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Results WP5: Translational Research

Biomarkers In CVID

The use of b2MG and sIL2R as well as free light chains as biomarkers were evaluated retrospectively in a cohort of 100 CVID patients. Both b2MG and sIL2R correlate highly significant with the appearance of splenomegaly, lymphadenopathy, autoimmune disease and granulomatous disease. At the same time there is a significant association with cellular alterations of B and T cells most pronounced in CVID Ia patients. The value of these markers at the time of malignant transition in CVID patients with secondary lymphoma is under current evaluation.

Definition of protective role of IgM memory B cells and IgM anti PnPS antibodies in PAD

We defined that IgM anti PnPS antibodies in PAD correlate with the capacity to differentiate into plasmablasts. The residual capacity to produce IgM anti PnPS antibodies correlated with the risk to develop pneumonia and chronic lung disease. B cell subsets and their functional development have been also analysed in 74 newborns from birth to 6 months of life. Moreover, we evaluated natural antibody production in vitro. The results documented a predominance of naive B-lymphocytes at all time-points evaluated, decreasing from birth to 6 months (p=0.009). The percentages of CD27+IgD+ and CD27+IgDneg memory B-cells were very low at birth and significantly increased only at 6 months (p=0.02 and o less than 0.001, respectively). We found a significant increase only in in vitro stimulated IgG production at 6 months as compared to birth (p less than 0.001). Moreover, a lower secretion of anti-Pn IgM antibodies up to 6 months of age, as compared to controls was observed. Our results underline that the susceptibility and severe course of infection in the neonate can be attributed, at least in part, to the lack of pre-existing immunological memory and competent adaptive immunity.

The role of IgM memory B cells in the protection from encapsulated bacteria

In this project we evaluated B-cell subsets and their functional development in 74 newborns from birth to 6 months of life. Moreover, we evaluated natural antibody production in vitro. The results documented a predominance of naive B-lymphocytes at all time-points evaluated, decreasing from birth to 6 months (p=0.009). The percentages of CD27+IgD+ and CD27+IgDneg memory B-cells were very low at birth and significantly increased only at 6 months (p=0.02 and p less than 0.001, respectively). We found a significant increase only in in vitro stimulated IgG production at 6 months as compared to birth (p less than 0.001). Moreover, a lower secretion of anti-Pn IgM IgM antibodies up to 6 months of age, as compared to controls was observed. Our results underline that the susceptibility and severe course of infection in the neonate can be attributed, at least in part, to the lack of pre-existing immunological memory and competent adaptive immunity.

Definition of B cell stimulation as prognosis indicator in PAD

Reduced proliferative response to CpG correlates to increased lung infection rate. Due to reduced proliferative response, CpG induced plasmablast differentiation is reduced in patients with CD21low cells to <10%.

Development of prototype ELISA for IgM anti PnPs response

The aim of the subproject is the development of a reliable and easy-to-use ELISA for anti-pneumococcal polysaccharide (PnPS) antibodies of IgM isotype. The assay is intended to assess the function of B cells in patients with antibody deficiency syndrome in vivo. The project was conducted in co-operation with our industrial partner, The Binding Site (TBS), Schwetzingen, Germany. N = 20 volunteers were vaccinated with a 23 valent pneumococcal polysaccharide vaccine (Pneumovax©). Subsequently, serum and cellular components were obtained at 8 time points within 2 months. The vaccination study was performed to i) determine the optimal time point for assessment of the anti PnPS-IgM-antibody contents required for the ELISA development. The anti-PnPS-IgG-ELISA was developed on the basis of a commercial anti-PnPS-IgG-ELISA provided by our industrial partner TBS. The assay was optimised for sensitivity and retest-reliability by serial variations of IgG removing agents and crossreactive antibodies binding substances. The ELISA is now ready for definitive measurements of the IgM immune response of the healthy vaccines. The antibody immune response can be compared to the detection of PnPS specific B-cells at the same time points.

Evaluation of IgM anti PnPS response in PAD patients after vaccination

The serum level of IgM antibodies against different pneumococcal serotypes (type 1, 3, 4, 14) has been analysed according to the D. Goldbatt (WHO) recommendations before and after vaccination with unconjugated pneumococcal vaccine. In cooperation with The Binding Site (Germany) an easy-to-use ELISA for anti-PnPS antibodies of IgA and IgM isotype has been developed. This test is based on a commercially available 23-valent anti-PnPS ELISA for IgG antibodies (Pneumococcal Capsular Polysaccharide (PCP) Assay, The Binding Site). Outcome: A good correlation of the IgM 23-valent anti-PnPS with each single serotypes IgM titre (type 1, 3, 4, 14) was observed. The serum level of specific antibody IgM and IgA against single serotypes (type 1, 3, 4, 14) and the IgM and IgA 23-valent anti-PnPS have been correlated with the B lymphocyte subsets (according to the IPIDNET panel). A response to at least two serotypes, doubling the IgM or IgA level was considered a good response to vaccination. Based on this definition, a good IgM response was significantly related to higher numbers of switched memory B cells and IgM memory B cells and with lower levels of CD21low B cells. Instead a good IgA response to vaccination was significantly related to higher numbers of switched memory B cells and with lower levels of CD21low B cells. The clinical evaluation included the clinical history (frequency of pneumonias), the chest CT scan for the detection of bronchiectasis and other structural changes and the pulmonary and the pulmonary function test (FEV1 of less than 80%[predicted]) over a period of 36 months. An inverse correlation was found between the response to the vaccination and the presence of bronchiectasis at the chest CT scan. Conversely, no significant correlation was found between the response to immunisation and the pulmonary function test (FEV1). Observing the distribution of the response to vaccination with specific IgA and IgM 23-valent anti PnPS production, a cut-off level has been suggested in order to define a response to vaccination in subjects with PAD: IgM >20 U/mL and IgA>0.15 ug/mL. Based on this definition, three groups of patients have been described: Patients with good IgM and IgA response (complete responders), patients with no response at all (non-responders) and patients with IgM response only (IgM only). The latter group of patients (IgM only) has defined intermediate level of switched memory B cells (lower than complete responders but higher than non-responders) and lower numbers of CD21low B cells than complete responder patients. Comparing the response to vaccination with IgA and IgM 23-valent anti-PnPS production to the clinical findings, a stronger inverse correlation was found between the complete response to the vaccination and the frequency of pneumonia and between the response to the vaccination and the presence of bronchiectasis at the chest CT scan, while an intermediate inverse correlation was observed in the IgM only group.

Assessing antibody response in patients with hypogammaglobulinaemia by ELISPOT

Hypogammaglobulinaemic patients are often started on immunoglobulin substitution therapy before antibody production is adequately evaluated. In such a situation, it is difficult to segregate transferred from antigen-induced specific antibody. Therefore we characterised changes in B cell subpopulations in hypogammaglobulinaemic patients, including plasmablasts, in peripheral blood by flow cytometry after in vivo antigen challenge. We investigated the specificity of antibody production on the B cell level by ELISPOT, which is independent of substitution therapy. Hypogammaglobulinaemic patients and healthy volunteers were immunised with Tetanus toxoid (T-cell dependent) and pneumococcal polysaccharide (T-cell independent) vaccines. Specific antibody levels were measured by ELISA. ELISPOT was used before and on day 7 after vaccination. B-cell subpopulations were examined by flow cytometry. In the control group, an increase in circulating plasmablasts (IgD-CD27++, IgM-CD38++ gated from CD19+ B cells) on day 7 post immunisation corresponded with the appearance of spot forming cells. In contrast, patients with Common Variable Immunodeficiency (CVID) who are characterised by hypogammaglobulinaemia and impaired antibody production failed to increase plasmablasts significantly in peripheral blood after antigen challenge. Our observation that the majority of CVID patients lack antigen specific spot forming B cells and fail to increase circulating plasmablasts following in vivo challenge provides a rapid screening test to demonstrate defective antibody responses in CVID patients. Clinical Implications and Conclusion: Identification of circulating plasmablasts after vaccination is a new simple flow based test to assess antibody responses in hypogammaglobulinaemic patients, even if on immunoglobulin (IVIG or SCIG) replacement therapy.

Development of an IgM-specific anti PnPS ELISA based on 23 serotypes from S. pneumonia and assessment of the IgM and IgG anti PnPS antibody response after vaccination with pneumococcal unconjugated vaccine in PAD patients

As an immunodiagnostic company the role of The Binding Site in the EUROPADnet project was to translate the observations from primary antibody deficient (PAD) patients into a medical device so the data and results could be utilised in patient care. Since there is a high risk of bacterial infections in patients suffering from Commmon Variable Immunodeficiency (CVID) we supported the investigation for the hypothesis that IgA and IgM responses to pneumovax may improve the determination of the risk of pulmonary morbidity in PAD patients. Along with the study group we have facilitated the development of both PCP IgM and IgA ELISAs. The assays have been validated and the IgM and IgA responses to pneumovax have been determined in normal healthy subjects. The development of both PCP IgM and IgA ELISAs has involved the identification, location and preparation of suitable materials. The optimisations of assay conditions and clinical validation have been achieved in collaboration with the study group. Both assays have been initiated as new products and entered into the New Product Development system at the Binding Site. This process involves full product specification and commercial justification established through the EUROPADnet group. Working with the same team of experts we are currently evaluating evaluating the measurement of both the IgA and IgM responses to pneumovax using these ELISAs in broader and wider clinical applications such as in patients diagnosed with IgA deficiency and those with combined IgA/IgG subclass deficiencies.

Predicting the risk of infusion reactions to anti-IgA antibodies

The occurrence of anti-IgA antibodies at high titre is a risk factor associated with anaphylactoid reaction in hypogammaglobulinaemic patients. The presence or absence of anti-IgA antibodies is also considered important basic information in the European ESID database of primary immunodeficiencies. Screening for anti-IgA antibodies in patients lacking IgA may be helpful for determining the best therapeutic strategy, including the route of administration of immunoglobulin substitution therapy or the transfusion of IgA-free blood products. From a haematological point of view, tested IgA-deficient blood components without anti-IgA antibodies are a precious resource that should be allocated with care and reserved for IgAD patients at risk of anaphylactoid reaction. The combined use of quantitative ELISA for detection of anti-IgA antibodies and the measurement of serum IgA concentration by nephelometry provides an effective and safe strategy for the diagnosis and prevention of IgA anaphylactic transfusion reactions. We found a significant amount of anti-IgA antibodies in 10% of patients with the most common primary hypogammaglobulinaemia (selective IgA deficiency and common variable immunodeficiencies) where the serum IgA level was lower than 0.05g/L. Analysis of anti-IgA response in PAD patients and the assessment of biomarker predicting the risk of infusion reaction in PAD demonstrated that Common Variable Immunodefiency (CVID) patients who are seropositive for anti-IgA antibodies have a predisposition for anaphylactoid reactions to intravenous immunoglobulin replacement therapy (IVIG). A new anti-IgA ELISA has been established www.biovendor.com

Development of a combined clinical, phenotypic and functional classification of PAD

This subproject represents the main translational research target of WP5 and might be completed in the following months. This is a final goal of WP5. Earlier than anticipated, and thanks to the sterling work of EUROPADnet, the new classification of PAD is already in progress. Major changes of the new proposal compared to the current criteria present the definition of absolute immunoglobulin levels as cut off, the definition of vaccine responses, a more precise definition of exclusion criteria especially defining patients with combined immunodeficiency.