1 exposure during the first five years of life, cat-specific IgE value and FeI d 1 -specific IgG value derived from the multivariate logistic regression analysis. Higher exposure to cat is associated with higher level of IgE and IgG antibody.

Figure 4. Fitted predicted probability curve for the association between cumulative FeI d 1 exposure during the first five years of life, cat-specific IgE value and FeI d 1 -specific IgG4 value derived from the multivariate logistic regression analysis. Higher exposure to cat is associated with higher level of IgE and IgG4 antibody.

Figure 5: Schematic drawing of an apparatus according to the fifth aspect of the invention.

Definitions Allergen source should in the context of the present invention be construed as the organism from which an allergen is derived, such as cat, dog, birch etc.

Allergen should in the context of the present invention be construed as a hyposensitizing environmental agent derived from an allergen source, such as an extract from grass pollen or dog dander etc.

Allergen component should in the context of the present invention be construed as an isolated or recombinantly produced molecule being a component of an allergen, such as FeI d 1 from cat, PhI p 1 from timothy grass etc.

Detailed description of the invention

The invention is further explained in the following example wherein the allergen is derived from cat, the immunoglobulins are IgG, IgG₄ and IgE, and the symptoms related to hypersensitivity is wheeze. This example shall not be considered as limiting the scope of the invention, which is that defined by the appended claims.

Examples

Example 1

METHODS Study Population The Manchester Asthma and Allergy Study is an unselected population-based birth cohort study described in detail elsewhere (12, 13). Subjects were recruited from the antenatal clinics when all pregnant women (first trimester of pregnancy) were screened for eligibility. Both parents completed a questionnaire and skin prick testing. Children were followed prospectively and attended review clinics at age three and five years (±four weeks). The study was approved by the Local Research Ethics Committee. Written informed consent was obtained from subjects' families, and children gave their assent.

Although retention in this population-based observational birth cohort is excellent, we analyzed data from approximately half of the subjects (mostly because the child refused venepuncture) . We emphasize that there was no difference between children excluded or included in the analysis in any relevant parameter. Furthermore, the prevalence of allergic sensitization amongst the parents of the children is similar to that of young adults in the UK²¹, suggesting the subjects are representative of the general population.

Outcomes

Symptoms: A validated questionnaire (14) was interviewer-administered to collect information on parentally reported symptoms, physician-diagnosed illnesses and treatments received. Children were defined as having current wheeze if they had wheezing in the previous 12 months at age five years. In addition, we assigned children into different wheeze phenotypes according to history of wheeze at the two follow-ups (15): No wheezing -no wheeze during the first three years of life, no wheezing ever by age five;

Transient early wheeze -wheezing during the first three years, no wheezing in the previous 12 months at age five; Late-onset wheezing -no wheeze during the first three years, wheezing in the previous 12 months at age five;

Persistent wheezing -wheezing during the first three years, wheezing in the previous 12 months at age five.

Antibody measurement

Allergen specific antibody levels were determined using ImmunoCAP™ assay (Phadia AB, Uppsala, Sweden) according to the manufacturer. For the IgE antibody determinations the commercially available reagents of whole allergen extract for mite and cat were used. Consistency, reproducibility and precision of the assays are reported to be very good (18).

For the specific IgG antibody measurements pure rFel d 1 Allergen ImmunoCAP were used. rFel dl was produced by Phadia AB and subsequently coupled to the solid phase of flexible, hydrophilic, cellulose sponge polymer matrix activated by CNBr chemistry. The purified components were covalently coupled to the activated solid phase via the amino groups of the proteins according to standard in house methodology as previously described (18).

Environmental exposures

We visited homes immediately after birth and at the age of 3 years and 5 years. Dust samples from the child's bed, the child's bedroom floor, the parental bed and the lounge floor were collected on each occasion by vacuuming 1 m² areas for 2 minutes in a standardised fashion. FeI d 1 was assayed using enzyme- linked immunoassays as previously described (21). The results were expressed as allergen concentration (μg/g). We estimated cumulative allergen exposure over the first 5 years of life as a sum of allergen levels in four sites at three time points (i.e. 12 separate measurements of allergen exposure). Statistical methods

The primary outcome measure was "Persistent wheeze" at age five years. The relationship between antibody responses and outcome measure was analyzed using logistic regression. Odds ratios (OR) were estimated using the regression models and 95% confidence intervals (95% CI) were generated according to

WaId, using a p-value of 0.05 as significant. Fitted predicted probability curves were plotted using the results from the logistic regression. The levels of specific antibodies were subject to a logarithmic transformation prior to analysis; OR are presented for different antibody levels expressing the increased or decreased risk associated with increasing antibody levels. Since a logarithmic transformation was used all calculations were done on the logarithmic scale, i.e. the OR was estimated as exp(r*b) and r, the distance between a certain antibody level and the level indicating the absence of antibody, was defined as r=ln(x2)-ln(xl). The relationship between FeI d 1 exposure and antibody levels was ascertained using linear regression analysis. Computerized statistical analysis was carried out using SAS System V8.01.

RESULTS

Of 121 1 couples who initially agreed to take part, 1085 had a successful full- term pregnancy (>36 weeks gestation; child did not need intensive care) and gave consent to a further follow-up. Of those, 128 were prenatally randomized to environmental control (19, 20) (excluded from this analysis), and 957 children were followed in the observational cohort. Of the children in the observational cohort, 840 (87.8%) attended the 5 year follow-up. AU of these children had questionnaire data, and 540 provided blood sample for antibody measurement (62%).

Children excluded did not differ from those included in terms of family history, parental smoking, maternal age, socioeconomic status, gestational age, birth weight, history of wheeze and skin test results. Current wheeze at age five years was reported by the parents of 1 14 children

(21.9%); 257 children never wheezed, 107 had transient early wheezing, 26 late- onset wheezing and 70 persistent wheezing (61 children could not be classified into one of the four wheeze phenotypes) . Multivariate

However, this effect was significantly modified by FeI d 1 -specific IgG antibodies. The magnitude of the effect of specific IgE decreased with increasing FeI d 1 - specific IgG; Figure 3. For example, predicted probability of persistent wheezing with cat IgE of 50kUA/L was 0.82, corresponding to an OR of 7.4 (95% CI 5.9 - 9.1) when IgG to FeI d 1 was low, 2 mg/L; this decreased to an OR of 2.5 (95% CI 1.5 - 4.4) when the IgG antibody level to FeI d 1 increased to 6mg/L. FeI d 1- specific IgG4 did not modify the relationship between the level of cat-specific IgE and persistent wheezing (Figure 4) .

DISCUSSION

It is well known that exposure to allergen can lead to IgE, IgG and IgG4 antibody production. In the susceptible individual the IgE antibody level is mostly associated with clinical disease triggered by exposure to the allergen. However, it is also well known that extensive exposure can lead to IgG and IgG4 antibody formation but no expression of disease. The present invention establishes quantitatively the negative impact of IgG, but not IgG4, antibody on the association between the presence of IgE antibody and clinical disease.

Claims

1. Method for obtaining a parameter for assessing if symptoms in a subject are related to a hypersensitivity to an allergen or allergen component, comprising the steps

- obtaining from said patient a body fluid sample containing immunoglobulins;

- determining the amount in the sample of a first type of immunoglobulin binding specifically to said allergen or allergen component;

2. Method according to claim 1 , wherein said first and second types of immunoglobulins are selected from the group consisting of IgA, IgD, IgE, IgG and IgM and subclasses thereof.

3. Method according to claim 2, wherein said first and second types of immunoglobulins are selected from the group consisting of IgG and IgE.

4. Method according to any of claims 1-3, wherein one of said first and second types of immunoglobulins is of subtype IgG₄.

5. Method according to any of claims 1-4, wherein the step of relating the amount of said first type of immunoglobulin to the amount of said second type of immunoglobulin in said sample comprises the formation of a ratio between the amounts of said first and second immunoglobulin, respectively.

6. Method according to any of claims 1-5, wherein the amount of said first and/or second type of immunoglobulins, respectively, is a sum of serum immunoglobulins .

7. Method according to any of claims 1-6, wherein the allergen or allergen component is a respiratory allergen or allergen component.

8. Method according to any of claims 1-7, wherein the allergen or allergen component is derived from an allergen source selected from the group consisting of animals and plants, such as cat, cockroach, dog, horse, mouse, grass pollen, tree pollen, , olive pollen, house dust mites and moulds .

9. Method according to any of claims 1-8, wherein the symptoms are selected from the group consisting of persistent wheeze, asthma, rhinitis, rhino- conjunctivitis, eczema or other allergy related or allergy like symptoms..

10. Method for assessing if symptoms in a subject are related to a hypersensitivity to an allergen, comprising the steps

- obtaining a value for a parameter according to the method according to any of claims 1-9;

- assigning a probability for hypersensitivity to said allergen for said patient by relating the obtained value of said parameter to a predetermined correlation between said parameter and hypersensitivity to said allergen.

1 1. . Method for assessing if symptoms in a subject are related to a hypersensitivity to an allergen, comprising the steps

- obtaining a parameter according to the method according to any of claims 1-9

- comparing the value of the obtained parameter to at least one previously determined value of said parameter, which previously determined value is in turn coupled to a probability score for the symptoms to be related to a hypersensitivity to the allergen; and

12. Computer program product directly loadable into the internal memory of a processing means associated with an apparatus for analysis of binding assays, comprising software code means adapted for controlling the steps of the method according to any of claims 1-1 1.

13. Apparatus for analysis of binding assays, comprising