Assessment of the Humoral Immune System in Adults with Respiratory Tract Disease

Assessment of the humoral immune system in adults with respiratory tract disease

Diana van Kessel Assesment of the humoral immune system in adults with respiratory tract disease D.A. van Kessel

Thesis University Utrecht, the Netherlands ISBN: 978-94-90329-35-8 NUR: 870

Cover illustration: Henk ten Have, Zoelen, the Netherlands Cover design and lay-out: emjee | grafische vormgeving, Varik, the Netherlands Print: Veldhuis Media BV, Raalte, the Netherlands

© 2017, D.A. van Kessel All rights are reserved. No part of this publication may be reproduced without written permission of the author. The copyright of articles that already have been published has been transferred to the respective journals. Assessment of the humoral immune system in adults with respiratory tract disease

Evaluatie van het humorale afweersysteem bij volwassenen met een aandoening van de ademhalingswegen

(met een samenvatting in het Nederlands)

Proefschrift ter verdediging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. G.J. van der Zwaan, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op dinsdag 7 november 2017 des middags te 12.45 uur

door

Dirkje Anna van Kessel

geboren op 13 december 1955 te Culemborg Promotoren: Prof.dr. J.C. Grutters Prof.dr. G.T. Rijkers

Copromotor: Dr. P. Zanen Paranimfen: Thijs Hoffman Joost Jacobs Dedicated to Frans Jozef†, Isabelle en Joost Jacobs Table of contents

Chapter 1: General introduction, aims and outline of the thesis 3

Part I: Immunological screening of patients with 21 recurrent respiratory tract infections Chapter 2: Response to pneumococcal vaccination in mannose- 23 binding lectin-deficient adults with recurrent respiratory tract infections Chapter 3: Clinical and immunological evaluation of patients 39 with mild IgG1 deficiency Chapter 4: Impaired pneumococcal antibody response in patients 53 with bronchiectasis of unknown aetiology

Part II: Immune status in patients before and after lung 71 transplantation Chapter 5: Immune status assessment in adult lung transplants 73 candidates Chapter 6: Long-term follow up of humoral immune status in 85 adult lung transplant recipients

Part III: Antibody replacement therapy in patients with 101 humoral immunodeficiency Chapter 7: Antibody replacement therapy in primary antibody 103 deficiencies and iatrogenic hypogammaglobulinemia Chapter 8: Long-term clinical outcome of antibody replacement 131 therapy in humoral immunodeficient adults with respiratory tract infections

Part IV: Remarkable clinical cases 153 Chapter 9: Defective antibody response against pneumococcal 155 polysaccharide serotype 9V in a patient with a single episode of pneumonia Chapter 10: An unusual presentation of severe 163 hypogammaglobulinemia Chapter 11: Summary and general discussion 181

Chapter 12: Nederlandse samenvatting 195

Dankwoord 209 Curriculum vitae 213

CHAPTER 1

General introduction, aims and outline of the thesis

General introduction, aims and outline of the thesis 5

1. Introduction

The lung is a vital organ that is in constant contact with the outside world. This causes life-long continuous exposure to potential pathogenic microorganisms. Recurrent respiratory tract infections are a common complaint in the daily clinical practice at the outpatient pulmonology department. This can have various causes but importantly it is also the most common presentation of a primary immunodeficiency.1-4

Long-term complications of primary immunodeficiency include bronchiectasis, immune-mediated diseases such as granulomatous-lymphocytic interstitial lung disease and lymphoma5-7 It is therefore important for the clinician to recognize this possible cause of recurrent respiratory tract infections, so that treatment can be started early and complications can be prevented.

Normally, there are several defence mechanisms that protect the respiratory system against infections.8, 9 The first line of defence is formed by the mucosa itself. This compromises the internal body surfaces that are lined by a mucus-secreting epithelium, including the upper and lower respiratory tract. The mucociliary system of the respiratory tract forms a mechanical, biochemical and biological barrier for microorganisms. The epithelial cells lining the airways can secrete host-defence proteins that increase the efficacy of the immune system in clearing pathogens from the respiratory tract.10

1.1 Immunological defences of the lung

1.1.1 Innate immunity

The immune system can be divided into innate and adaptive immunity (Figure 1). The innate immune system is a fast reacting system, recognizing molecular patterns on microorganisms with limited or no immunologic memory.8 It has a cellular and humoral component. The cellular component consists of phagocytes like monocytes, macrophages and granulocytes. These cells recognize pathogens through pattern recognition receptors on the membrane. The humoral component consists of several circulating proteins like lactoferrin, de- fensins, collectins and an elaborate system of different cytokines, as well as the complement system. 6 Chapter 1

Figure 1. The immune system can be divided into innate and adaptive immunity. On encountering a pathogen, both an innate and an adaptive immune response will be mounted in the host.

The main function of the complement system is to neutralize and/or kill microorganisms.8 The complement system can be activated through 3 pathways: the classical pathway, the lectin pathway and the alternative pathway (Figure 2). The classical pathway is activated by antibodies. Complement factor 1 (C1) binds to antibodies on pathogen surfaces. C1 then activates complement factors further downstream in the complement cascade. The lectin pathway is activated by mannose binding lectin (MBL) and ficolins, both soluble pattern recognition molecules. MBL and ficolins bind to mannose-containing or other carbohydrates on the surface of yeast and encapsulated bacteria, such as pneumococci. The alternative pathway works through low-level spontaneous activation. Spontane- ously activated complement factor C3 can also bind to pathogen surfaces.

Activation of the complement system is mostly through the classical pathway. But, against microorganisms with repetitive carbohydrate structures on their surface, the lectin pathway can also be used for complement activation. The alternative pathway functions mainly as an amplifying loop for the classical and lectin pathways.8 Interestingly, in the MBL2 gene, the gene that encodes the MBL protein, a number of genetic polymorphisms can be found. Some of these variants have functional consequences. Patients carrying these polymorphisms have decreased serum levels of MBL. Polymorphisms leading to very low or absent circulating MBL can be found in 10-25% of the population.11 The reason for General introduction, aims and outline of the thesis 7

the high prevalence of these polymorphisms is not yet known. The association between decreased MBL levels and the risk of infections with encapsulated bacteria is still unclear.12

Figure 2. The complement system can be divided in three pathways. Activation of the complement system can be initiated via the classical, MBL, and alternative pathway. For the classical pathway, when IgG or IgM is bound to surface antigens on microorganisms, a C1q molecule can be recruited to the Fc region of the antibody. C1s and C1r bind to C1q becoming an enzymatic active complex that causes cleavage of C4 and C2 into C4a and C4b and C2a and C2b, respectively. C4b associates with C2a on the pathogen surface, and this complex functions as a C3 convertase. C3 is cleaved into C3a and C3b. C3a is released from the site and act as a soluble factor, whereas C3b, when bound to complement factor B (which is activated by complement factor D), remains bound to the pathogen surface. C3b then activates complement proteins that are further downstream in the complement cascade, causing the for- mation of the so-called membrane-attack complex (MAC). The MAC creates holes in the pathogen membrane, causing death of the pathogen by lysis. The MBL pathway (representing the lectin pathway in this Figure) can be initiated by binding of MBL to mannose containing carbohydrates on bacteria or viruses. Bound MBL then complexes with the MBL-associated serine protease (MASP), which has similar functionality as the C1q-C1r-C1s complex. The MASP-complex can activate C2 and C4, further initiating the same cascade as described above for the classical pathway. The alternative pathway is initiated when a spontaneously activated C3 molecule binds to the surface of a pathogen. The C3b molecule itself can function as a C3 convertase as well, causing low level activity when spontaneously activated and amplifying the response when the classical or lectin pathways are activated. Figure and legend based on Leerboek immuno- logie8 (page 33 and 35). 8 Chapter 1

1.1.2 Adaptive immunity

The cellular component of the adaptive immune system consists of lymphocytes and antigen presenting cells.8 (Figure 3) There are two major groups: T- and B-lymphocytes. These cells are characterized by the presence of T- or B-cell receptors on their surface. These receptors are highly polymorphic, and each T- or B-cell that is produced in the thymus or bone marrow, respectively, has a different receptor. These receptors form the binding sites for antigens, either autoantigens or alloantigens. In the normal selection process within the thymus and bone marrow, most T- and B-cells with receptors that bind autoantigens are selected out, and these will not be released into the circulation. The extreme high variability in T- and B-cell receptors serves to match the (potential) variation in antigens expressed by pathogens that can be encountered by the immune system. In theory there is a high-affinity T- and B-cell receptor for every imaginable antigen.

Figure 3. Humoral and cellular immune responses of the adaptive immune system. The adaptive immune system can be divided in a humoral and a cellular part. The humoral immune response begins with the binding of an antigen to a specific B-lymphocyte. This B-lymphocyte divides and differentiates within the germinal center of a lymph node, and becomes an antibody producing plasma cell. The cellular immune response begins with specific, indirect recognition of an antigen by a T-helper lymphocyte (Th) or a cytotoxic T lymphocyte (Tc). This antigen is present within the molecules of the major histocompatibility complex (MHC) on antigen presenting cells. T-lymphocytes di- vide and differentiate to effector-helper-T-lymphocytes or effector-cytotoxic T-lymphocytes. The- ef fector T-helper lymphocytes produce cytokines. Cytokines are small peptides that regulate immunological processes as phagocytosis- and killing capacity of macrophages. Effector-T-lymphocytes and cytokines amplify activation and differentiation of B-lymphocytes. Effector-cytotoxic-T-lymphocytes recognize virus proteins in the MHC of infected cells causing cell death. Not all cells that participate in a humoral or cellular immune response differentiate into an effector cell. A part of the cells differentiates into memory cells (memory-B-lymphocytes and memory-T-lymphocytes (helper and cytotoxic)). Figure and legend based on Leerboek immunologie 8 (page 11). General introduction, aims and outline of the thesis 9

The T-lymphocytes form the cellular component of the adaptive immune system. After interaction with an antigen-presenting cell, naive T-cells can become activated and develop into effector or memory T-cells. Effector T-cells include helper and cytotoxic T-lymphocytes. Cytotoxic T-lymphocytes can directly induce cellular apoptosis in target cells, whereas T-helper cells produce cytokines that influence cytotoxic T-lymphocyte responses, but can also stim- ulate B-lymphocyte responses. B-lymphocytes are the precursors of antibody-producing cells. When a B-lymphocyte encounters an antigen, it can differentiate into an antibody-producing plasma blast, an antibody producing plasma cell, or a memory B-cell that can become activate upon encountering the same antigen at a later time. The latter cell type is responsible for immunologic memory, together with memory T-cells, and causes the antibody response to an antigen to be faster and more effective when encountering it for a second time. This principle forms the basis for the efficacy of vaccination. The major steps in B-cell development are depicted in Figure 4.

1.1.2.1 Antibodies Antibodies are glycoproteins consisting of four protein chains: two heavy chains and two light chains, which are linked by disulphide bounds.8 The complete antibody molecule is more-or-less Y-shaped. Each antibody molecule has two identical antigen-binding sites, which are formed by the variable regions of the heavy and light chain combined: the V-region. This part of the antibody molecule is also called the Fab region (fragment of antigen binding). The constant part of the antibody molecule, the C-region is less variable than the V-region and is the part that interacts with effector cells and molecules. This part of the antibody is also called the Fc region (fragment constant). In humans, five different classes of antibody molecules can be distinguished. The class of an antibody is determined by the structure of the heavy chain. The five different classes of immunoglobulins are IgM, IgD, IgG, IgE and IgA (Figure 5). Each antibody molecule can bind to specific antigenic determinants, and this is the primary function of the antibody. Upon binding to antigen, the Fc regions of the antibodies then mediate effector functions like fixation of complement and binding (via Fc receptors) to different cell types such as phagocytic cells, lymphocytes, platelets, mast cells and basophils. 10 Chapter 1

Figure 4. B-cell development. In the bone marrow, B-lymphocyte development starts with differentiation of progenitor B cells into pro-B cells. These cells then differentiate into pre-B cells which can be characterized by a rearranged heavy chain which is expressed with a surrogate light chain and the Ig-a and -b proteins in the form of a pre-BCR (B-cell receptor). Upon successful rearrangement of the light chain, pre-B cells differentiate into immature B-cells, which express the complete surface IgM molecule in the context of the BCR. Immature B-cells leave the bone marrow compartment and via transitional B-cells differentiate into naïve mature B-cells, which present both IgM and IgD on their surface. Naïve mature B-cells migrate to secondary lymphoid tissues. Upon encountering specific antigen, the B-cell can be activated, in most cases with the help of T-cells. Activated B-cells either differentiate into short-lived plasma cells that produce IgM antibodies or the activated B-cells migrate to the follicular zone of lymphoid follicles. There they proliferate rapidly and undergo somatic hypermutation. The resulting B-cell clones with the highest antigen specificity are stimulated by follicular dendritic cells and follicular T-cells to proliferate and undergo immunoglobulin class switching. These cells differentiate into either long- lived plasma cells or circulating memory B-cells. Long-lived plasma cells home in the bone marrow and produce antibodies of any class over prolonged periods. Circulating memory B-cells either reside in the circulation or home to the bone marrow. They become active upon renewed antigen stimulation.13 Figure and legend based on Leerboek immunologie 8 (page 14). General introduction, aims and outline of the thesis 11

Figure 5. Major immunoglobulin classes in humans.

Immunoglobulin M normally exists as a pentamer in serum. IgM is the first immunoglobulin made during development and also is the first immunoglobulin produced by B-cells in older children and adults when stimulated by an antigen during a primary immune response. IgM has 10 antigen binding sites due to its pentameric configuration but has low affinity for binding an antigen. Neverthe- less binding to the antigen can be effective, since the avidity can be high because of the multiple binding sites. Phagocytic cells do not have a receptor for the Fc part of IgM. An IgM- antigen complex can however very effectively bind to complement factor C1q and activate the complement cascade. Subsequently this antigen-IgM-complement complex can be phagocytised via complement receptors expressed on phagocytic cells.

Immunoglobulin D is primarily expressed on the cell surface of naive mature B-cells (next to IgM) as antigen receptor but is also present in low concentrations in serum. The antibody functions of IgD are not very clear but IgD is thought to support mucosal immunity in the lungs, and is also capable of activating other cells of the immune system, such as basophil granulocytes.14

Immunoglobulin G is the most abundant immunoglobulin in blood (> 75%). IgG is found in monomeric form in blood and is capable of carrying out many different effector functions. IgG can be divided in four subclasses with different Fc regions, each having their own function. The subclasses are numbered in order of the quantities that are present in normal serum. IgG1 is the dominant subclass in the antibody response to soluble and membrane protein antigens and the most abundant subclass in healthy individuals. IgG2 is the predominant antibody that is produced in response to bacterial capsular polysaccharide antigens. IgG3 antibodies are effective in the induction of effector functions such as complement activation. IgG3 antibodies are mostly produced in combination with other subclasses. IgG4 antibodies are also mostly produced in combina- 12 Chapter 1

tion with other subclasses. An IgG4 dominated response can be seen with certain parasitic infections.15

IgG can be measured in serum but can also be present in the interstitial fluid. The neonatal Fc-receptor (FcRn) plays an important role in IgG metabolism by actively transporting IgG through endothelial cells and recycling of IgG (Figure 6). Because via the latter mechanism IgG can be rescued from degradation, it has a longer half-life than IgA and IgM.16 This does not apply to IgG3, which does not bind to FcRn.17 FcRn is also important for the transport of IgG through epithelial cells; for example the IgG transport through the placenta provides protective maternal antibodies for the child.16

Figure 6. IgG recycling through FcRn. Left: Antigen bound by IgG molecules is removed from the circulation (1) by endocytosis in vascular endothelial cells. IgG dissociates from the antigen and is bound to FcRn (2). The IgG FcRn complexes are sorted out in recycling endosomes (3) and through exocytosis return to the blood (5) where disso- ciation of IgG occurs from the FcRn. The IgG FcRn complexes can also be transported to the intersti- tium (6). The antigens in the endosomes are disintegrated here. Right: In the syncytiotrophoblast of the placenta the FcRn (1) is located in the endosomes. Maternal IgG is bound to FcRn (2) and transported (3) and secreted into the foetal blood (4). Figure and legend based on Leerboek immunologie8 (page 199).

Immunoglobulin E antibodies are important for defence against helminths and other parasites and pathologically in allergic reactions (IgE mediated hypersensitivity reaction).

Immunoglobulin A is mainly important for local mucosal immunity. IgA is found as a monomer in blood, but as a dimer in secretions. Two subclasses of IgA exist: IgA1 and IgA2. In saliva, tears and colostrum predominantly IgA1 is found. In the mucosa of the respiratory and digestive system 60% of the IgA is General introduction, aims and outline of the thesis 13

of the IgA2 subclass. IgA plays an important role preventing direct penetration of antigens and toxins into the body.

1.2 Primary antibody deficiencies

A defect in the humoral immune system can lead to recurrent or unusual respiratory tract infections. Most commonly, these infections are caused by encapsulated bacteria such as Streptococcus pneumoniae and Haemophilus influenzae.18 Respiratory tract infections (including sinusitis) are the most common type of infection, but patients can present with other infections, such as gastro- enteritis, meningitis, and osteomyelitis as well.18 To differentiate immunological from non-immunological causes of recurrent respiratory tract infections, immune status investigation can be performed. This is indicated after exclusion of other common causes of recurrent respiratory tract infections such as chronic sinusitis due to anatomical abnormalities, suboptimally treated asthma, gastric reflux or bronchiectasis (of known aetiology (e.g. tuberculous sequelae)). Nowadays, these investigations are performed according to the guideline “recurrent respiratory tract infections” from the Dutch Society of Pulmonol- ogy and Tuberculosis19 and ESID guidelines.20 Immune investigation is indicated when the above causes are excluded (Table 1).

Table 1. Indications for immune status investigation in adult patients.

Indications for immune status investigation in adult patients Recurrent (bacterial) respiratory tract infections More than one severe infection; e.g. meningitis, pneumonia Atypical presentation or unusual course of infection Bronchiectasis of unknown aetiology Unstable asthma Family history of immunodeficiency

1.2.1 Diagnosis

The conditions of absent or low circulating antibodies are termed agammaglobulinemia and hypogammaglobulinemia, respectively. These conditions are the hallmark of primary antibody deficiencies (PADs). In the spring of 1952 Col. Ogden C. Bruton described the first case of agammaglobulinemia in a child with recurrent respiratory tract infections.21 This 8-year-old boy presented with a history of recurrent bacterial sinopulmonary infections. No gammaglobulins were detected in his serum. This patient was successfully treated with monthly injec- tions of immunoglobulins, which resulted in a significant decrease of infections. 14 Chapter 1

This was the first description of a primary antibody deficiency and treatment with antibody replacement.22 Since Bruton’s first description, many more PADs have been discovered.23 The different types of PAD have different underlying causes, and vary in severity. The estimated prevalence for PADs ranges from 1.3- 2.9/100,000 persons in Europe, although the actual prevalence is likely higher due to underdiagnosis of PADs and underrepresentation of mild PADs in these estimates.24

The diagnosis of a PAD can be made when other causes of recurrent respiratory tract infections are excluded and the results of the immune status investigation are abnormal. If the immune status investigation shows an immunological defect, and a patient is exposed to known immunocompromising factors, this is termed secondary antibody deficiency. Known immunocompromising factors include: immunosuppressive drugs (e.g. for the treatment of auto-immune disease or for organ-transplant recipients)25-27, haematological malignancy27, and asplenia.28 There are various categories of PAD, as shown in Table 2. Classifica- tion of PADs is largely based on the results of immune status investigations.23 Immune status investigation is always carried out in a stepwise manner, with the next step depending on the results of the previous step. The used schedule is shown in Figure 7. Investigations include: measurement of blood cell lines, serum immunoglobulins, IgG subclasses, complement components, and response to pneumococcal polysaccharide vaccination.

1.2.1.1 Response to pneumococcal vaccination The immune response to the pneumococcal polysaccharide vaccine reflects the ability of the patient to mount adequate antibody titers against infections with encapsulated bacteria. For assessment of the immune response the 23-valent pneumococcal polysaccharide response is used. Pneumococcal antibody titers are measured before and three to six weeks after vaccination. Nowadays only IgG titers are measured. Factors like immunosuppressive therapy or underlying disease such as malignancy can influence the immunologic response. The definition of an adequate antibody response is mainly based on recommendations of an expert working group.23 Pneumococcal specific serotype titers are considered by consensus to be protective when the IgG concentration is ≥ 1.3 microgram/ml. An exact level of protectiveness is difficult because every serotype may have its own level of protection based on avidity, age or exposure.23 The definition of an adequate response has varied over the years,29, 30 but the current definition is: (1) two-fold increase of post vaccination titers over the prevaccination titers for ≥ 70% of the tested serotypes; and (2) antibody levels ≥ 1.3 microgram/ml for ≥ 70% of the tested serotypes. General introduction, aims and outline of the thesis 15

Table 2. Clinical and laboratory manifestations of the most common PADs, with indications for antibody replacement therapy.23, 31

Clinical Laboratory Indication for Disorder manifestations characteristics gammaglobulin X-linked Bacterial infections, Low or absent IgM, Always agammaglobulinemia autoimmune disease, IgG and IgA malignancy Impaired response to vaccination Autosomal recessive Bacterial infections, Low or absent IgM, Always agammaglobulinemia autoimmune disease, IgG and IgA malignancy Impaired response to vaccination Common variable Bacterial infections, Low IgG, as well as Always immunodeficiency viral infections, au- low IgA, and/or IgM (CVID) toimmune disease, Impaired response to malignancy vaccination Hyper IgM syndrome Bacterial infections, Low IgA, IgG and IgE Always autoimmune disease, levels with normal or malignancy high IgM levels Impaired response to vaccination Selective IgM Bacterial infections, Low IgM No, only in case of deficiency viral infections, au- severe recurrent in- toimmune disease fections and impaired vaccination response Selective IgA Bacterial infections, Low IgA No, only in case of deficiency autoimmune disease severe recurrent infections and impaired vaccination response Selective Bacterial infections Low IgG1, IgG2, IgG3 Only in case of severe immunoglobulin or IgG4 recurrent infections subclass deficiency and impaired vaccination response Selective Bacterial infections Impaired specific an- Only in case of severe polysaccharide tibody production in recurrent infections antibody deficiency response to polysac- (SAD) charide vaccination 16 Chapter 1

Figure 7. European Society for Immunodeficiencies protocol for immune status investigations in patients with recurrent respiratory tract infections.20

1.2.2 Therapy

The main therapy for PAD is substitution therapy with immunoglobulins.32, 33 This has not changed since its first recognition more than half a century ago by Col. Ogden C Bruton21 and is still based on the repeated infusion of antibodies from pooled donor plasma, which is also known as antibody, immunoglobulin or gammaglobulin replacement therapy. Despite the absence of randomized controlled trials there is broad consensus to provide antibody replacement therapy in case of hypo- of agammaglobulinemia.34 Also in secondary hypogammaglobulinemia studies have been published.35, 36 In these populations, gammaglobulin therapy has been shown to be effective in reducing infection frequency and improving quality of life. In mild humoral immunodeficiency (e.g. IgG subclass deficiency (IgGSD) or specific antibody deficiency (SAD) there is less evidence supporting antibody replacement therapy. The most extensive study is a retrospective study in IgGSD, where treatment with subcutaneously given immunoglobulin resulted in a significant decrease of respiratory tract infections requiring antibiotic treatment.37 General introduction, aims and outline of the thesis 17

The use of antibody replacement therapy in PAD is defined in international guidelines.23, 34 Immunoglobulins can be given intravenously or subcutaneously. Intrave- nous immunoglobulins (IVIG) can be given monthly at the outpatient department of the hospital or at home, whereas subcutaneous immunoglobulins (SCIG) can be (self-)administered daily or weekly at home.38 A choice between administration routes is made on an individual basis. The preferred administration route is IVIG in case of CVID. The starting dose depends on serum IgG levels. In case of SAD the preferred route is SCIG. Dose adjustments and changes in administration route can be made based on IgG trough levels and infection frequency. There is a great variation in IgG trough levels required to prevent infections.39, 40 This supports the concept of biological through levels, meaning that each patient requires an individual through level to prevent infections, and that IgG dosage should be individualized.38

2. Aims and outline of this thesis

This thesis evaluates the value of humoral immune status assessment in patients with respiratory tract disease and lung transplant patients and the treatment of primary antibody deficiency with immunoglobulin replacement therapy in clinical practice. The thesis is divided into four parts.

Part I describes immunological screening in patients with recurrent respiratory tract infections. This part comprises descriptions of three cohorts: a general cohort of patients with recurrent respiratory tract infections, as well as two smaller selected subgroups of patients with recurrent respiratory tract infections. The immune status in the respective cohorts is described, and the roles of MBL-deficiency, IgG1 deficiency and an impaired pneumococcal antibody response in these patients are investigated.

Part II gives the results of immunological screening in a cohort of lung transplant candidates, as well as follow up of humoral immune status after lung transplantation in a cohort of lung transplant recipients.

Part III provides an overview of the literature on antibody replacement therapy in primary antibody immunodeficiency and iatrogenic hypogammaglobulinemia. The outcome of long-term antibody replacement therapy in our own cohort of patients with humoral immunodeficiency is described using infection frequency, use of antibiotics and hospital admissions as parameters of efficacy.

Part IV describes two cases that had an uncommon clinical presentation. These cases illustrate the components of immunological screening and sometimes remarkable findings in daily clinical practice. 18 Chapter 1

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24. Edgar JD, Buckland M, Guzman D, et al. The united kingdom primary immune deficiency (UK- PID) registry: Report of the first 4 years’ activity 2008-2012. Clin Exp Immunol. 2014;175(1):68-78. 25. Samson M, Audia S, Lakomy D, Bonnotte B, Tavernier C, Ornetti P. Diagnostic strategy for patients with hypogammaglobulinemia in rheumatology. Joint Bone Spine. 2011;78(3):241-245. 26. Mawhorter S, Yamani MH. Hypogammaglobulinemia and infection risk in solid organ transplant recipients. Curr Opin Organ Transplant. 2008;13(6):581-585. 27. Mouthon L, Fermand JP, Gottenberg JE. Management of secondary immune deficiencies: What is the role of immunoglobulins? Curr Opin Allergy Clin Immunol. 2013;13 Suppl 2:S56-67. 28. Rubin LG, Schaffner W. Clinical practice. care of the asplenic patient. N Engl J Med. 2014;371(4):349-356. 29. Bonilla FA, Bernstein IL, Khan DA, et al. Practice parameter for the diagnosis and management of primary immunodeficiency. Ann Allergy Asthma Immunol. 2005;94(5 Suppl 1):S1-63. 30. Paris K, Sorensen RU. Assessment and clinical interpretation of polysaccharide antibody responses. Ann Allergy Asthma Immunol. 2007;99(5):462-464. 31. Picard C, Al-Herz W, Bousfiha A, et al. Primary immunodeficiency diseases: An update on the classification from the international union of immunological societies expert committee for primary immunodeficiency 2015.J Clin Immunol. 2015;35(8):696-726. 32. Orange JS, Ochs HD, Cunningham-Rundles C. Prioritization of evidence-based indications for intravenous immunoglobulin. J Clin Immunol. 2013;33(6):1033-1036. 33. Bonilla FA, Khan DA, Ballas ZK, et al. Practice parameter for the diagnosis and management of primary immunodeficiency.J Allergy Clin Immunol. 2015;136(5):1186-205.e1-78. 34. Orange JS. Hossny EM. Weiler CR. Ballow M. Berger M. Bonilla FA. Buckley R. Chinen J. El- Gamal Y. Mazer BD. Nelson RP Jr. Patel DD. Secord E. Sorensen RU. Wasserman RL. Cunningham-Rundles C. Primary Immunodeficiency Committee of the American Academy of Allergy,Asthma and Immunology. Use of intravenous immunoglobulin in human disease: A review of evidence by members of the primary immunodeficiency committee of the american academy of allergy, asthma and immunology. J Allergy Clin Immunol. 2006;117(4 Suppl):S525-53. 35. Duraisingham SS, Buckland M, Dempster J, Lorenzo L, Grigoriadou S, Longhurst HJ. Primary vs. secondary antibody deficiency: Clinical features and infection outcomes of immunoglobulin replacement. PLoS One. 2014;9(6):e100324. 36. Roberts DM, Jones RB, Smith RM, et al. Immunoglobulin G replacement for the treatment of infective complications of rituximab-associated hypogammaglobulinemia in autoimmune disease: A case series. J Autoimmun. 2015;57:24-29. 37. Olinder-Nielsen AM, Granert C, Forsberg P, Friman V, Vietorisz A, Bjorkander J. Immuno globulin prophylaxis in 350 adults with IgG subclass deficiency and recurrent respiratory tract infections: A long-term follow-up. Scand J Infect Dis. 2007;39(1):44-50. 38. Bonagura VR. Using intravenous immunoglobulin (IVIG) to treat patients with primary immune deficiency disease. J Clin Immunol. 2013;33 Suppl 2:S90-4. 39. Lucas M, Lee M, Lortan J, Lopez-Granados E, Misbah S, Chapel H. Infection outcomes in patients with common variable immunodeficiency disorders: Relationship to immunoglobulin therapy over 22 years. J Allergy Clin Immunol. 2010;125(6):1354-1360.e4. 40. Quinti I, Soresina A, Guerra A, et al. Effectiveness of immunoglobulin replacement therapy on clinical outcome in patients with primary antibody deficiencies: Results from a multicenter prospective cohort study. J Clin Immunol. 2011;31(3):315-322.

PART I

IMMUNOLOGICAL SCREENING OF PATIENTS WITH RECURRENT RESPIRATORY TRACT INFECTIONS

CHAPTER 2

Response to pneumococcal vaccination in mannose-binding lectin-deficient adults with recurrent respiratory tract infections

D.A. van Kessel1,2 T.W. Hoffman1 H. van Velzen-Blad3 P. Zanen1,2 G.T. Rijkers3,4 J.C. Grutters1,2

1Department of Pulmonology, St. Antonius Hospital, Nieuwegein, The Netherlands 2Division of Heart and Lungs, University Medical Center Utrecht, Utrecht, The Netherlands 3Department of Medical Microbiology and Immunology, St. Antonius Hospital, Nieuwegein, The Netherlands 4Science Department, University College Roosevelt, Middelburg, The Netherlands

Clin Exp Immunol 2014 Jul; 177(1):272-9 24 Chapter 2

Abstract

Mannose-binding lectin (MBL)-deficiency is associated with an increased susceptibility to pneumococcal infections and other forms of disease. Pneumococ- cal vaccination is recommended in MBL-deficient patients with recurrent respiratory tract infections (RRTI). The response to pneumococcal vaccination in MBL-deficient individuals has not yet been studied in detail. An impaired response to pneumococcal polysaccharides in MBL-deficient patients might explain the association between MBL deficiency and pneumococcal infections. This study investigates the antibody response to pneumococcal vaccination in MBL-deficient adult patients with RRTI. Furthermore, we investigated whether there was a difference in clinical presentation between MBL-deficient and -sufficient patients with RRTI.

Eighteen MBL-deficient and 63 MBL-sufficient adult patients with RRTI were all vaccinated with the 23-valent pneumococcal polysaccharide vaccine and antibodies to 14 pneumococcal serotypes were measured on a Luminex platform. There were no differences observed in the response to pneumococcal vaccination between MBL-sufficient and -deficient patients. Forty-three MBLsuffi- cient patients could be classified as responders to pneumococcal vaccination and 20 as low responders, compared to 15 responders and three low responders in the MBL-deficient patients.

We found no clear difference in clinical, radiological, lung function and medication parameters between MBL-sufficient and -deficient patients. In conclusion, our study suggests that MBL-deficient adults with RRTI have a response to a pneumococcal capsular polysaccharide vaccine comparable with MBL-sufficient patients. Moreover, we did not find a clear clinical role of MBL deficiency in adults with RRTI. As MBL deficiency is associated with an increased susceptibility to pneumococcal infections, pneumococcal vaccination might be protective in MBL-deficient patients with RRTI. Pneumococcal vaccination in MBL-deficiency 25

Introduction

Primary immunodeficiencies (PID) are an uncommon cause of recurrent respiratory tract infections (RRTI) in adults. However, a major number of adults with manifest PID present with RRTI.1, 2 Early diagnosis and initiation of treatment are important for the prognosis of patients with primary immunodeficiencies,3, 4 and therefore many efforts have been made to develop a structured guideline for the diagnosis of patients with PID. This has led to a multi-stage diagnostic protocol developed by the European Society for Immunodeficien- cies (ESID).5 The ESID protocol was first published in 2006 and subsequently updated in 2012.6

The diagnostic work-up for patients with recurrent ear, nose, throat and airway infections includes measuring the serum activity of the alternative and classical complement pathways and the option to determine the activity of mannose-binding lectin (MBL), a protein that plays a major role in the third complement pathway (i.e. lectin pathway).

MBL is a pattern recognition receptor that can bind mannose and N-acetyl- glucosamine oligosaccharides, which are present on a wide variety of micro organisms. MBL facilitates opsonization of these microorganisms and can activate the complement system via the lectin pathway. Human MBL is encoded by the MBL2 gene on chromosome 10, which comprises four exons. Structural polymorphisms in exon 1 of MBL2 influence the functionality of the MBL protein, whereas polymorphisms in the promoter region quantitatively affect serum MBL levels.7 Genetic variants that cause MBL deficiency are common in the general population. Whether or not MBL deficiency leads to a survival dis advantage remains a matter of debate.8

For instance, MBL-deficient children are at an increased risk for invasive pneumococcal disease, and also nasopharyngeal carriage of Streptococcus pneumoniae is twice as high in MBL-deficient children compared to MBL-sufficient children.9, 10 In adults, MBL deficiency is associated with a more severe course of pneumococcal infection and an increased risk for invasive pneumococcal disease.11, 12 In two independent cohort studies, no association between MBL deficiency and susceptibility to community-acquired pneumococcal pneumonia was found.13, 14 Little is known about the involvement of MBL in the initiation and the effector phase of the antibody response to S. pneumoniae. MBL is considered to have only a minor role in the opsonization of S. pneumoniae.15, 16 Di- rect in-vitro demonstration of MBL binding to pneumococci is limited (our own unpublished observations) and no differences in phagocytosis of pneumococci were seen between MBL-sufficient and -deficient patients.17 26 Chapter 2

It has been suggested that MBL deficiency, especially in the context of a subop- timal function of other components of the immune system, may contribute to an increased susceptibility for infections.18, 19 From this viewpoint, it is reason- able that international guidelines recommend the administration of pneumococcal vaccines to MBL-deficient patients with recurrent infections.20 The response to a pneumococcal capsular polysaccharide vaccine in MBLdeficient individuals has not been studied in detail. We hypothesize that the association between MBL deficiency and pneumococcal infections can be explained by an impaired polysaccharide responsiveness in MBL-deficient compared to MBL-sufficient people.

Therefore, the aim of this study was to investigate the ability of MBL-deficient adults with RRTI to respond to pneumococcal vaccination. To that end, we evaluated the response to pneumococcal vaccination in patients with RRTI. Furthermore, we compared the clinical presentation and immunological laboratory parameters between MBLsufficient and -deficient adults with RRTI who were evaluated according to the ESID protocol.

Materials and methods

Patient population

All patients were from St Antonius Hospital, Nieuwegein, one of the largest non-academic teaching hospitals in the Netherlands and a referral centre for patients with pulmonary diseases. All patients referred to the out-patient clinic pulmonary diseases for analysis of recurrent respiratory tract infections in the period September 2009–December 2011 were subjected to an immunological work-up based on the ESID protocol for evaluation of recurrent respiratory tract infections. The work-up included administration of a pneumococcal capsular polysaccharide vaccine (PVX) with measurement of immunoglobulin (Ig)G antibody concentrations to 14 pneumococcal serotypes before and after vaccination (as detailed below). Measurement of functional complement activity, including the MBL pathway (MP), was performed in all patients. Genetic analysis was performed if the MP ≤ 10%.

Data collection

In total, 117 patients were evaluated prospectively according to the ESID protocol (version 2006). Patients who met the criteria for RRTI, defined as three or more infectious episodes of the respiratory tract per year, were included in the present study. Pneumococcal antibody concentration measurement had to be Pneumococcal vaccination in MBL-deficiency 27

performed 3–6 weeks after vaccination. Other exclusion criteria were previous lung transplantation, previous pneumococcal vaccination or haematological malignancies. Eventually, 81 patients were included in the present study. A consort diagram of the patient selection procedure is presented in Figure 1. Patient characteristics were collected retrospectively from the patients’ medical records.

Classification of clinical characteristics

Infectious episodes were categorized as sinusitis, bronchitis or pneumonia. Si- nusitis was defined as symptomatic inflammation of the paranasal sinuses lasting no longer than 6–8 weeks. Bronchitis was defined as having an excessive mucous secretion.21 A sputum culture was carried out when possible. Pneumonia was defined as a new infiltrate on a chest X-ray in combination with two of six clinical signs of pneumonia (i.e. cough, sputum production, signs of consolidation on auscultation, leucocytosis or leucopenia, temperature < 35°C or > 38°C or > 10% rods in the differential count and C-reactive protein three times above the upper limit of normal). Pulmonary function testing was carried out according to the standards of the European Respiratory Society.22 Lung function scores were assigned based on the Global Initiative for Chronic Obstructive Lung Disease 23 (GOLD) guidelines. Based on forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) measurement, patients are classified as GOLD stage 0 (no obstruction), stage I (mild obstruction), stage II (moderate obstruction), stage III (severe obstruction) or stage IV (very severe obstruction). Computed tomography (CT)-scan characteristics were derived from patients’ radiology reports. Medica- tion use and smoking status were extracted from internal patient reports. Patients who were classified as receiving prednisone therapy started this at least 2 weeks before vaccination.24, 25 A patient was classified as an ex-smoker in case of smoking cessation for at least 6 months prior to vaccination.

Laboratory investigations

Standard laboratory investigations in all subjects included a blood leucocyte count with differentiation, serum alpha-1 anti-trypsin, serum immunoglobulin and serum IgG subclass-level measurement. Sputum was cultured using standard microbiological techniques.

A commercially available enzyme immunoassay was used to determine the functionality of the classical, alternative and lectin complement pathways (Wiel- isa®; Wieslab, Lund, Sweden).26 Any pathway activity ≤ 10% of normal can be an indication of a complement deficiency and needs additional confirmation. MBL2 genotyping was performed if MP ≤ 10% of normal to confirm a MBL deficiency. 28 Chapter 2

Combined haplotypes of functional single nucleotide polymorphisms (SNP) in the promoter region (Y/X) and exon 1 of MBL2 (the coding SNPs B, C and D are denoted as ‘0’ and A is wild-type) were determined using a denaturating gradi- ent gel electrophoresis (DGGE) assay, as described previously.13 MBL genotypes O/O and XA/O were considered to be MBL-deficient.

Anti-capsular pneumococcal polysaccharide antibody assay

Patients were vaccinated intramuscularly with one dose of 23-valent pneumococcal polysaccharide vaccine (Pneumovax 23; Merck, Rahway, NJ, USA) containing 25 μg purified type-specific capsular polysaccharides of 23 pneumococcal serotypes (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F: Danish nomenclature). Blood samples were drawn before and 3–6 weeks after vaccination. Serum samples were stored at −80°C until use.

IgG antibodies against 14 pneumococcal polysaccharides were measured on a Luminex platform (Luminex Corporation, Austin, TX, USA), using a quantitative multiplex immunoassay: the XMAP pneumococcal immunity panel.27 This assay identifies serotype-specific anti-capsular polysaccharide IgG antibodies to the pneumococcal serotypes 1, 3, 4, 6B, 7F, 8, 9N, 9V, 12F, 14, 18C, 19A, 19F and 23F. All antibody concentrations were calibrated on the international Food and Drug Administration (FDA) reference preparation 89-SF. Samples were analysed as singlets; pre- and post-vaccination sera were analysed in pairs.

Patients were classified as responder or low responder based on their post- vaccination pneumococcal polysaccharide antibody response profile. A positive immune response to a given serotype was defined as a postvaccination antibody concentration ≥ 1·3 μg/ml or as a ≥ 4-fold antibody concentration increase between the preand post-vaccination serum samples.28 A patient was considered to be a responder if at least 70% of the antibody responses to the serotypes tested (i.e. 10 of 14 tested serotypes) were positive.28

Statistical analysis

Groups were compared with Fisher’s exact test, McNemar and analysis of co- variance (ancova) where appropriate. Statistical analyses were performed using spss. A p-value < 0·05 was considered to represent a statistically significant difference. Pneumococcal vaccination in MBL-deficiency 29

Figure 1. Consort diagram of the patient selection procedure; patients that were initially included were evaluated according to the European Society for Immunodeficiencies (ESID) protocol for diagnosing primary immunodeficiencies (PID) in the period September 2009–December 2011 and a genotypic analysis of MBL2 was performed if the complement mannose-binding lectin (MBL) pathway (MP) was ≤ 10% of normal.

Results

The study group comprised 81 patients, in 20 patients of whom functional MBL pathway activity was less than 10%. Subsequent genotyping showed that 18 patients were MBLdeficient (14 XA/O and 4 O/O genotypes). No indication was found for a complement factor deficiency in the alternative or classical complement pathway. The patient characteristics of the total study group and the MBL-sufficient and -deficient subgroups are provided in Table 1. Sex, age and smoking status did not differ between both subgroups. The majority of the patients have an underlying disease, mainly chronic obstructive pulmonary disease (COPD) or asthma. A GOLD score of 0 on a lung function test was significantly more prevalent in the MBL-deficient group (p = 0·0463), but pulmonary pathology on CT-scan images did not differ between both subgroups. Medica- tion use, in terms of gammaglobulin or prednisone, was comparable in both subgroups. Twenty-six patients were using prednisone at the time of their vaccination, with a mean dosage of 10·5 mg/day (range 2·5–50, median dosage 6·25). 30 Chapter 2

Table 1. Clinical characteristics of complement mannose-binding lectin (MBL)-sufficient and -deficient patients.

MBL-sufficient MBL-deficient Total Patients p-value Patients (%) Patients (%) (%)

Total 63 (78) 18 (22) 81 (100) Male 23 (37) 9 (50) 0.4129 32 (40) Median Age 62.2 60.1 61.3 Low responder 20(32) 3(17) 0.2515 23(28)

Smoking Status Never smoker 20 (32) 9 (50) 0.1731 29 (36) Ex smoker 34 (54) 7 (39) 0.2951 41 (51) Current smoker 9 (14) 2 (11) 1.0000 11 (14)

Underlying Disease Asthma 19 (30) 7 (39) 0.5698 26 (32) COPD 27 (43) 4 (22) 0.1692 31 (38) Allergy 15 (24) 6 (33) 0.5425 21 (26) Immune Deficiency 4 (6) 0 (0) 0.5704 4 (5)

Lung Function 59 (94) 17 (94) 76 (94) GOLD 0 18 (29) 10 (56) 0.0463 28 (35) GOLD I 8 (13) 1 (6) 0.6743 9 (11) GOLD II 10 (16) 1 (6) 0.4383 11 (14) GOLD III 15 (24) 4 (22) 1.0000 19 (23) GOLD IV 8 (13) 1 (6) 0.6743 9 (11)

CT scan 56 (89) 17 (94) 73 (90) Small Airway Disease 12 (19) 3 (17) 1.0000 15 (19) Bronchopathy 8 (13) 5 (28) 0.1656 13 (16) Bronchiectasis 23 (37) 7 (39) 1.0000 30 (37) Emphysema 14 (22) 4 (22) 1.0000 18 (22) Fibrotic Lesions 5 (8) 1 (6) 1.0000 6 (7) Fibrosis 1 (2) 1 (6) 0.4140 2 (2)

Medication Gammaglobulin 9 (14) 3 (17) 0.7235 12 (15) Prednisone 21 (33) 5 (28) 0.7788 26 (32) p-values were calculated using Fisher’s exact test, based upon the number of patients for whom data regarding a specific parameter was available. Percentages are based on the total number of patients per subgroup. Gammaglobulin substitution therapy was initiated after analysis of the response to pneumococcal vaccination. Prednisone use was scored if the therapy was initiated at least 2 weeks before pneumococcal vaccination. COPD, chronic obstruactive pulmonary disease; CT, computerized tomogtaphy; GOLD, Global Initiative for Chronic Obstructive Lung Disease. Pneumococcal vaccination in MBL-deficiency 31

Figure 2. Serotype specific antibody concentrations in complement mannose-binding lectin-sufficient (red dots) and MBL-deficient (blue dots) patients measured 3–6 weeks after vaccination with the 23-valent pneumococcal polysaccharide vaccine. Geometric means are indicated with black bars. The horizontal black line indicates the threshold antibody concentration of 1·3 μg/ml.

All patients were vaccinated with the 23-valent pneumococcal polysaccharide vaccine and their antibody response was measured 3–6 weeks after vaccination (median 26 days, interquartile range 23–34 days, range 19–43 days). There were four patients who could already be categorized as responders in the prevaccination stage, two of whom were MBL-deficient. Overall, 58 patients were categorized as a responder to PVX and 23 patients as having a low response. Subgroup analysis showed that 15 of the 18 MBL-deficient patients were responders and three were low responders, compared to 43 responders and 20 low responders in the MBL-sufficient patients. The frequency of response to PVX thus did not differ significantly between the MBL-sufficient and -deficient patient groups (p = 0·2515). The MBL genotypes of the three MBL-deficient patients with a low response to pneumococcal vaccination were XA/YB (n = 2) and XA/YC (n = 1). The MBL genotypes of the 15 MBL-deficient patients with a normal response to pneumococcal vaccination were XA/YD (n = 5), XA/YB (n = 6), YD/YB (n = 2), YD/YC (n = 1) and YB/YB (n = 1).

In order to further analyse the response to pneumococcal vaccination, a detailed comparison was made of serum antibodies to 14 pneumococcal serotypes before and after vaccination. There were no significant differences in prevaccination concentrations between both MBL-sufficient and -deficient patient groups. No significant differences were observed between both groups in the geometric mean antibody levels per serotype after vaccination (Figure 2), 32 Chapter 2

except for the mean antibody level for pneumococcal serotype 4, which was just barely significantly higher in the MBL-deficient patients (p = 0·047 when post-vaccination concentrations are corrected for prevaccination concentrations). There was considerable interpatient variation in the number of serotypes against which patient had protective post-vaccination antibody concentrations. Of the 23 low responders, 16 had a response to fewer than 50% of the serotypes that were measured (i.e. seven serotypes).

The above data indicate that in our cohort of patients with RRTI, 28% can be diagnosed with a (mild) specific polysaccharide antibody deficiency. Four of those patients had an underlying immunodeficiency according to the International Union of Immunological Societies (IUIS) criteria:29 two patients with hypogammaglobulinaemia (patient 1: IgG = 2·67 g/l and IgA = 0·03 g/l; patient 2: IgG = 3·47 g/l and IgA = 0·45 g/l), one patient with an IgA deficiency (IgA = 0·03 g/l) and one patient with an IgG2 deficiency (IgG = 6·80 g/l and IgG2 = 0·5 g/l). All four patients had a low response to pneumococcal vaccination and were MBL-sufficient.

When comparing the patients with an adequate response to pneumococcal vaccination to patients with a low response for the parameters in Table 1, independent of MBL status, several significant differences are observed for lung function parameters, medication use and serum immunoglobulin levels. All four patients with a humoral immunodeficiency were in the low responder group (p = 0·0053). A lung function score of GOLD 0 was more common in the responder group (26 of 55 responders and two of 21 low responders, p = 0·0029), while a GOLD 2 score was more common in the low responder group (four of 55 responders and seven of 21 low responders, p = 0·0079). Prednisone use was more common in the low responder group (13 of 58 responders and 13 of 23 low responders, p = 0·0072).

Significantly fewer patients receiving prednisone treatment responded with protective antibodies concentrations (≥ 1·3 μg/ml) to four pneumococcal serotypes (1, 8, 14, 23F) compared to the patients without prednisone. Furthermore, the mean post-vaccination concentration was significantly lower in the prednisone user group for pneumococcal serotype 8 (p = 0·004, when post-vaccination concentrations are corrected for prevaccination concentrations).

Serum immunoglobulin levels, including IgG subclass levels, were retrieved for all patients for whom they were available, and shown in Table 2. The levels for each patient were evaluated against the reference lower-level values for the respective immunoglobulin (sub)classes30 (IgM = 0·40 g/l; IgA = 0·70 g/l; IgG = 7·0 g/l; IgG1 = 4·90 g/l; IgG2 = 1·50 g/l; IgG3 = 0·20 g/l and IgG4 = 0·08 g/l) and Pneumococcal vaccination in MBL-deficiency 33

Table 2. Immunoglobulin levels compared between complement mannose-binding lectin (MBL)- sufficient and -deficient patients.

MBL-sufficient MBL-deficient Total Patients p-value Patients (%) Patients (%) (%) Total 63 (78) 18 (22) 81 (100)

IgM 59 (94) 17 (94) 76 (94) N < 0.40 g/l 5 (8) 3 (17) 0.3674 8 (10) Median (g/l) 0.95 0.72 0.89

IgA 61 (97) 17 (94) 78 (96) N < 0.70 g/l 8 (13) 1 (6) 0.6746 9 (11) Median (g/l) 1.97 2.64 2.13

IgG 60 (95) 16 (89) 76 (94) N < 7.0 g/l 20 (32) 3 (17) 0.3633 23 (29) Median (g/l) 8.19 9.98 8.31

IgG1 62 (98) 18 (100) 80 (99) N < 4.90 g/l 25 (40) 7 (39) 1.0000 32 (40) Median (g/l) 5.6 6.2 5.6

IgG2 62 (98) 18 (100) 80 (99) N < 1.50 g/l 24 (38) 4 (22) 0.2657 28 (35) Median (g/l) 2.1 2.7 2.2

IgG3 62 (98) 18 (100) 80 (99) N < 0.20 g/l 15 (24) 6 (33) 0.5441 21 (26) Median (g/l) 0.4 0.4 0.4

IgG4 62 (98) 18 (100) 80 (99) N < 0.08 g/l 13 (21) 0 (0) 0.0336 13 (16) Median (g/l) 0.2 0.4 0.2 p-values were calculated using Fisher’s exact test, based upon the number of patients for whom data regarding a specific parameter was available. Percentages are based on the total number of patients per subgroup. Only measurements that were carried out before patients received gammaglobulin are included. Ig, immunoglobulin.

the number of patients with reduced serum levels was determined. No significant differences in immunoglobulin and IgG subclass levels were observed between MBL-sufficient and -deficient patient groups except for serum IgG4 levels, which were reduced only in the MBL-sufficient group (p = 0·0336). 34 Chapter 2

Immunoglobulin and IgG subclass levels were also compared between responders and low responders to PVX. There were significantly more low responders with serum IgG levels below the lower-level value (7·0 g/l, 12 of 56 responders and 11 of 20 low responders, p = 0·0028). The same applies to the number of patients with serum IgG1 levels < 4·90 g/l (19 of 58 responders and 13 of 22 low responders, p = 0·0421) and to the number of patients with serum IgG2 levels < 1·50 g/l (14 of 58 responders and 14 of 22 low responders, p = 0·0015).

In addition, several differences between prednisone users and non-prednisone users for immunoglobulin and IgG subclass levels were observed. IgG levels below the lowerlevel value were more frequent in prednisone users (p = 0·0031), as were IgG1 and IgG2 levels below the lowerlevel values (p = 0·0306 and p = 0·0010, respectively).

When patients who had reduced IgM, IgA, IgG, IgG1 or IgG2 levels, or were low responders, were excluded for a subgroup analysis, a group of 33 patients remained. This was carried out in order to study the role of MBL deficiency in patients where it was the sole immunological defect. Within this group, there were 25 MBL-sufficient patients and eight MBL-deficient patients. The clinical characteristics of these MBL-sufficient and -deficient patient groups did not differ.

Discussion

This study is the first to suggest that MBL-deficient patients with RRTI do not demonstrate a significantly lower response to a pneumococcal capsular polysaccharide vaccine compared to MBL-sufficient patients. The only difference in response that was observed between both groups was for the response to pneumococcal serotype 4, which was just significantly higher in MBL- deficient patients. This is most probably a chance finding, and does not suggest a lesser response to pneumococcal serotype 4 in MBL-deficient individuals.

Pneumococcal vaccination in MBL-deficient patients with RRTI is justifiable, based on this quantitative assessment of antibody production in response to vaccination. However, a limitation of the present study is that we studied pneumococcal vaccination only in a diagnostic setting. We evaluated the ability to produce specific antibodies, and not the functionality of those antibodies. Lit- tle is known about the effect of MBL on pneumococcal opsonophagocytosis, and this needs to be investigated further, as well as the effect of pneumococcal vaccination on the frequency of pneumococcal infections in MBL-deficient patients. Pneumococcal vaccination in MBL-deficiency 35

There were no significant differences in medication use and clinical and immunological laboratory characteristics between MBL-sufficient and -deficient patients in our cohort, except that a GOLD 0 score on lung function tests was significantly more prevalent in MBL-deficient patients and that there were no MBL-deficient patients with reduced IgG4 levels. When we investigated the latter further, it appeared to have been a chance finding.When patients with immunological laboratory abnormalities were excluded, there were no significant differences between MBLsufficient and -deficient patients.

Our results do not support a clear role for MBL deficiency in adult patients with RRTI, as a different pattern does not emerge in the clinical presentation of MBLdeficient compared to MBL-sufficient patients. Furthermore, the prevaccination antibody concentrations did not differ between both groups, indicating that invasive exposition to pneumococci was not higher in MBL-deficient patients. However, our cohort is somewhat small, and it would therefore be inappropriate to make any definitive conclusions.

Moreover, we could not validate the suggestion that MBL deficiency has an effect only in patients with concomitant immune defects, as there were only three MBL-deficient patients with a low response to pneumococcal vaccination. Also, our cohort comprised only four patients with underlying immune defects, apart from selective antibody deficiencies, who were all MBL-sufficient.

While multiplex techniques have made quantification of the serotype specific antibody response to pneumococcal capsular polysaccharide vaccination easier, conversion of individual antibody concentrations into overall responsiveness is still a subject of debate. We have used the threshold of antibody concentrations ≥ 1·3 μg/ml according to international guidelines.28 However, the value of 1·3 μg/ml in the guidelines is based on quantification by enzyme-linked immunosorbent assay (ELISA), and has not been validated for Luminex xMAP technologies. Lu- minex xMAP technology appears to estimate higher concentrations of IgGs to pneumococcal capsular polysaccharides compared to ELISA.31. Because this has not been shown for all pneumococcal serotypes, it was not possible to correct for the use of Luminex xMAP technology in the classification of antibody responses.

The frequency of low responders to pneumococcal vaccination was comparable with the frequency we found in a previous cohort of patients with recurrent respiratory tract infections.32 We could not find a suitable cohort of patients in the literature measured by a Luminex multiplex assay with which we could compare the antibody response to pneumococcal vaccination in our patients. The group of low responders as a whole showed a lesser response to all individual pneumococcal serotypes studied compared to the group of responders. 36 Chapter 2

We observed considerable interserotype variation in post-vaccination antibody concentrations in both groups, which is in accordance with previous studies on the serotype-specific antibody response to pneumococcal vaccination.33, 34

A comparison between patients with an adequate response to pneumococcal vaccination and patients with a low response (irrespective of their MBL status) revealed significant differences between both groups for lung function, prednisone use and serum immunoglobulin levels. These differences were compatible with the low responder status, and indicate that patients with a low response to pneumococcal vaccination are predisposed to a more severe clinical presentation than patients with an adequate response.

Significant differences between prednisone users and non-prednisone users for several clinical characteristics indicate that patients who require prednisone have a clinical presentation that sets them apart from other patients. Although prednisone has been widely recognized as a cause of reduced immunoglobulin levels,25, 35 a relation between prednisone use and a low response to pneumococcal vaccination has not been established previously.34, 36, 37 In this study it was found that prednisone use at the time of vaccination was significantly more prevalent in patients with a low overall response to their pneumococcal vaccination. A low response to pneumococcal vaccination may predispose to a clinical presentation that requires prednisone treatment or, alternatively, prednisone use can affect patients’ antibody response negatively. The present study is not capable of discriminating between any of these two options, and further investigation will be needed to find definitive answers.

In summary, this study suggests that MBL status does not play a role for IgG responses to pneumococcal capsular polysaccharide vaccination. Moreover, we did not find a clear clinical role of MBL deficiency in adults with RRTI. As MBL deficiency has generally been associated with an increased susceptibility to pneumococcal infections, pneumococcal vaccination might be protective in MBL-deficient patients with RRTI. The data support other data suggesting that human MBL may be largely redundant for protective immunity in humans.38 Pneumococcal vaccination in MBL-deficiency 37

References

1 Pasternak MS. Approach to the adult with recurrent infections. In: Basow DS, ed. Uptodate. Waltham, MA: UpToDate, 2013. 2 Sicherer SH, Winkelstein JA. Primary immunodeficiency diseases in adults. JAMA 1998;279 :58–61. 3 Seymour B, Miles J, Haeney M. Primary antibody deficiency and diagnostic delay. J Clin Pathol 2005; 58:546–7. 4 Blore J, Haeney MR. Primary antibody deficiency and diagnostic delay. BMJ 1989;298 :516–7. 5 de Vries E. Clinical Working Party of the European Society for Immunodeficiencies (ESID). Patient-centred screening for primary immunodeficiency: a multi-stage diagnostic protocol designed for non-immunologists. Clin Exp Immunol 2006; 145:204–14. 6 de Vries E. European Society for Immunodeficiencies (ESID) members. Patient-centred screening for primary immunodeficiency, a multi-stage diagnostic protocol designed for nonimmunologists: 2011 update. Clin Exp Immunol 2012; 167: 108–19. 7 Minchinton RM, Dean MM, Clark TR, Heatley S, Mullighan CG. Analysis of the relationship between mannose-binding lectin (MBL) genotype, MBL levels and function in an Australian blood donor population. Scand J Immunol 2002; 56:630–41. 8 Heitzeneder S, Seidel M, Forster-Waldl E, Heitger A. Mannanbinding lectin deficiency – good news, bad news, doesn’t matter? Clin Immunol 2012; 143:22–38. 9 Valles X, Roca A, Lozano F et al. Serotype-specific pneumococcal disease may be influenced by mannose-binding lectin deficiency. Eur Respir J 2010; 36:856–63. 10 Vuononvirta J, Toivonen L, Grondahl-Yli-Hannuksela K et al. Nasopharyngeal bacterial colonization and gene polymorphisms of mannose-binding lectin and Toll-like receptors 2 and 4 in infants. PLOS ONE 2011; 6:e26198. 11 Eisen DP, Dean MM, Boermeester MA et al. Low serum mannosebinding lectin level increases the risk of death due to pneumococcal infection. Clin Infect Dis 2008; 47:510–6. 12 Brouwer MC, Baas F, van der Ende A, van de Beek D. Genetic variation and cerebrospinal fluid levels of mannose binding lectin in pneumococcal meningitis patients. PLOS ONE 2013; 8:e65151. 13 Endeman H, Herpers BL, de Jong BA et al. Mannose-binding lectin genotypes in susceptibility to community-acquired pneumonia. Chest 2008; 134:1135–40. 14 Garcia-Laorden MI, Sole-Violan J, Rodriguez de Castro F et al. Mannose-binding lectin and mannose-binding lectin-associated serine protease 2 in susceptibility, severity, and outcome of pneumonia in adults. J Allergy Clin Immunol 2008; 122:368–74. 374.e1–2. 15 Neth O, Jack DL, Dodds AW, Holzel H, Klein NJ, Turner MW. Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition. Infect Immun 2000; 68:688–93. 16 Krarup A, Sorensen UB, Matsushita M, Jensenius JC, Thiel S. Effect of capsulation of opportunistic pathogenic bacteria on binding of the pattern recognition molecules mannan-binding lectin, L-ficolin, and H-ficolin. Infect Immun 2005; 73:1052–60. 17 Brouwer N, Dolman KM, van Houdt M, Sta M, Roos D, Kuijpers TW. Mannose-binding lectin (MBL) facilitates opsonophagocytosis of yeasts but not of bacteria despite MBL binding. J Immunol 2008; 180:4124–32. 18 Gadjeva M, Takahashi K, Thiel S. Mannan-binding lectin – a soluble pattern recognition molecule. Mol Immunol 2004; 41:113–21. 19 Worthley DL, Bardy PG, Mullighan CG. Mannose-binding lectin: biology and clinical implications. Intern Med J 2005; 35:548–55. 20 Bronkhorst MW, Bouwman LH. Mannose-binding lectin deficiency. In: Basow DS, ed. Uptodate. Waltham, MA: UpToDate, 2013. 21 Celli BR, MacNee W. ATS/ERS Task Force. Standards for the diagnosis and treatment of patients with COPD: a summary of the ATS/ERS position paper. Eur Respir J 2004; 23:932–46. 22 Quanjer PH, Tammeling GJ, Cotes JE, Pedersen OF, Peslin R, Yernault JC. Lung volumes and forced ventilatory flows. Report Working Party for Standardization of Lung Function Tests, European Community for Steel and Coal, Official Statement of the European Respiratory Soci- ety. Eur Respir J Suppl 1993; 16:5–40. 38 Chapter 2

23 Rabe KF, Hurd S, Anzueto A et al. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med 2007; 176:532–55. 24 Settipane GA, Pudupakkam RK, McGowan JH. Corticosteroid effect on immunoglobulins. J Allergy Clin Immunol 1978; 62:162–6. 25 Posey WC, Nelson HS, Branch B, Pearlman DS. The effects of acute corticosteroid therapy for asthma on serum immunoglobulin levels. J Allergy Clin Immunol 1978; 62:340–8. 26 Seelen MA, Roos A, Wieslander J et al. Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA. J Immunol Methods 2005; 296:187–98. 27 Pickering JW, Martins TB, Greer RW et al. A multiplexed fluorescent microsphere immunoassay for antibodies to pneumococcal capsular polysaccharides. Am J Clin Pathol 2002; 117:589–96. 28 Bonilla FA, Bernstein IL, Khan DA et al. Practice parameter for the diagnosis and management of primary immunodeficiency. Ann Allergy Asthma Immunol 2005;5 (Suppl 1):S1–63. 29 Geha RS, Notarangelo LD, Casanova JL et al. Primary immunodeficiency diseases: an update from the international union of immunological societies primary immunodeficiency diseases classification committee. J Allergy Clin Immunol 2007;120 :776–94. 30 Vlug A, Nieuwenhuys EJ, van Eijk RV, Geertzen HG, van Houte AJ. Nephelometric measurements of human IgG subclasses and their reference ranges. Ann Biol Clin (Paris) 1994; 52:561–7. 31 Whaley MJ, Rose C, Martinez J et al. Interlaboratory comparison of three multiplexed bead- based immunoassays for measuring serum antibodies to pneumococcal polysaccharides. Clin Vaccine Immunol 2010; 17:862–9. 32 van Kessel DA, van Velzen-Blad H, van den Bosch JM, Rijkers GT. Impaired pneumococcal antibody response in bronchiectasis of unknown aetiology. Eur Respir J 2005; 25:482–9. 33 Jackson LA, Gurtman A, van Cleeff M et al. Immunogenicity and safety of a 13-valent pneumococcal conjugate vaccine compared to a 23-valent pneumococcal polysaccharide vaccine in pneumococcal vaccine-naive adults. Vaccine 2013; 31:3577–84. 34 Chen M, Hisatomi Y, Furumoto A et al. Comparative immune responses of patients with chronic pulmonary diseases during the 2-year period after pneumococcal vaccination. Clin Vaccine Immunol 2007; 14:139–45. 35 Butler WT, Rossen RD. Effects of corticosteroids on immunity in man. I. Decreased serum IgG concentration caused by 3 or 5 days of high doses of methylprednisolone. J Clin Invest 1973; 52:2629– 40. 36 de Roux A, Schmidt N, Rose M, Zielen S, Pletz M, Lode H. Immunogenity of the pneumococcal polysaccharide vaccine in COPD patients. The effect of systemic steroids. Respir Med 2004; 98:1187–94. 37 Steentoft J, Konradsen HB, Hilskov J, Gislason G, Andersen JR. Response to pneumococcal vaccine in chronic obstructive lung disease – the effect of ongoing, systemic steroid treatment. Vaccine 2006; 24:1408–12. 38 Casanova JL, Abel L. Human mannose-binding lectin in immunity: friend, foe, or both? J Exp Med 2004; 199:1295–9. CHAPTER 3

Clinical and immunological evaluation of patients with mild IgG1 deficiency

D.A. van Kessel1 P.E. Horikx†2 A.J. van Houte3 C.S. de Graaff2 H. van Velzen-Blad4 G.T. Rijkers5

1Department of Pulmonology, St. Antonius Hospital, Nieuwegein, The Netherlands 2Department of Pulmonology, Medical Center Alkmaar, Alkmaar, The Netherlands 3Department of Clinical Chemistry, Hematology and Immunology, Medical Center Alkmaar, Alkmaar, The Netherlands 4Department of Medical Microbiology and Immunology, St. Antonius Hospital, Nieuwegein, The Netherlands 5Department of Immunology, Wilhelmina Children’s Hospital, Utrecht, The Netherlands

Clin Exp Immunol 1999 Oct; 118(1):102-7 40 Chapter 3

Abstract

Serum IgG subclass concentrations were determined in patients visiting, the pulmonology out-patient clinic with chronic respiratory tract problems. A total of 24 patients with a serum IgG1 concentration < 4·9 g/l (i.e. below the reference range) and normal values for IgG2, IgM and IgA were included. Patients with a selective IgG1 deficiency were vaccinated with a 23-valent pneumococcal polysaccharide vaccine. There were nine patients with a poor antibody response to pneumococcal capsular polysaccharide antigens. Responsive- ness to protein antigens was intact in all patients. Patients with pneumonia showed a significantly lower anti-polysaccharide response in the IgG2 subclass than patients without pneumonia. Patients with recurrent sinusitis showed a significantly lower response in the IgA isotype after vaccination with pneumococcal polysaccharide vaccine compared with non-sinusitis patients. It can be concluded that patients with recurrent sinopulmonary infections and a mild IgG1 subclass deficiency have an impaired IgG1 anti-polysaccharide response, which can extend to decreased IgG2 and IgA anti-polysaccharide responses. Mild IgG1 deficiency 41

Introduction

Pneumococci are a leading cause of bacterial upper and lower respiratory tract infections. Laboratory investigation in patients with recurrent sinopulmonary infections may show a normal serum immunoglobulin level, but an IgG subclass deficiency can be found.1

Pneumococcal polysaccharide antibodies in adults are mostly of the IgG2 subclass.2, 3 In (paediatric) patients with a selective IgG2 deficiency and recurrent sinopulmonary infections a pneumococcal antibody deficiency is frequently found. 4–7 These data indicate the importance of IgG2 in clearing pneumococci. In young children, the antibody response to pneumococcal capsular polysaccharides belongs predominantly to the IgG1 subclass, yet in children with dysimmuno globulinaemia a defective IgG2 antipolysaccharide antibody response is found.8

There is no consensus in the literature on the clinical relevance of an isolated IgG1 deficiency in adult patients with a normal serum level of IgG2.9 Within an adult patient population with chronic bacterial respiratory infections, an isolated IgG1 subclass deficiency can be found,10–14 but normal IgG1 levels are also described in these patients.15, 16

In order to determine the impact of a selective IgG1 deficiency, we studied the serum antibody response after immunization with a 23-valent pneumococcal vaccine in a group of 24 patients with a history of recurrent respiratory infections with encapsulated bacteria and decreased serum levels of IgG1.

Patients and methods

Patient population

Adult patients were referred to the out-patient pulmonary department of the Sint Antonius Hospital Nieuwegein or the Medical Centre Alkmaar with the primary complaint of recurrent sinopulmonary infections, defined as three or more infectious episodes a year. Pneumonia was diagnosed by a positive sputum culture and confirmed by a chest x-ray. Sinusitis was defined as symptomatic inflammation of the paranasal sinuses lasting no longer than 6–8 weeks and occurring fewer than four times a year (acute) or more than 8 weeks at a time and four or more times a year (chronic).17

Patients were included in the study when serum IgG1 levels were below the reference range (< 4·9 g/l)18 on at least two occasions. Other IgG subclasses and 42 Chapter 3

immunoglobulin isotypes should be normal. Patients with bronchiectasis, cystic fibrosis or a1-anti-trypsin α( 1-AT) deficiency were excluded.

Patients were evaluated by reporting their clinical history, including the number of respiratory infections, dyspnoea, allergy, use of maintenance antibiotics, use of corticosteroid therapy and smoking habits. Pulmonary function testing was carried out according to the standards of the European Respiratory Society.19, 20 At entry into the study a blood leucocyte count with differentiation was performed, and serum α1-AT determined. Serum immunoglobulins and IgG subclasses were determined by nephelometry or radial immunodiffusion.18

In all patients, blood group isohaemagglutinins and diphtheria/tetanus/ poliomyelitis (DTP) antibody titres were determined. In selected patients, DTP antibody titres pre- and post-vaccination were measured (see below). A high resolution computed tomography was performed to exclude bronchiectasis. A sweat test was used to exclude cystic fibrosis. Sputum was cultured using standard microbiological techniques.

A mild IgG1 deficiency was defined as the condition in which on two subsequent occasions IgG1 levels were < 4·9 g/l. IgG2 levels as well as IgA and IgM had to be within normal range (1·5–6·4, 0·7–4·0 and 0·4–2·3 g/l, respectively).

All patients in the study were immunized intramuscularly with a 23-valent pneumococcal vaccine (Pneumovax; Merck and Co., Rahway, NJ) containing 25 µg of purified type-specific capsular polysaccharides of 23 pneumococcal serotypes (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F; Danish nomenclature). Before and 3 weeks after immunization blood samples were drawn. Serum samples were stored at –20°C until use.

Anti-capsular polysaccharide antibody assays

Total antibodies to capsular polysaccharides of Streptococcus pneumoniae serotypes 3, 4 and 9 in pre- and post-immunization serum samples were measured by ELISA.21, 22 In addition, antibodies of the IgA, IgG1 and IgG2 isotype/subclass to capsular polysaccharides of S. pneumoniae serotypes 3, 4, 6B, 9V, 14, 19F and 23F were measured in post-immunization samples only.21 Pre- and post- immunization serum samples were preincubated with excess (50 µg/ml) pneumococcal common cell wall polysaccharide (CPS) overnight at 4°C before analysis to block anti-CPS antibodies.2, 23 Serum from a normal non-vaccinated adult was included in each ELISA run to control for interassay variability. Antibody concentrations in patient samples were expressed relative to a reference adult hyperimmune plasma pool.24 This plasma pool contains 2648 ng Mild IgG1 deficiency 43

antibody N/ml for serotype 3; 1196 ng antibody N/ml for serotype 4; 539 ng antibody N/ml for group 6; 927 ng antibody N/ml for group 9 and 440 ng antibody N/ml for group 19.24 Antibody concentrations in the plasma pool were assigned 100 U/ml (100%) for each isotype and each serotype.

A group of 34 adults (age range 30–72 years) was vaccinated with a 23-valent pneumococcal polysaccharide vaccine and antibody levels to pneumococcal serotypes 3, 4 and 9 were determined before and 14 days after vaccination. Post-vaccination antibody titres were 87 (35–220) (geometric mean (x/: s.d.) for serotype 3, 53 (21–148) for serotype 4 and 68 (18–393) for serotype 9. Based on criteria previously used,25 the minimum post-vaccination titre required to qualify as a responder would be 35, 21 and 18 U/ml, respectively. For the sake of uniformity and in line with previous publications from our group,21, 22 we have used a threshold value of 20 U/ml for every serotype.

Patients were thus classified as responder or non-responder on the basis of their total pneumococcal polysaccharide antibody response after vaccination. A responder was defined as having a post-vaccination titre > 20 U/ml and at least a two-fold increase for two of the three pneumococcal serotypes tested. To allow for statistical analysis, the IgA, IgG1 and IgG2 antibody levels to pneumococcal serotypes 3, 4, 6B, 9V, 14, 19F and 23F in postimmunization samples were converted into positive (> 50 U/ml) or negative (< 50 U/ml).

To analyse further the immune response in patients with decreased serum IgG1 levels,26 patients with DTP antibody titres below protective levels were vaccinated (DTP; National Institute of Public Health and the Environment, Bil- thoven, The Netherlands) and antibody levels were determined 3 weeks later.27

Statistical analysis

Antibody concentrations were log converted and expressed as geometric means. Comparisons of the geometric mean antibody levels of responder and non-responder patients were made by the unpaired Student’s t-test. The total antibody titres to three pneumococcal serotypes and the IgG1, IgG2 and IgA antibody titres to seven pneumococcal serotypes in the various patient subgroups were converted into positive or negative and subsequently compared with the Fisher’s exact test. p < 0·05 was considered statistically significant. 44 Chapter 3

Results

Serum IgG subclasses were determined in patients with recurrent sinopulmonary infections, irrespective of the total serum IgG concentration. In the period from 1992 to 1996, 24 patients showed a serum IgG1 concentration below the lower limit of the reference range and fulfilled the inclusion criteria (Figure 1). The median total IgG level was 7·7 g/l (range 4·9–11·1 g/l); 9/24 patients had a total serum IgG level below the reference range (7·0–16·0 g/l). None of the patients showed decreased serum IgM or IgA levels (Figure 1). The median IgG1 level in the patients was 3·9 g/l (range 2·4–4·6 g/l); the reference range for serum IgG1 in normal healthy adults is 4·9–11·4 g/l.18 In all patients serum IgG2 levels were within the reference range (1·5–6·4 g/l). Median values for serum IgG3 and IgG4 were within the normal reference range, although two patients showed decreased serum IgG3 and two decreased serum IgG4 (data not shown).

All 24 patients were vaccinated with a 23-valent pneumococcal polysaccharide vaccine and antibodies to pneumococcal serotypes 3, 4 and 9F were determined in sera obtained before and 2–3 weeks post-vaccination. A responder was defined as having a postvaccination titre > 20 U/ml and at least a two-fold increase for two of the three serotypes tested. According to these criteria nine patients were classified as non-responders and 15 as responders. There were no differences in prevaccination anti-serotype 4 or anti-serotype 9F titres between responder and non-responder patients (Figure 2). A low prevaccination titre against serotype 3, however, was indicative for non-responsiveness because all six patients with low anti-serotype 3 titres before vaccination turned out to be non-responders.

Having defined pneumococcal polysaccharide vaccine responder and nonresponder patients, we subsequently investigated whether the hyporespon siveness to pneumococcal polysaccharides also extends to protein antigens. To that end, anti-DTP antibodies were determined, and when antibody titres were below the considered protective levels patients were vaccinated with DTP. All patients showed a positive antibody response to diphtheria toxoid (geometric mean fold-increase in antibody titre: ×23), tetanus toxoid (×19) and poliomyelitis type 1 (×14), type 2 (×21), and type 3 (×37). There was no difference in the anti-DTP antibody response between pneumococcal polysaccharide vaccine responders (n=4) and non-responders (n=6; p > 0·05 in all cases).

From these data it can be concluded that responsiveness to protein antigens appears to be intact in all patients. The clinical characteristics of both pneumococcal polysaccharide vaccine responding and non-responding patients are shown in Table 1. There was no difference in IgG1 concentrations, age distribution, sex ratio, atopic constitution, degree of obstructive lung disease (scored as percent forced expiratory volume in 1 s (FEV1) of predicted), incidence of pneumonia, smoking habits and corticosteroid or antibiotic use between the two groups. Mild IgG1 deficiency 45

Figure 1. Serum IgM, IgG, IgA and IgG subclass levels (g/l ). Adult reference ranges are boxed

Figure 2. Anti-pneumococcal polysaccharide antibody responses to vaccination in IgG1-deficient patients. Antibodies to capsular polysaccharides of pneumococcal serotypes 3, 4 and 9V were determined before and 2–3 weeks after vaccination with 23-valent pneumococcal polysaccharide vaccine. To that end, polysaccharide-coated ELISA wells were incubated with serial dilutions of patient sera, washed and developed with anti-human immunoglobulin reagents. Each symbol represents an individual patient; correspon- ding pre- and post-immunization data points are connected with a drawn line. Patients who are classified as responders are indicated by closed symbols (n=15); open symbols represent non-responders (n=9). 46 Chapter 3

Table 1. Clinical characteristics of pneumococcal polysaccharide vaccine responding and non-responding patients

All patients Non-responders Responders 24 (100%) 9 (100%) 15 (100%) 3·9 3·8 4·0 IgG1 (g/l) range 2·4–4·6 range 2·4–4·4 range 2·6–4·6 Male/female 13/11 7/2 6/9 49 49 49 Age (years) range 33–70 range 33–67 range 35–70 Allergy 7 (29) 3 (33) 4 (27)

FEV1 as percentage of normal Normal > 80% 6 (25) 3 (33) 3 (20) Mild ≤ 70% to ≤ 79% 6 (25) 4 (44) 2 (13) Moderate > 50% to ≤ 69% 7 (28) 1 (11) 6 (40) Severe ≤ 50% 5 (21) 2 (22) 3 (20) Pneumonia 12 (50) 5 (55) 7 (47) Sinusitis 14 (58) 5 (55) 9 (60) Smoking 8 (33) 3 (33) 5 (33) Corticosteroids 10 (42) 5 (55) 5 (33) Antibiotics 5 (21) 2 (22) 3 (20) Streptococcus pneumoniae 9 (38) 5 (55) 4 (27

In order to define better pneumococcal polysaccharide vaccine responsiveness and non-responsiveness, pre- and post-immunization sera were subsequently analysed for IgG1, IgG2 and IgA antibodies against capsular polysaccharides of seven different pneumococcal serotypes (Figure 3). As expected, in the majority of patients there was no or a very low increase of IgG1 antibodies, and for pneumococcal serotypes 3, 4, 14 and 19F especially, low antibody titres were found (Figure 3). More unexpected was the finding that there was also a low IgG2 and/or a low IgA response in a number of patients. Previously we have defined an anti-capsular polysaccharide antibody deficiency as the condition in which, following vaccination, antibody levels were < 50 U/ml for at least five out of seven pneumococcal serotypes.21 According to this definition 16 out of 24 patients were IgG1 antibodydeficient; five were IgG2 antibody-deficient, of whom two were IgA antibody-deficient as well.

The nine patients with one or more positive sputum cultures of S. pneumoniae in their clinical history demonstrated a significantly lower IgA antibody response than patients with consistently negative cultures (p < 0·01; mean number of pneumococcal serotypes with a post-vaccination IgA antibody titre > 50 U/ml 4·2 and Mild IgG1 deficiency 47

Figure 3. IgA, IgG1 and IgG2 anti-pneumococcal polysaccharide antibody titres 2–3 weeks after immunization with 23-valent pneumococcal polysaccharide vaccine of 24 patients with recurrent respiratory tract infections and decreased serum IgG1. IgA, IgG1 and IgG2 antibodies against pneumococcal serotypes 3, 4, 6B, 9V, 14, 19F and 23F were determined by ELISA. Post-immunization titres < 50 U/ml are indicated in black. Patients are numbered consecutively; patients 1–12 (upper panels) did not have pneumonia, patients 13–24 (lower panels) did have one or more cases of diagnosed pneumonia in their clinical history. *Patient with a history of chronic or acute sinusitis.

6·0, respectively). Patients with an isohaemagglutinin titre < 1:16 showed a lower IgA antibody response (p < 0·01; mean number of pneumococcal serotypes with a post-vaccination IgA antibody titre > 50 U/ml 4·1 and 5·9, respectively) and a lower IgG2 response (p < 0·01; mean number of pneumococcal serotypes with a postvaccination IgG2 antibody titre > 50 U/ml 2·8 and 5·5, respectively).

Twelve patients had a documented pneumonia in their clinical history. The IgG2 antibody response in the pneumonia subgroup was significantly lower than in patients without pneumonia (p < 0·5; mean number of pneumococcal serotypes with a post-vaccination IgG2 antibody titre > 50 U/ml 3·8 and 5·2, respectively). The IgG1 and IgA antibody response between pneumonia and non-pneumonia patients did not differ. Of the 24 patients who were vaccinated with pneumococcal polysaccharide vaccine, 14 patients had recurrent sinusitis. The IgA antibody response of these patients was significantly lower than in the remaining 10 patients (p < 0·05; mean number of pneumococcal serotypes with a post-vaccination IgA antibody titre > 50 U/ml 5·0 and 6·1, respectively). The IgG1 and IgG2 antibody response between sinusitis and non-sinusitis patients did not differ. 48 Chapter 3

Discussion

Pneumococci are a leading cause of bacterial upper and lower respiratory tract infections. The results presented in this study show that patients with a mild IgG1 deficiency and recurrent respiratory tract infections have a defective IgG1 antibody response to polysaccharide antigens that can extend to the IgG2 and/or IgA response. It should be noted that the latter hyporesponsiveness could not be predicted by serum IgG2 or IgA levels, because these were within the normal range. Our data show that an antipolysaccharide antibody deficiency can extend beyond the deficient IgG subclass deficiency in itself. These data are compatible with findings in patients with a selective IgA deficiency, where a defective IgG2 anti-polysaccharide response is frequently observed.22

Selective IgG1 deficiency has been described mainly in adults over 40 years.28 Also in our patient population, the median age is 49 years and 19/24 patients are > 40 years old. The relation between onset of clinical symptomatology and onset of IgG1 deficiency is not clear in all patients.

There is no consensus in the literature whether IgG1 deficiency can be considered a separate entity or only occurs in combination with a deficiency of other IgG subclasses. It therefore remains a matter of debate whether the term IgG1 deficiency should be used for this condition. Furthermore, quantitatively the IgG1 deficiency in our patients was mild because residual IgG1 concentrations varied between 49% and 94% of the lower limit of the reference range. By comparison, IgA deficiency is defined by an IgA concentration in serum < 0·02 g/l, which is < 5% of the lower limit of the reference range. In a large series of patients described by Lacombe et al. this mild form of IgG1 deficiency is the most common.9 IgG1 antibody deficiency, however, appears to be much more severe than immunoglobulin deficiency: for most pneumococcal polysaccharides IgG1 antibody levels are below 10% of normal. As indicated above, in addition to the more or less general IgG1 anti-polysaccharide hyporesponsiveness, a number of patients also display a poor IgG2 and/or IgA antibody response. The patients with documented cases of pneumonia had a significant lower pneumococcal polysaccharide IgG2 response, where the pneumococcal poly saccharide IgA response in patients with sinusitis were significantly decreased.

The polyvalent pneumococcal vaccine was given with a diagnostic purpose, i.e. to study responsiveness to polysaccharide antigens. In view of the poor antibody response it is questionable whether the vaccine would result in clinical protection against pneumococcal acquired pneumonia. Other patient groups at risk for invasive pneumococcal disease because of a poor antibody response to capsular polysaccharide antigens may benefit from pneumococcal conjugate Mild IgG1 deficiency 49

vaccination. A prerequisite is that unresponsiveness is restricted to T cell- independent type 2 (polysaccharide) antigens and does not extend to protein antigens. The patients with low pneumococcal polysaccharide responses also responded poorly to meningococcal C polysaccharide (data not shown) and had low isohaemagglutinin titres to blood group polysaccharide antigens. Therefore they may have a wider defect than can be overcome with a pneumococcal polysaccharide conjugate. Responsiveness to protein antigens appears to be intact in all our patients.

In a recent study it has been demonstrated that children with recurrent respiratory tract infections who failed to respond to the conventional pneumococcal polysaccharide vaccine were able to respond to the pneumococcal conjugate vaccine.29 We are currently vaccinating our patient group with the heptavalent CRM197 pneumococcal conjugate vaccine.

We did not find a correlation between the degree of obstructive lung dysfunction (%FEVl predicted) and the anti-pneumococcal antibody response or with the frequency of invasive infections. Apparently the emphysematous changes of the lung parenchyma do not constitute an independent variable predictive for severe pulmonary infections. On the other hand, the data presented suggest that there is a relation between the magnitude of the IgG2 antibody response and the severity of the clinical condition. While this suggests a prominent role for IgG2 antibodies in defence against respiratory tract infections, it should be noted that this relation is found against the background of a generalized defective IgG1 antibody response. In a recent publication it has been shown that 80–94% (depending on pneumococcal serotype) of IgG anti-pneumococcal antibodies are of the IgG2 subclass.30

A deficient IgG1 antibody response may therefore not show up when evaluating the total anti-polysaccharide antibody response. Indeed, our data show that 16 patients were IgG1 antibodydeficient, while on the basis of total antibody nine patients were classified as non-responders. The clinical impact of a selective IgG1 anti-polysaccharide antibody deficiency may be limited. Patients with recurrent sinopulmonary infections can have a mild serum IgG1 deficiency, even when total IgG levels are within the reference range. Upon vaccination with pneumococcal polysaccharide vaccine, approximately 40% of patients show decreased antibody responses. When vaccination is not possible, a low prevaccination anti-pneumococcal polysaccharide serotype 3 antibody titre (< 20 U/ml) and low isohaemagglutinins (< 1:16) are indicative of non-responsiveness to pneumococcal polysaccharides. While the severity of the clinical condition correlates with antibody responsiveness, this correlation is not absolute: patients with an apparently sufficient antibody response may show serious infections. 50 Chapter 3

Apart from the magnitude of the anti-polysaccharide response, the functional quality (in opsonophagocytosis) of the antibodies produced and the expression of polymorphic Fcg receptors IIa (CD32) and IIIb (CD16) on phagocytes determine the effectiveness of the humoral immune response, and thus the clinical outcome of an infection with encapsulated bacteria.

References

1. Popa V, Kim K, Heiner DC. IgG deficiency in adults with recurrent respiratory infections. Ann Allergy 1993; 70:418–24. 2. Musher DM, Luchi MJ, Watson DA, Hamilton R, Baughn RE. Pneumococcal polysaccharide vaccine in young adults and older bronchitics: determination of IgG responses by ELISA and the effect of adsorption of serum with non-type-specific cell wall polysaccharide. J Infect Dis 1990; 161:728–35. 3. Barrett DJ, Ayoub EM. IgG2 subclass restriction of antibody to pneumococcal polysaccharides. Clin Exp Lmmunol 1986; 63:127–34. 4. Sorensen RU, Hidalgo H, Moore C, Leiva LE. Post-immunization pneumococcal antibody titers and IgG subclasses. Pediatr Pulmonol 1996; 22:167–73. 5. Umetsu DT, Ambrosino DM, Quinti I, Siber GR, Geha RS. Recurrent sinopulmonary infection and impaired antibody response to bacterial capsular polysaccharide antigen in children with selective IgG-subclass deficiency. N Engl J Med 1985; 313:1247–51. 6. Rynnel-Dagoo B, Freijd A, Hammarstrom L, Oxelius V, Persson MA, Smith CI. Pneumococcal antibodies of different immunoglobulin subclasses in normal and IgG subclass deficient individuals of various ages. Acta Otolaryngol (Stockh) 1986; 101:146–51. 7. Geha RS. IgG antibody response to polysaccharides in children with recurrent infections. Monogr Allergy 1988; 23:97–102. 8. Lane PJ, MacLennan IC. Impaired IgG2 anti-pneumococcal antibody responses in patients with recurrent infection and normal IgG2 levels but no IgA. Clin Exp Immunol 1986; 65:427–33. 9. Lacombe C, Aucouturier P, Preud’homme JL. Selective IgG1 deficiency. Clin Immunol Immu- nopathol 1997; 84:194–201. 10. Gross S, Blaiss MS, Herrod HG. Role of immunoglobulin subclasses and specific antibody de- terminations in the evaluation of recurrent infection in children. J Pediatr 1992; 121:516–22. 11. Jiang LP, Yang XQ, Li CR, Zhang YW, Wang U, Shen J. Immunoglobulin G subclass deficiency in children with recurrent respiratory tract infections. Chin Med J (Engl) 1991; 104:119–23. 12. Noyes J, Woodmansee D, Chervinsky P. IgG subtype abnormalities with normal total IgG in a clinical allergy practice. Ann Allergy 1986; 57:273–5. 13. Feldman C, Weltman M, Wadee A, Sussman G, Smith C, Zwi S. A study of immunoglobulin G subclass levels in black and white patients with various forms of obstructive lung disease. S Afr Med J 1993; 83:9–12. 14. Kelsey SM, Lowdell MW, Newland AC. IgG subclass levels and immune reconstitution after T cell-depleted allogeneic bone marrow transplantation. Clin Exp Immunol 1990; 80:409–12. 15. DeBaets F, Kint J, Pauwels R, Leroy J. IgG subclass deficiency in children with recurrent bronchitis. Eur J Pediatr 1992; 151:274–8. 16. Loftus BG, Price JF, Lobo-Yeo A, Vergani D. IgG subclass deficiency in asthma. Arch Dis Child 1988; 63:1434–7. 17. Senior BA, Kennedy DW. Management of sinusitis in the asthmatic patient. Ann Allergy Asthma Immunol 1996; 77:6–15. 18. Vlug A, Nieuwenhuys EJ, van Eijk RV, Geertzen HG, van Houte AJ. Nephelometric measurements of human IgG subclasses and their reference ranges. Ann Biol Clin (Paris) 1994; 52:561–7. 19. Siafakas NM, Vermeire P, Pride NB et al. Optimal assessment and management of chronic obstructive pulmonary disease (COPD). The European Respiratory Society Task Force. Eur Respir J 1995; 8:1398–420. Mild IgG1 deficiency 51

20. Quanjer PH, Tammeling GJ, Cotes JE et al. Symbols, abbreviations and units. Working Party standardization of lung function tests, European Community for Steel and Coal. Eur Resp J Suppl 1993; 16:85–100. 21. Sanders EAM, Rijkers GT, Kuis W et al. Defective anti-pneumococcal polysaccharide antibody response in children with recurrent respiratory tract infections. J Allergy Clin Immunol 1993; 91:110–9. 22. Sanders LA, Rijkers GT, Tenbergen-Meekes AM, Voorhorst-Ogink MM, Zegers BJ. Immuno- globulin isotype-specific antibody responses to pneumococcal polysaccharide vaccine in patients with recurrent bacterial respiratory tract infections. Pediatr Res 1995; 37:812–9. 23. Goldblatt D, Levinsky RJ, Turner MW. Role of cell wall polysaccharide in the assessment of IgG antibodies to the capsular polysaccharides of Streptococcus pneumoniae in childhood. J Infect Dis 1992; 166:632–4. 24. Siber GR, Ambrosino DM, McIver J et al. Preparation of human hyperimmune globulin to Hae- mophilus influenzae b, Streptococcus pneumoniae and Neisseria meningitidis. Infect Immun 1984; 45:248–54. 25. Shackelford PG, Granoff DM, Polmar SH et al. Subnormal serum concentrations of IgG2 in children with frequent infections associated with varied patterns of immunologic dysfunction. J Pediatr 1990; 116:529–38. 26. Maslanka SE, Gheesling LL, LiButti DE et al. Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays. The Multilaboratory Study Group. Clin Diagn Lab Immunol 1997; 4:156–67. 27. Hendriksen CF, van der Gun JW, Kreeftenberg JG. Combined estimation of tetanus and diphtheria antitoxin in human sera by the in vitro Toxin-Binding Inhibition (ToBI) test. J Biol Stand 1989; 17:191–200. 28. Aucouturier P, Lacombe C, Preud’homme JL. Serum IgG subclass level determination: meth- odological difficulties and practical aspects. Ann Biol Clin (Paris) 1994;52 :53–56. 29. Sorensen RU, Leiva LE, Giangrosso PA et al. Response to a heptavalent conjugate Streptococcus pneumoniae vaccine in children with recurrent infections who are unresponsive to the polysaccharide vaccine. Pediatr Infect Dis J 1998; 17:685–91. 30. Soininen A, Seppala I, Wuorimaa T, Kayhty H. Assignment of immunoglobulin GI and G2 concentrations to pneumococcal capsular polysaccharides 3, 6B, 14, 19F, and 23F in pneumococcal reference serum 89-SF. Clin Diagn Lab Immunol 1998; 5:561–6.

CHAPTER 4

Impaired pneumococcal antibody response in patients with bronchiectasis of unknown aetiology

D.A. van Kessel1 H. van Velzen-Blad2 J.M.M. van den Bosch†1 G.T. Rijkers3

1Department of Pulmonology, St. Antonius Hospital, Nieuwegein, The Netherlands 2Department of Medical Microbiology and Immunology, St. Antonius Hospital, Nieuwegein, The Netherlands 3Department of Immunology, Wilhelmina Children’s Hospital, Utrecht, The Netherlands

Eur Respir J 2005 Mar; 25(3):482-9 54 Chapter 4

Abstract

As a defective anti-polysaccharide response can exist in the absence of an immunoglobulin deficiency, a series of 26 patients with bronchiectasis of unknown aetiology was vaccinated with a 23-valent pneumococcal polysaccharide vaccine. All patients suffered from recurrent respiratory tract infections.

When measuring total antibody levels to pneumococcal serotypes 3, 4 and 9, a normal polysaccharide antibody response was found in 22 patients. However, only 11 of these subjects showed a normal pneumococcal antibody response within the IgA and/or IgG2 subclass, and thus could be classified as true responders, while 15 patients did not respond in either the IgA class or in the IgG2 subclass.

When analysing differences between the responder (n=11) and nonresponder (n=15) groups, the latter demonstrated higher frequencies of respiratory tract infections and more severe lung pathology, as revealed by the presence of more bronchi visualised in the peripheral third of the lung by high-resolution computed tomography scanning. Moreover, nonresponders needed extensive lung surgery more often in order to control their disease (number of resected segments eight versus five).

In conclusion, an important fraction of patients presenting with idiopathic bronchiectasis is associated with a selective anti-polysaccharide response deficiency and this subgroup appears to represent a more severe clinical phenotype. Therefore, it can be regarded as a separate clinical entity with possible therapeutic targets. In order to identify IgA and IgG2 anti-polysaccharide non responders, all patients presenting with bronchiectasis of unkown aetiology should be immunised with a pneumococcal polysaccharide vaccine, and IgA and IgG2 isotype responses should be evaluated as well as the total antibody response. Pneumococcal vaccination in bronchiectasis 55

Introduction

Bronchiectasis is defined as chronic dilatation of bronchi, which is presently best diagnosed by high-resolution computed tomography (HRCT) of the chest.1 Patients with bronchiectasis can be asymptomatic, but many suffer from recurrent respiratory tract infections, with productive cough, dyspnoea and occasionally haemoptysis. Disease severity can range from mild disease requiring only antibiotics on demand, to severe forms, which may evolve into end-stage lung disease, ultimately requiring lung transplantion.2

There are many known causes of bronchiectasis, which can be grouped into extrinsic (postinfectious) and intrinsic (noninfectious) causes. In the past, bronchiectasis was most commonly a post-infectious complication.3 However, since the introduction of antibiotics, effective tuberculostatic drugs and the widespread implementation of childhood vaccination programmes, the incidence of post-infectious bronchiectasis has dramatically decreased. At present, bacterial infections in cystic fibrosis (CF) and allergic bronchopulmonary aspergillosis (ABPA) are amongst the most frequent forms of post-infectious bronchiectasis encountered in pulmonary medicine.4, 5

Intrinsic causes of bronchiectasis include α1- antitrypsin deficiency, mucociliary clearance defects, rheumatoid arthritis and various forms of (humoral) immunodeficiency. In particular, patients with an impaired antibody response to encapsulated bacteria are thought to be at risk for recurrent respiratory tract infections, which may lead to bronchiectasis.

A selective anti-polysaccharide antibody deficiency can be present in individuals with normal total immunoglobulin levels. Ambrosino et al.6 were the first to describe this phenomenon, which was later confirmed by others.7 Therefore, currently, a selective anti-polysaccharide antibody deficiency is a well-recognised entity in the context of recurrent respiratory tract infections.

Anti-pneumococcal antibody responses predominantly reside in the IgA and IgG2 subclasses. It has previously been demonstrated by the current authors that defective IgA antibody responses can be found after vaccination with a 23- valent pneumococcal polysaccharide vaccine in patients with recurrent sinusitis, despite normal total serum immunoglobulin levels.8 In addition, in the same paper, defective IgG2 antibody responses were reported upon pneumococcal polysaccharide vaccination in patients presenting with pneumonia.8 Therefore, these results prompted an extension of the study to include antibody responses to pneumococcal polysaccharide vaccination in patients with idiopathic bronchiectasis and normal total serum immunoglobulin levels. 56 Chapter 4

Patients and methods

Patient population

The study was designed as a consecutive clinical attendees’ cohort study, which included 26 patients who were recruited at the outpatient pulmonary department of the Sint Antonius Hospital (Nieuwegein, The Netherlands). All patients complained of recurrent respiratory tract infections caused by bronchiectasis. Twenty-three patients were female, which is a common finding in idiopathic bronchiectasis.9 Subjects were included when serum levels of immunoglobulins, IgG1 and G2 subclasses, and IgE were within the normal range: IgA 0.5– 3.7 g/l, IgM 0.4–2.3 g/l, IgG 8–17 g/l, IgG1 4.9–11.4 g/l, IgG2 1.5–6.4 g/l, IgE,100 kU/L.

Bronchiectasis patients with a history of tuberculosis or exposure to Myco bacterium tuberculosis (based on a positive tuberculin skin test), those with indications for aspiration or with severe respiratory infections during childhood were excluded. CF was excluded by a sweat test and/or genotyping the CF transmembrane regulator gene (two patients).

As hypersensitivity reactions to Aspergillus fumigatus can play a role in the development of bronchiectasis,10 precipitating antibodies to A. fumigatus were determined. All but two patients scored negative in this test. The two patients with precipitins to A. fumigatus did not meet the other clinical and laboratory parameters for the diagnosis of ABPA (asthma, elevated total serum IgE, positive immediate skin test to A. fumigatus, serum IgE antibodies, blood eosino- 11 philia, increased serum IgE and central bronchiectasis). α1-Antitrypsin deficiency was excluded by normal serum levels of α1- antitrypsin. Primary ciliary dyskinesia was excluded by histological examination of nasal or bronchial biopsies in 10 patients or by analysis of mucociliary transport using 99mTc labelled albumen in the remainder.

The respiratory tract infection frequency of each patient was carefully assessed and based on reported infectious episodes. Infectious episodes were catego- rised as sinusitis, bronchitis or pneumonia. Sinusitis was defined as symptomatic inflammation of the paranasal sinuses lasting no longer than 6–8 weeks. Bronchitis was defined as having excessive mucous secretion.12 A sputum culture was performed if possible. Chest radiography was always used to confirm the clinical diagnosis of pneumonia.

Pulmonary function testing was carried out according to the standards of the European Respiratory Society.13, 14 Pneumococcal vaccination in bronchiectasis 57

Laboratory investigations

Standard laboratory investigations in all subjects included a blood leukocyte count with differentiation, serum α1- antitrypsin measurement, and determination of serum levels of immunoglobulins and IgG subclasses, which was performed by nephelometry or radial immunodiffusion.15 Sputum was cultured using standard microbiological techniques.

All patients in the study were immunised intramuscularly with a 23-valent pneumococcal vaccine (Pneumovax; Merck and Co., Rahway, NJ, USA), containing 25 µg of purified typespecific capsular polysaccharides of 23 pneumococcal serotypes (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F; Danish nomenclature). Blood samples were drawn before and 3 weeks after immunisation. Serum samples were stored at -20°C until use.

Anti-capsular polysaccharide antibody assays

Antibodies to capsular polysaccharides of Streptococcus pneumonia serotypes 3, 4 and 9V in pre- and post-immunisation serum samples were measured by ELISA.16 In addition, isotype-specific antibodies (IgA, IgG1 and IgG2) to sero types 4, 6B, 9V, 14, 19F and 23F were measured in pre- and postimmunisation samples. All antibody titres were calibrated on the international reference prep- eration 89-SF.16–18 Antibody concentrations in the reference preparation were assigned 100 U/ml for each isotype and each serotype (Figures 1 and 2).

Patients were classified as total responders or nonresponders on the basis of their total pneumococcal polysaccharide antibody response after vaccination. A responder was defined as having a post-vaccination titre > 20 U/ml and at least a two-fold increase for two of the three pneumococcal serotypes tested.

To allow for statistical analysis, the IgA and IgG2 antibody levels to pneumococcal serotypes 4, 6B, 9V, 14, 19F and 23F in post-immunisation samples were converted into positive (> 50 U/ml) or negative (< 50 U/ml). An isotype responder was defined as having a positive IgA and/or IgG2 antibody response to more than four out of six of the serotypes tested. 58 Chapter 4

Figure 1. Antibody response to pneumococcal serotypes 3, 4 and 9V. Antibody titres were measured before (pre) and 21 days after (post) vaccination with 23-valent pneumococcal polysaccharide vaccine. An antibody concentration of 100 U/ml corresponds with 7.29 µg/ml anti-serotype 3, 6.63 µg/ ml antiserotype 4 and 10.26 µg/ml anti-serotype 9V. : nonresponders; : responders.

Radiological classification

The presence, severity and extent of bronchiectasis were determined by bronchog- raphy19 in two patients and by HRCT in 24 patients. HRCT scanning was carried out with a Philips SR 7000 CT-scanner (Philips, Eindhoven, The Netherlands). Pa- tients were put into the supine position. No (intravenous) contrast material was used. Measurements were made at end-inspiratory volume. Slices 1.5 mm thick were obtained at 10-mm intervals from the costophrenic angles to the lung apices and reconstructed with a high-spatial algorithm. Scanning parameters included a voltage of 140 kV of 175 mA with a scan time of 1 s. For images, a window width of 1500 HU, a window level of -750 HU/1500 HU and volumetric scanning were used.

The HRCT scans were analysed according to the criteria described by McGuinness et al.,1 Naidich et al.20 and Reiff et al.21 This classification system includes three categories: cylindrical, cystic and varicose bronchiectasis. Addi- tionally, another analysis was performed, according to the criteria described by Diederich et al.22 The major items scored in this system were as follows. 1) Bronchial dilatation as compared with the diameter of the adjacent pulmonary artery was divided into three categories: normal < 110%, mild 110– 150% and severe > 150%. 2) The luminal diameter of the bronchus as compared with the total diameter of the bronchus was also divided into three categories: normal > 80%, mild 80 – 50% and severe < 50%. 4) The bronchial contents was divided into two categories: air or mucus. 5) The cysts in cystic bronchiectasis were placed into three groups: < 1 cm, 1–2 cm or > 2 cm in size. Pneumococcal vaccination in bronchiectasis 59

Figure 2. a) IgG1, b) IgG2 and c) IgA antibody responses to pneumococcal serotypes 4, 6B, 9V, 14, 19F and 23F. Antibody titres were measured before (pre) and 21 days after (post) vaccination with 23-valent pneumococcal polysaccharide vaccine. An antibody concentration of 100 U/ml corresponds with 0.48 µg/ml IgG1, 3.59 µg/ml IgG2, 1.18 µg/ml IgA anti-serotype 4; 0.66 µg/ml IgG1, 10.54 µg/ml IgG2, 1.52 µg/ml IgA anti-serotype 6B; 0.80 µg/ml IgG1, 6.10 µg/ml IgG2, 1.70 µg/ml IgA anti-serotype 9V; 2.87 µg/ml IgG1, 21.23 µg/ml IgG2, 1.94 µg/ml IgA anti-serotype 14; 1.12 µg/ml IgG1, 10.09 µg/ml IgG2, 1.98 µg/ml IgA anti-serotype 19F; 0.73 µg/ml IgG1, 6.66 µg/ml IgG2, 1.29 µg/ml IgA anti-serotype 23F. 60 Chapter 4

Other signs of severity of disease were recorded as either present or absent: vis- ualisation of bronchi in the peripheral third of the lung, volume loss, pulmonary consolidation, emphysema, interstitial lines and nodules. The extent of abnormal bronchi per lobe was recorded in four categories: < 25% involvement, 26–50% involvement, 51–75% involvement and > 75% involvement.

Statistics

The IgG2 and IgA antibody titres to six pneumococcal serotypes in the various patient subgroups were converted into positive or negative and subsequently compared with the Fishers exact test. A p-value < 0.05 was considered statistically significant.

Results

A series of 26 patients was included in the study. In 24 subjects the diagnosis bronchiectasis was established based on HRCT of the chest and in two patients based on bronchography.

After pneumococcal polysaccharide vaccination, 22 patients showed a post- vaccination total antibody titre > 20 U/ml and at least a two-fold increase of two of the three pneumococcal serotypes tested (Figure 1). Therefore, on the basis of this test, 22 patients would have to be classified as responders and four as nonresponders.

However, determination of IgA and IgG2 antibodies to pneumococcal poly saccharides of serotypes 4, 6B, 9V, 14, 19F and 23F largely changed this classification (Figure 2). Defining an individual with a positive IgA and/or IgG2 antibody response for more than four out of six serotypes classified them as an isotype responder, an adequate response to the vaccine was observed in only 11 out of 22 responders in the total antibody evaluation. Three of these patients had both an IgA and IgG2 response. The remaining 11 patients had an inadequate IgA and IgG2 response, despite initially being classified as a total responder (Table 1).

Thus, based on measurement of the post-vaccination total antibody titre and the selective antibody response against pneumococcal polysaccharide in the IgA and IgG2 subclasses, the nonresponder group consisted of four total nonresponders and 11 isotype nonresponders. The clinical characteristics of the nonresponder group (n=15) and that of the responder group (n=11) are summarised in Table 2. Pneumococcal vaccination in bronchiectasis 61

Table 1. Selective defects in antibody response against pneumococcal polysaccharide in IgA and IgG subclasses

Responder Nonresponder Total p-value Patients 11 15 26 IgA anti-PPS response 4 0 4 0.0221 IgG2 anti-PPS response 10 0 10 < 0.001

IgA anti-serotype 4 6 2 8 0.0384 6B 0 0 0 9V 9 13 22 14 10 14 24 19F 6 3 9 23F 2 0 2

IgG2 anti-serotype 4 9 2 11 < 0.001 6B 0 0 0 9V 10 6 16 0.0143 14 11 13 24 19F 11 5 16 < 0.001 23F 7 0 7 < 0.001

Data are presented as n patients. PPS: pneumococcal polysaccharides. IgA and/or IgG2 isotype responder is defined as a post-vaccination titre > 50 U·ml−1 for more than four out of six pneumococcal serotypes tested. 62 Chapter 4

Table 2. Clinical characteristics of the nonresponder and responder groups

Responder Nonresponder Total p-value Patients 11 15 26 Female/male 11/0 12/3 23/3 Median age yrs 50(20-69) 49(25-69) 49(20-69)

FEV1% pred Normal ≥ 80% 5 9 14 Mild 70-79 1 2 3 Moderate 50-69 4 2 6 Severe < 50 1 2 3 Pneumonia 10 12 22 Sinusitis 8 13 21 Allergy 4 7 11 Smoking 1 1 2 Corticosteroids 1 0 1 Antibiotics 1 4 5 IgG4 deficiency 1 1 2 Infections per yr 5(3-12) 8(4-12) 8(3-12) 0.078 S. pneumonia 4 8 12 H. influenzae 7 12 19 M. catarrhalis 2 7 9 0.06 Surgery 4 8 12 Resected segments mean 3 8 0.029

Data are presented as n patients or median (range). FEV1: forced expiratory volume in one second. Fishers exact test was used to compare the two groups.

Comparison of clinical characteristics between responders and nonresponders

In both groups there was a remarkable predominance of females, i.e. 11 in the responder and 12 in the nonresponder group (p < 0.01). Median age was 49 yrs in the responder versus 50 yrs in the nonresponder group. No difference was observed in distribution of smoking behaviour, allergic constitution and use of antibiotics or corticosteroids.

Analysis of lung function showed normal values of forced expiratory volume in one second in five individuals (45%) of the responder group and in nine (60%) of the nonresponder group. In total, nine patients had a mild-to-moderate impairment and only three patients showed severe obstructive lung disease. Pneumococcal vaccination in bronchiectasis 63 Patients n Patients

Infections per year

Figure 3. Infection frequency in pneumococcal polysaccharide IgA or IgG2 antibody responders ( ) and nonresponders ( ).

Statistical analysis of these results showed no significant differences.

The number of respiratory tract infections ranged from three to 12 per year. The nonresponder group showed a higher number of infections, as illustrated in Figure 3.

Four patients underwent surgery in the responder group versus eight patients in the nonresponder group. Importantly, the nonresponders required more extensive surgery than the responders. These data indicate that patients with a defective pneumococcal antibody response show a higher infection frequency and require more extensive surgery.

Radiological evaluation of reponders and nonresponders

Results of evaluation of radiological characteristics in responder and non responder bronchiectasis patients are summarised in Table 3. Cylindrical bronchiectasis was the predominant finding in both groups. Three patients with cystic bronchiectasis were present in each group. No differences were found in scoring for bronchial contents and bronchial thickening, volume loss and consolidation. 64 Chapter 4

Table 3. Radiological characteristics

Responder Nonresponder Total Patients 11 13 24 Bronchiectasis Mild dilatation 11 13 24 Severe dilatation 0 0 0 Type of bronchiectasis Cylindrical 6 6 12 Cystic 1 2 3 Varicose 0 1 1 Indeterminate 0 0 0 More than one type 4 4 8 Cystic bronchiectasis None 10 12 22 1–2 cm average size 1 1 2 Bronchial wall thickening Mild thickening > 50% normal 10 13 23 Severe thickening < 50% normal 1 0 1 Bronchial dilatation and wall thickening None 1 1 2 Yes 10 12 22 Bronchial contents Air only 8 10 18 Air and fluid 3 3 6 Bronchi visualised in the peripheral third of the lung No 9 5 14 Yes 2 8 10# Volume loss No 10 9 19 Yes 1 4 5 Consolidation No 5 8 13 Yes 6 5 11 Emphysema No 10 10 20 Yes 1 3 4 Interstitial lines No 9 6 15 Yes 2 7 9

Data are presented as n patients. #: p=0.0472. According to the criteria as described by Diederich et al.22 Pneumococcal vaccination in bronchiectasis 65

Figure 4. High-resolution computed tomography (HRCT) analysis of bronchial abnormalities. The extent of radiological abnormalities is calculated as percentage category of abnormal bronchi in right (R) or left (L), upper (U), middle (M) or lower (L) lobes and in the lingual (LIN): a) RUL, b) LUL, c) RMl, d) LIN, e) RLL, f) LLL. : responders; : nonresponders.

Signs of emphysema were equally seen in both groups. There were significantly more visible bronchi in the peripheral third of the lung in the nonresponder group (p=0.0472), indicating more severe bronchiectasis.23

The extent of areas with abnormal bronchi per lobe was determined in all available HRCT scans. In agreement with previous data in the literature, the lower lobes tended to be more severly affected, and this was most prevalent in the nonresponding group (Figure 4).2

In 11 patients a follow-up HRCT scan was performed because of the high infection frequency. Ten of them were nonresponders. In four patients the HRCT scan showed progression of bronchiectasis. 66 Chapter 4

Discussion

It is shown in the present study that ~50% of the patients with idiopathic bronchiectasis and normal total serum immunoglobulin levels had a defective IgA and IgG2 anti-polysaccharide response. Clinically, these nonresponders demonstrated a higher rate of respiratory tract infections and showed more severe lung pathology as assessed by HRCT scanning. Humoral immunodeficiencies, -in cluding common variable hypogammaglobulinaemia and IgA or IgG2 subclass deficiency, are well-recognised aetiological risk factors for bronchiectasis.2

As antibodies to pneumococcal polysaccharides predominantly reside in the IgA and IgG2 subclasses, it could be anticipated that bronchiectasis patients with defective antipolysaccharide antibody responses show insufficient levels of anti-polysaccharide IgA and IgG2 upon vaccination with pneumococcal- derived polysaccharide. Selective antipolysaccharide antibody deficiency is associated with a variable clinical spectrum of recurrent and/or severe respiratory tract infections.7, 24 Bronchiectasis develops because respiratory pathogens are not handled adequately due to this humoral immunodeficiency, which results in permanent damage of lung tissue.

It is well recognised that patients with dysimmunoglobulinaemia have a defective anti-polysaccharide antibody response.8 In addition, in patients with bronchiectasis, in the context of dysimmunoglobulinaemia deficient, anti- polysaccharide antibody responses have been found upon vaccination. 4 How ever, this study now clearly shows that an antibody response deficiency may exist even in bronchiectasis patients with normal total immunoglobulin levels. Therefore, measurement of IgA and IgG subclass levels cannot be used as a substitute for a specific antibody response estimation.25 Consequently, it is advocated that (pneumococcal) vaccination is used to reveal specific antibody response deficiencies in apparently normal total immunoglobulin subclasses.

Pneumococcal vaccination in patients with bronchiectasis of unknown cause has been carried out in several studies and the response to vaccination has been measured by determination of total antibody concentrations to a mixture of 23 polysaccharides.4, 26, 27 In all these studies the vast majority of patients showed a normal response and, therefore, it was recommended that such screening is not required.22 Our initial screening procedure for anti-polysaccharide antibody deficiency also comprised determination of total antibody levels to pneumococcal serotypes 3, 4 and 9, which represent strong, intermediate and poor immunogenic polysaccharides, respectively. When using this approach a normal polysaccharide antibody response was found in 22 out of 26 patients studied. Hence, using the above screening only, 11 out of 15 of the non- Pneumococcal vaccination in bronchiectasis 67

responder patients would have been missed. The extended screening procedure for specific IgA and IgG2 antibody levels identified a significant number of nonresponders who initially had a normal total polysaccharide antibody response. Apparently, low levels of IgA and IgG2 antibodies are quantitatively compensated by other isotypes, notably IgM. Identification of the antibody deficiency required testing the response to six pneumococcal serotypes (in the IgG2 and IgA class). The failure to respond was not associated with any particular serotype lack of response (Table 1). Notably, there were no antibody responders to serotype 6B in either group; almost all patients had an IgG2 and IgA response to serotype 14.

From the clinical management point of view, it is important to recognise that nonresponder patients have a higher infection frequency, show more radiological abnormalities and have more extensive lung pathology. Of note, the radiological evaluation of bronchiectasis used in this study was based on the methodology as described by Naidich et al.,20 combined with criteria formulated by Diederich et al.22 This approach enabled the scoring of severity and extension of lung pathology. In the current series, cylindrical, mild bronchiectasis was mainly found, and only one patient showed progression of bronchiectasis on follow-up HRCT scan. No relationship was found between the clinical symptoms and disease progression on HRCT scan. This is in agreement with the fact that there is, to date, no satisfactory radiological scoring system for severity and progression of bronchiectasis.28

In the authors opinion, a sizeable fraction of patients with bronchiectasis of unknown cause can now be classified as bronchiectasis associated with polysaccharide antibody response deficiency, which might have important clinical implications for treatment. In patients with hypogammaglobulinaemia, immunoglobulin replacement therapy reduces the infection episodes and prevents further damage of the bronchi.29, 30 In this present patient group with a defective IgG2 antibody response, immunoglobulin replacement might supplement the polysaccharide antibodies, but this would require screening of batches with sufficient polysacharide antibody titres, as well as monitoring antibody levels in patients on substitution therapy.

In conclusion, all patients with bronchiectasis of unknown aetiology should be immunised with a pneumococcal polysaccharide vaccine. In addition, specific antibody responses within the IgA and IgG2 subclass should be evaluated as well as the total antibody response to identify this potentially new brochiecta- sis phenotype. 68 Chapter 4

References

1. McGuinness G, Naidich DP, Leitman BS, McCauley DI. Bronchiectasis: CT evaluation. AJR Am J Roentgenol 1993; 160: 253–259. 2. Barker AF. Bronchiectasis. N Engl J Med 2002; 346: 1383–1393. 3. Barker AF, Bardana EJ Jr. Bronchiectasis: update of an orphan disease. Am Rev Respir Dis 1988; 137: 969–978. 4. Pasteur MC, Helliwell SM, Houghton SJ, et al. An investigation into causative factors in patients with bronchiectasis. Am J Respir Crit Care Med 2000; 162: 1277–1284. 5. Shah PL, Mawdsley S, Nash K, Cullinan P, Cole PJ, Wilson R. Determinants of chronic infection with Staphylococcus aureus in patients with bronchiectasis. Eur Respir J 1999; 14: 1340–1344. 6. Ambrosino DM, Siber GR, Chilmonczyk BA, Jernberg JB, Finberg RW. An immunodeficiency characterized by impaired antibody responses to polysaccharides. N Engl J Med 1987; 316: 790–793. 7. Rijkers GT, Sanders LA, Zegers BJ. Anti-capsular polysaccharide antibody deficiency states.Im - munodeficiency 1993; 5: 1–21. 8. Van Kessel DA, Horikx PE, Van Houte AJ, De Graaff CS, Velzen-Blad H, Rijkers GT. Clinical and immunological evaluation of patients with mild IgG1 deficiency. Clin Exp Immunol 1999; 118: 102–107. 9. Keistinen T, Saynajakangas O, Tuuponen T, Kivela SL. Bronchiectasis: an orphan disease with a poorly-understood prognosis. Eur Respir J 1997; 10: 2784–2787. 10. Barker AF, Craig S, Bardana EJ Jr. Humoral immunity in bronchiectasis. Ann Allergy 1987; 59: 179–182. 11. Patterson R, Greenberger PA, Harris KE. Allergic bronchopulmonary aspergillosis. Chest 2000; 118: 7–8. 12. Fletcher CM, Pride NB. Definitions of emphysema, chronic bronchitis, asthma, and airflow obstruction: 25 years on from the Ciba symposium. Thorax 1984; 39: 81–85. 13. Siafakas NM, Vermeire P, Pride NB, et al. Optimal assessment and management of chronic obstructive pulmonary disease (COPD). The European Respiratory Society Task Force. Eur Respir J 1995; 8: 1398–1420. 14. Quanjer PH, Tammeling GJ, Cotes JE, et al. Symbols, abbreviations and units. Working Party Standardization of Lung Function Tests, European Community for Steel and Coal. Eur Respir J 1993; 6: Suppl. 16, 85–100. 15. Vlug A, Nieuwenhuys EJ, van Eijk RV, Geertzen HG, Van Houte AJ. Nephelometric measurements of human IgG subclasses and their reference ranges. Ann Biol Clin (Paris) 1994; 52: 561–567. 16. Sanders EA, Rijkers GT, Kuis W, et al. Defective antipneumococcal polysaccharide antibody response in children with recurrent respiratory tract infections. J Allergy Clin Immunol 1993; 91: 110–119. 17. Quataert SA, Kirch CS, Wiedl LJ, et al. Assignment of weight-based antibody units to a human antipneumococcal standard reference serum, lot 89-S. Clin Diagn Lab Immunol 1995; 2: 590–597. 18. Soininen A, Seppala I, Wuorimaa T, Kayhty H. Assignment of immunoglobulin G1 and G2 concentrations to pneumococcal capsular polysaccharides 3, 6B, 14, 19F, and 23F in pneumococcal reference serum 89-SF. Clin Diagn Lab Immunol 1998; 5: 561–566. 19. Silverman PM, Godwin JD. CT/bronchographic correlations in bronchiectasis. J Comput Assist Tomogr 1987; 11: 52–56. 20. Naidich DP, McCauley DI, Khouri NF, Stitik FP, Siegelman SS. Computed tomography of bronchiectasis. J Comput Assist Tomogr 1982; 6: 437–444. 21. Reiff DB, Wells AU, Carr DH, Cole PJ, Hansell DM. CT findings in bronchiectasis: limited value in distinguishing between idiopathic and specific types. AJR Am J Roentgenol 1995; 165: 261–267. 22. Diederich S, Jurriaans E, Flower CD. Interobserver variation in the diagnosis of bronchiectasis on high-resolution computed tomography. Eur Radiol 1996; 6: 801–806. 23. Diederich S, Roos N, Thomas M, Peters PE. [Diagnostic imaging in bronchiectases. Value of CT and HRCT]. Radiologe 1996; 36: 550–559. Pneumococcal vaccination in bronchiectasis 69

24. Ambrosino DM, Umetsu DT, Siber GR, et al. Selective defect in the antibody response to Hae- mophilus influenzae type b in children with recurrent infections and normal serum IgG subclass levels. J Allergy Clin Immunol 1988; 81: 1175–1179. 25. Maguire GA, Kumararatne DS, Joyce HJ. Are there any clinical indications for measuring IgG subclasses? Ann Clin Biochem 2002; 39: 374–377. 26. Snowden N, Moran A, Booth J, Haeney MR, Swinson DR. Defective antibody production in patients with rheumatoid arthritis and bronchiectasis. Clin Rheumatol 1999; 18: 132–135. 27. Stead A, Douglas JG, Broadfoot CJ, Kaminski ER, Herriot R. Humoral immunity and bronchiectasis. Clin Exp Immunol 2002; 130: 325–330. 28. Kang EY, Miller RR, Muller NL. Bronchiectasis: comparison of preoperative thin-section CT and pathologic findings in resected specimens. Radiology 1995; 195: 649–654. 29. Cunningham-Rundles C, Bodian C. Common variable immunodeficiency: clinical and immunological features of 248 patients. Clin Immunol 1999; 92: 34–48. 30. Roifman CM, Lederman HM, Lavi S, Stein LD, Levison H, Gelfand EW. Benefit of intravenous IgG replacement in hypogammaglobulinemic patients with chronic sinopulmonary disease. Am J Med 1985; 79: 171–174.

PART II

IMMUNE STATUS IN PATIENTS BEFORE AND AFTER LUNG TRANSPLANTATION

CHAPTER 5

Immune status assessment in adult lung transplant candidates

D.A. van Kessel1,2 T.W. Hoffman1 H. van Velzen-Blad3 E.A. van de Graaf2 J.C. Grutters1,2 G.T. Rijkers3,4

1Department of Pulmonology, St. Antonius Hospital, Nieuwegein, The Netherlands 2Division of Heart and Lungs, University Medical Center Utrecht, Utrecht, The Netherlands 3Department of Medical Microbiology and Immunology, St. Antonius Hospital, Nieuwegein, The Netherlands 4Department of Science, University College Roosevelt, Middelburg, The Netherlands

Transpl Immunol 2017 Feb; 40:31-4 74 Chapter 5

Abstract

Background: Lung transplant recipients have an increased susceptibility to a variety of infections due to immunosuppressive therapy. Current guidelines recommend pneumococcal and other vaccinations, prior to lung transplantation to protect against post-transplant infections, but measurement of the antibody response to vaccination is not advised. Immune status investigation in lung transplant candidates, including the response to pneumococcal poly saccharide vaccination, has not been described.

Methods: Immune status investigation, including measurement of immunoglobulins, complement and the response to 23-valent pneumococcal polysaccharide vaccination (23vPPV) was performed in 81 adult lung transplant candidates.

Results: Eighteen patients had low IgG levels and 32 patients had low IgG1 and/or IgG2 levels. After vaccination with 23vPPV the median antibody concentration of all serotypes increased significantly. Fifty-two patients had protective IgG-post- vaccination antibody levels to at least 10 serotypes. Twenty-nine patients had an impaired response to 23vPPV.

Conclusions: In conclusion, a significant proportion of our cohort of lung transplant candidates had one or more abnormalities in the immune status. It is likely that these patients have an increased risk for infections after transplantation. Revaccination, including measurement of antibody response, and possibly antibody replacement therapy should be considered to minimize infection risk. Immune status in lung transplant candidates 75

Introduction

Solid organ transplant recipients have an increased susceptibility to infections caused by a broad spectrum of pathogens.1 Pre-transplant screening and management are designed to minimize infection risk after transplantation. After transplantation, all patients receive immunosuppressive medication and can be considered immunodeficient. For optimal infection prevention it is advised to update vaccinations in transplant candidates prior to transplantation. Recommended vaccinations include 23-valent pneumococcal polysaccharide vaccination (23vPPV), 13-valent pneumococcal conjugate vaccine (13cPCV) and other (childhood) vaccinations.2, 3

Pneumococcal polysaccharide vaccination can be used to prevent pneumococcal disease but can also be used to measure the status of the humoral immune system and therefore is part of the immune investigation protocol in patients with suspected immunodeficiency.4 In current guidelines immune status investigation, including the response to pneumococcal polysaccharide vaccination, prior to lung transplantation is not included in the standard screening protocol. Hu- moral immunodeficiency is not a contraindication to lung transplantation,5 but some reports suggest that immunodeficient patients have a complicated post-transplant course.6

Lung transplant candidates suffer from end-stage lung disease. Chronic immunosuppressive therapy might influence immune status and vaccination responses in this population. It is of clinical importance to knowwhether these patients have an impaired immune status as this can increase infection risk after transplantation. As far as we are aware, this has not been studied before.

Therefore, we performed immune status investigation including evaluation of the polysaccharide antibody response in a group of lung transplant candidates.

Objective

The objective of this study is to assess the immune status of lung transplant candidates, including the responsiveness to pneumococcal vaccination. It therefore can provide the rational arguments for the existing vaccination guidelines. 76 Chapter 5

Materials and methods

The St. Antonius Hospital, Nieuwegein, the Netherlands, is a tertiary referral hospital for lung transplantation in collaboration with the University Medical Center in Utrecht. Patients are seen at the St. Antonius Hospitalwith all indications for lung transplantation except cystic fibrosis. This study reports on all patientswho underwent screening for lung transplantation in the St. Antonius Hospital in the period 2009–2012 and were placed on the waiting list.

Clinical characteristics, use of medication, immune status assessment at time of screening for lung transplantation and follow-up data were collected from patient records. All diagnoses were categorized according to the International Classification of Diseases 10 (ICD-10) used by Eurotransplant. Recurrent respiratory infections were defined as having three or more infectious periods per year. Infectious episodes were categorized as sinusitis, bronchitis or pneumonia. Medication use was reviewed and patients were considered to be on immunosuppressive therapy if this medication was used as maintenance therapy and had well-known immunosuppressive (side) effects.7

Standard immune investigation according to the protocol of the European Society of Immune Deficiencies (ESID)4 included immunoglobulins, IgG subclasses, isohaemaglutinins, and antibodies to protein antigens. Serum immunoglobulin and IgG subclass concentration were measured by nephelometry on an Immage 800 (Beckman-Coulter, Brea, CA, USA) and Siemens BN Prospec (Siemens Healthineers, Erlangen, Germany), respectively. Lower limits of normal for serum immunoglobulins and IgG subclasses were as follows: IgM0.4 g/l, IgG 7.0 g/l, IgA 0.7 g/l, IgG1 4.9 g/l, IgG2 1.5 g/l, IgG3 0.2 g/l, IgG40.08 g/l.8 Isohaemagglutinins were determined on (BG-0) test cells (Ortho-Diag- nostics, Turnhout, Belgium) in a 2-fold dilution series.

IgG antibodies to protein antigens of CMV, EBV, and Toxoplasma were determined on an automated immunoassay analyser (Liaison XL, DiaSorin, Saluggia, Italy). Complement activitywas determined by measuring AP (alternative pathway), CP (classical pathway) and MP (lectin pathway), using a commercially available enzyme immunoassay (Wielisa®;Wieslab, Lund, Sweden).9 When complementMP activity was < 10%, genotypingwas performed to confirm MBL deficiency. Genotypes O/O and XA/O were considered to be MBL deficient.

Patients were vaccinated intramuscularly with one dose of 23-valent pneumococcal polysaccharide vaccine (Pneumovax 23; Merck, Rahway, NJ, USA) containing 25 μg purified type-specific capsular polysaccharides of 23 pneumococcal serotypes. Blood samples were drawn before and 3–6 weeks after vaccination Immune status in lung transplant candidates 77

to evaluate the response to vaccination. Serum samples were stored at − 80°C until use. IgG antibodies against 14 different pneumococcal polysaccharides were measured on a Luminex platform (Luminex Corporation, Austin, TX, USA), using a quantitative multiplex immunoassay: the XMAP pneumococcal immunity panel. This assay allows measurement of serotype-specific anticap- sular polysaccharide IgG antibodies to the following serotypes: 1, 3, 4, 8, 9N, 12F, 14, 19F, 23F, 6B, 7F, 18C, 19A, and 9V (Danish nomenclature), correspond- ing to serotypes 1, 3, 4, 8, 9, 12, 14, 19, 23, 26, 51, 56, 57, and 68 according to the American nomenclature, respectively.

For categorization of the antibody response to pneumococcal polysaccharide vaccination, the 2015 AAAAI/ACAAI classification schedule was used.10 Protective antibody levels against an individual given pneumococcal serotype are defined as an antibody concentration N1.3 μg/ml. A positive immune response to a given serotype is defined as having a post-vaccination antibody concentration N1.3 μg/ml and a ≥ 2-fold antibody concentration increase between the pre- and postvaccination serum samples. A patient is considered to be a responder if at least 70% of the antibody responses to the serotypes tested (i.e. 10 of the 14 serotypes tested)were positive. According to this classification responses are categorized as 1) normal, 2) mildly impaired (antibody levels N1.3 μg/ml for ≥70% of serotypes, but ≥2-fold increase between pre- and post- vaccination antibody titers for < 70% of serotypes), 3) moderately impaired (antibody levels N1.3 μg/ml for < 70% of serotypes) or 4) severely impaired (antibody levels N1.3 μg/ml for ≤ 2 serotypes).

For statistical analyses the Fisher’s exact test, Mann-Whitney U test were used where appropriate. For data management and statistical analyses Microsoft Excel 2010 and IBM SPSS Statistics for Windows (version 22.0) were used. Graphsweremadewith GraphPad Prismversion 2.0. Differences were considered to be significant at the p-value < 0.05.

The local medical ethics committee approved the study. All patients gave formal written informed consent. 78 Chapter 5

Table 1. Clinical characteristics of 81 lung transplantation candidates

Patient Characteristics Total 81 Female 46 Median age (years) 52 Age range (years) 18-64 Eurotransplant Diagnosis Category COPD/Emphysema 43 Alpha-1 Antitrypsin Deficiency 8 Idiopathic Pulmonary Fibrosis 9 Other Pulmonary Fibrosis 11 Hypersensitivity Pneumonitis 2 Sarcoidosis 4 Lymphangiomyomatosis 1 Primary Pulmonary Hypertension 3 Infections Recurrent Respiratory Tract Infections (> 3/year) 29 Immunosuppressive Medication Use of maintenance corticosteroids ≥ 5mg/day only 26 Use of maintenance corticosteroids and other 11 immunosuppressive therapy Use of non-corticosteroid immunosuppressive 6 medication only

Results

Eighty-one patients on thewaiting list for lung transplantationwere included in this study (Table 1). 46 patients were female. The median age was 52 years. The majority of patients was diagnosed with emphysema or lung fibrosis. At time of screening for lung transplantation, 29 patients suffered fromrecurrent respiratory tract infections, 26 patients used ≥5mg prednisone daily as their only immunosuppressantmedication, 11 patients were on immunosuppressive therapy other than prednisone and 6 patients used a combination of immunosuppressive therapy including prednisone.

Immune status investigation showed 5 patients with a decreased serum IgM level, 4 patients with a decreased IgA and 18 patients with a decreased IgG (Table 2). Twenty-six patients had a decreased IgG1 and 17 patients had a decreased IgG2. In 11 of these patients, both IgG1 and IgG2 were decreased. Immune status in lung transplant candidates 79

Table 2. Immune status investigations at time of screening for lung transplantation and in 81 lung transplantation candidates

Immunoglobulins N < 0.40 g/l 5 IgM Median (g/l) 0.8 N < 0.70 g/l 4 IgA Median (g/l) 2.3 N < 7.0 g/l 18 IgG Median (g/l) 9.0 N < 4.90 g/l 26 IgG1 Median (g/l) 6.2 N < 1.50 g/l 17 IgG2 Median (g/l) 2.1 N < 0.20 g/l 2 IgG3 Median (g/l) 0.3 N < 0.08 g/l 10 IgG4 Median (g/l) 0.3 MBL status MBL deficiency 6 Antibodies against protein antigens CMV IgG Positive ≥15 AE/ml 49 EBV IgG Positive ≥1:100 79 Tox IgG Positive ≥3 IE/ml 28 Response to 23vPPV Normal Response 44 Mildly Impaired Response 8 Moderately Impaired Response 23 Severely impaired response 6

Six patients had a genotypically confirmed MBL deficiency, 3 had XA/O and 3 patients O/O genotype. No abnormalities in the classical and alternative complement pathway were found.

Eighty patients (99%) had IgG specific antibodies to at least one protein antigen (i.e. CMV, EBV or Toxoplasma), 56 (69%) patients to at least two protein antigens and 19 (23%) to all three protein antigens. 80 Chapter 5

Figure 1. Serotype-specific pre- and post-vaccination IgG antibody titers in 81 lung transplantation candidates. Green symbols indicate pre-vaccination titers and blue symbols indicate post-vaccination titers. The horizontal black line represents the 1.3 μg/ml threshold. Black bars indicate median antibody concentrations.

Response to 23-valent pneumococcal polysaccharide vaccination was measured when patients were placed on lung transplantation waiting list. The response to pneumococcal vaccinationwas categorized in accordance with the 2015 edition of the AAAAI/ACAAI “Practice parameter for the diagnosis and management of primary immunodeficiency”. MBL2 genotypes O/O and XA/O are considered to represent MBL deficiency.

IgG antibodies against CMV titer ≥15 AE/ml, against EBV VCA titer N 1:100, and against Toxoplasma N3 IE/m are positive; specific IgG antibodies were determined in 80 patients.

MBL status was available in 79 patients. Other laboratory parameters were available in 81 patients.

After vaccination with 23vPPV themedian antibody concentration of all sero types increased significantly (Figure 1). Fifteen patients had protective IgG post-vaccination antibody levels to all 14 pneumococcal serotypes tested and 52 patients had protective IgG-post-vaccination antibody levels to at least 10 serotypes. Forty-four patients could be classified as having a normal response, 8 patients had amildly impaired response, 23 a moderately impaired response and 6 a severely impaired response. Immune status in lung transplant candidates 81

Subgroup analyses showed that use of corticosteroid therapy combined with other immune suppressive medication was significantly more common in patients with a severely impaired response compared with patients with a normal response (3 of 6 and 5 of 44 patients, respectively, p = 0.042) as well as serum IgA levels < 0.7 g/l (3 of 6 and 1 of 44 patient(s), respectively, p = 0.004). Serum IgG2 levels < 1.5 g/l were significantly more common in all patients with an impaired response compared to patients with a normal response (13 of 37 and 4 of 44 patients, respectively, p = 0.006). Serum IgG3 levels were significantly lower in patients with an impaired response (median 0.3 vs 0.4 g/l, p = 0.036, MannWhitney test). In the group with an impaired response 13 of 37 patients suffered from recurrent respiratory tract infections. This did not significantly differ from the group with a normal antibody response.

In the total group of patients, 29 had recurrent respiratory tract infections. Re- current respiratory tract infectionswere significantly associated with the diagnosis emphysema (24/29 patients, p < 0.0001). IgG, IgG1, IgG2, IgG3 were significantly lower in patients with recurrent respiratory tract infection (p = 0.011, 0.015, 0.007 and 0.05, respectively, Mann Whitney test). Recurrent respiratory tract infectionswere not significantly associated with other variables, including medication use.

During followup, 38 patients were transplanted, 13 were still on the waiting list at the end of follow up, 15 patients died on the waiting list and 15 became ineli- gible for lung transplantation for various reasons and were therefore removed from the waiting list.

Discussion

We evaluated the immune status in 81 patients at the time of listing for lung transplantation. A significant part of the patient group showed abnormalities in the serumimmunoglobulin and/or IgG subclass levels. Approximately one fifth had low IgG levels, two fifths low IgG1 and/or IgG2 levels and half of the patients had an impaired polysaccharide antibody response. IgG levels to protein antigenswere unremarkable. Fifty patients (62% of the cohort) showed one or more abnormalities in IgM, IgA, IgG, IgG1 and/or IgG2 levels and/or antibody response to 23vPPV.

Subgroup analyses in our cohort revealed several significant associations. The use of corticosteroids and other immune suppressive medicationwas significantly associated with a severely impaired response to 23vPPV. This illustrates the importance of pre-transplantationmeasurement of the polysaccharide 82 Chapter 5

antibody response after pneumococcal vaccination since lung transplant recipients will be using even more intensive immune suppressive medication and on a lifelong basis. Immune status investigation also showed significant correlations of low levels of IgA, IgG2 and IgG3 with an impaired response to pneumococcal vaccination. Levels of IgG and IgG1 were also lower in patients with an impaired response in our cohort, but not significantly. Recurrent respiratory tract infections did not occur more in the groupwith an impaired response, but were significantly more present in emphysema patients and were significantly associatedwith lower IgG and IgG subclass levels.

After administration of 23vPPV there was a significant increase in the percentage of lung transplant candidates with protective antibody levels for all 14 tested serotypes, but in the majority of patients postvaccination antibody titers did not reach a protective level for all 14 serotypes tested. The internationally accepted protection level of 1.3 μg/l is a consensus definition and open for debate.10 It is plausible that the protection level varies for the various serotypes because of dif- fering immunogenicity. Another approach of evaluation the antibody response to 23vPPV, that includes serotype specific cut-off values derived from vaccination response in healthy controls, has been proposed.11 When interpreting the antibody levels and response categorizations of our study, it should also be kept in mind that antibody levels were measured using the Luminex xMap technique. Luminex assays have been shown to measure slightly higher concentrations of IgG compared to ELISA.12 The guidelines are based on ELISA and therefore the fraction of patients with protective antibody levels could be overestimated. The vaccination guidelines were recently updated. PCV13 should now be administered before 23vPPV and revaccination is recommended after five years.2 Re- sponsiveness to this vaccination schedule should be evaluated in future studies.

Solid organ transplant recipients have an increased susceptibility to invasive pneumococcal disease as well as other infections.13, 14 It would be of clinical relevance to know whether an impaired humoral immunity (in particular pneumococcal polysaccharide antibody response) is related to long-term outcome and infection risk after lung transplantation.

A part of the patient group showed low levels of immunoglobulins and/or IgG subclasses. Levels of immunoglobulins tend to decrease further in the post-transplantation period.15, 16 Post-transplant hypogammaglobulinemia is associated with increased mortality.17 In a recent retrospective cohort study on-demand immunoglobulin replacement therapy failed to reduce themortal- ity rate and development of bronchiolitis obliterans syndrome in lung transplant recipients.17 This study potentially suffered from selection bias and used only serum IgG levels as a single parameter of the immune status. As is stated in current guidelines for primary immune deficiency,10 response to polysaccha- Immune status in lung transplant candidates 83

ride vaccination should also be measured in assessing the immune status. Until the outcome of prospective studies will become available, immunoglobulin replacement therapy should be considered in lung transplant recipients when a humoral immune deficiency is diagnosed.

In conclusion, a significant proportion of our cohort of lung transplant candidates had one or more abnormalities in the humoral immune status. It is likely that these patients have an increased risk for infections after transplantation. (Re)Vaccination, including measurement of antibody response, and possibly antibody replacement therapy should be considered to minimize infection risk.

References

1. J.A. Fishman, Infection in solid-organ transplant recipients, N. Engl. J. Med. 357 (25) (2007) 2601–2614. 2. Centers for Disease Control and Prevention (CDC), Use of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine for adults with immunocompromising conditions: recommendations of the advisory committee on immunization practices (ACIP), MMWR Morb. Mortal. Wkly Rep. 61 (40) (2012) 816–819. 3. L. Danziger-Isakov, D. Kumar, AST infectious diseases community of practice. Vaccination in solid organ transplantation, Am. J. Transplant. 13 (Suppl. 4) (2013) 311–317. 4. E. de Vries, Clinical working party of the European Society for Immunodeficiencies (ESID). Patient-centred screening for primary immunodeficiency: a multi-stage diagnostic protocol designed for non-immunologists, Clin. Exp. Immunol. 145 (2) (2006) 204–214. 5. J.B. Orens, M. Estenne, S. Arcasoy, et al., International guidelines for the selection of lung transplant candidates: 2006 update—a consensus report from the pulmonary scientific council of the international society for heart and lung transplantation, J. Heart Lung Transplant. 25 (7) (2006) 745–755. 6. C.M. Burton, N. Milman, C.B. Andersen, H. Marquart, M. Iversen, Common variable immune deficiency and lung transplantation, Scand. J. Infect. Dis. 39 (4) (2007) 362–367. 7. T.W. Hoffman, D.A. van Kessel, H. van Velzen-Blad, J.C. Grutters, G.T. Rijkers, Antibody replacement therapy in primary antibody deficiencies and iatrogenic hypogammaglobulinemia, Expert. Rev. Clin. Immunol. 11 (8) (2015) 921–933. 8. A. Vlug, E.J. Nieuwenhuys, R.V. van Eijk, H.G. Geertzen, A.J. van Houte, Nephelometric measurements of human IgG subclasses and their reference ranges, Ann. Biol. Clin. (Paris) 52 (7–8) (1994) 561–567. 9. M.A. Seelen, A. Roos, J.Wieslander, et al., Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA, J. Immunol. Methods 296 (1–2) (2005) 187–198. 10. F.A. Bonilla, D.A. Khan, Z.K. Ballas, et al., Practice parameter for the diagnosis and management of primary immunodeficiency, J. Allergy Clin. Immunol. 136 (5) (2015) 1186, 205.e1-78. 11. H. Borgers, L. Moens, C. Picard, et al., Laboratory diagnosis of specific antibody deficiency to pneumococcal capsular polysaccharide antigens by multiplexed bead assay, Clin. Immunol. 134 (2) (2010) 198–205. 12. M.J. Whaley, C. Rose, J. Martinez, et al., Interlaboratory comparison of three multiplexed bead- based immunoassays for measuring serum antibodies to pneumococcal polysaccharides, Clin. Vaccine Immunol. 17 (5) (2010) 862–869. 13. D. Kumar, A. Humar, A. Plevneshi, et al., Invasive pneumococcal disease in solid organ transplant recipients—10-year prospective population surveillance, Am. J. Transplant. 7 (5) (2007) 1209–1214. 84 Chapter 5

14. A. Shigayeva,W. Rudnick, K. Green, et al., Invasive pneumococcal disease among immunocompromised persons: implications for vaccination programs, Clin. Infect. Dis. (2015). 15. A. Ohsumi, F. Chen, T. Yamada, et al., Effect of hypogammaglobulinemia after lung transplantation: a single-institution study, Eur. J. Cardiothorac. Surg. 45 (3) (2014) e61–e67. 16. N.H. Yip, D.J. Lederer, S.M. Kawut, et al., Immunoglobulin G levels before and after lung transplantation, Am. J. Respir. Crit. Care Med. 173 (8) (2006) 917–921. 17. A.B. Lichvar, C.R. Ensor, M.R. Morrell, et al., On-demand immunoglobulin-G replacement is not associated with benefits in lung transplant recipients with hypogammaglobulinemia, J. Heart Lung Transplant. 35 (4) (2016) S69. CHAPTER 6

Long-term follow up of humoral immune status in adult lung transplant recipients

D.A. van Kessel1,2 T.W. Hoffman1 J.M. Kwakkel-van Erp2 J.D. Oudijk1,2 P. Zanen1,2 G.T. Rijkers3,4 J.C. Grutters1,2

1Department of Pulmonology, St. Antonius Hospital, Nieuwegein, The Netherlands 2Division of Heart and Lungs, University Medical Center Utrecht, Utrecht, The Netherlands 3Department of Medical Microbiology and Immunology, St. Antonius Hospital, Nieuwegein, The Netherlands 4Department of Science, University College Roosevelt, Middelburg, The Netherlands

Transplantation 2017 Feb 14 [accepted for publication] 86 Chapter 6

Abstract

Background Lung transplant recipients have an increased risk for infections in the posttransplant period due to immunosuppressive therapy. Protection against infections can be achieved through vaccination, but the optimal vaccination schedule in lung transplant recipients is unknown. Data on long-term immunological follow up and vaccination responses after lung transplantation are scarce.

Methods Here we present long-term immunological follow up of a cohort of 55 lung transplant recipients. This includes detailed antibody responses after 23-valent pneumococcal polysaccharide vaccination (23vPPV).

Results All patients were vaccinated with 23vPPV prior to transplantation. Median follow-up after transplantation was 6.6 years (379 patient-years). Following transplantation, there is a significant decrease of all immunoglobulins, IgG subclasses and pneumococcal polysaccharide antibodies. After the first year posttransplantation there is a gradual increase of all immunoglobulins and IgG subclasses, but values were always significantly lower than in the pretransplant period. After a median of 4.4 years posttransplantation patients were revaccinated with 23vPPV. The pneumococcal polysaccharide antibody response was impaired in 87% of patients (ie, antibody titer above cutoff and 2-fold increase between pre and postvaccination values for < 70% of serotypes).

Conclusion We found that impairment of humoral immunity was most outspoken in the first year after lung transplantation. Immunoglobulin levels remain decreased several years after transplantation and the response to pneumococcal poly saccharide vaccine was significantly lower posttransplantation compared to the pretransplantation response. However, most patients did show a partial response to vaccination. Based on our results, revaccination with pneumococcal vaccines after transplantation should be considered 1 year after transplan tation. Humoral immune status in adult lung transplant recipients 87

Introduction

Lung transplant recipients require immunosuppressive therapy to prevent acute and chronic rejection of the transplanted organ1. This immunosuppressive therapy leads to an increased susceptibility to bacterial, viral and fungal infections. It is suggested that infectious episodes and/or lower airway colonization increase the risk of chronic lung allograft rejection, also known as bronchiolitis obliterans syndrome (BOS).2 BOS is the most important factor determining long- term survival posttransplantation.2 The risk for infections is likely highest in the early posttransplantation period, when immunosuppressive therapy is the most intensive.3 It has been shown that 60-73% of lung transplant recipients develop hypogammaglobulinemia (IgG level < 7 g/l) in the first year after transplantation. 4-8 Severe hypogammaglobulinemia (IgG level < 4.5 g/l) has been associated with shorter survival4,5,8 and hypogammaglobulinemia has been associated with BOS.9

Therefore, it is important to provide optimal protection against infections in the posttransplantation period. This can be achieved by updating routine immunizations and giving additional vaccinations prior to transplantation. Vaccinations should be repeated after 5 years.3,10-12 These vaccinations include the 23-valent pneumococcal polysaccharide vaccine (23vPPV),13 which should, since 2012, be preceded by the 13-valent pneumococcal conjugate vaccine (13vPCV).3,10-12 It is recommended to monitor specific antibody titers after transplantation in order to determine which patients should be revaccinated earlier,3, 11 but not to measure the response to vaccination as such. Therefore, there are few data available on vaccine responses in solid organ transplant recipients. Vaccination studies that have been performed in solid organ transplant candidates or recipients have included only relatively small patient populations and individual vaccines have been studied predominantly in the setting of a single organ.14

Because of the scarcity of evidence, it is not clear what the most effective vaccination schedule is in solid organ transplant candidates.10, 12 As far as we know, there are no studies reporting a detailed response to pneumococcal polysaccharide vaccination or long-term immunological follow-up after lung transplantation. In order to expand the evidence base for the use of pneumococcal vaccination in lung transplant-recipients and to increase knowledge on the effects of immunosuppressive therapy after lung transplantation, we report the long term immunological follow up and the response to 23vPPV vaccination in a large cohort of lung transplant recipients. 88 Chapter 6

Materials and Methods

This is a retrospective cohort study in lung transplant patients from the St. Antonius Hospital in Nieuwegein, the Netherlands. The hospital is a tertiary referral center for lung transplantation in collaboration with the University Medical Center in Utrecht. This study reports on all lung transplant recipients who were transplanted in the period 2003-2014 and vaccinated with 23vPPV after lung transplantation. All patients had been vaccinated prior to lung transplantation as well. The target vaccination interval was 5 years, but it was extended for patients who had already undergone transplantation at the time this interval was adapted.

Clinical characteristics, results of immune status assessment at time of screening for lung transplantation and follow-up data were collected from patient records. All diagnoses were categorized according to the International Classifica- tion of Diseases 10 (ICD-10) used by Eurotransplant. Immunoglobulins and IgG subclasses were determined by nephelometry.15 Lower limits of normal for serum immunoglobulins and IgG subclasses are as follows: 0.4 g/l for IgM, 7.0 g/l for IgG, 0.7 g/l for IgA, 4.9 g/l for IgG1, 1.5 g/l for IgG2, 0.2 g/l for IgG3 and 0.08 g/l for IgG4.

Patients were vaccinated intramuscularly with 1 dose of 23-valent pneumococcal polysaccharide vaccine (Pneumovax 23; Merck, Rahway, NJ, USA) containing 25 μg purified type-specific capsular polysaccharides of 23 pneumococcal serotypes. Blood samples were drawn before and 3–6 weeks after vaccination to evaluate the response to vaccination. Serum samples were stored at −80°C until use. Antibodies against pneumococcal serotypes were measured pretransplant and followed up in the posttransplant period. Until 2008 total antibodies against capsular polysaccharides of 2 or 3 Streptococcus pneumoniae serotypes (3, 4 and 9) were measured by ELISA as previously described.16 After 2008 IgG antibodies against 8, 13 or 14 different pneumococcal polysaccharides were measured on a Luminex platform as described previously.17-19

For categorization of the antibody response to pneumococcal polysaccharide vaccination, the 2015 AAAAI/ACAAI classification schedule was used.20 Protective antibody level against an individual given pneumococcal serotype is defined as an IgG antibody concentration > 1.3 μg/ml. A positive immune response to a given serotype is defined as having a postvaccination antibody concentration > 1.3 μg/ml and a ≥2-fold antibody concentration increase between the pre and postvaccination serum samples. Overall, a patient is considered to be a responder if at least 70% of the antibody responses to the serotypes tested were positive. According to this classification, responses are categorized as (1) normal, (2) mildly impaired (antibody levels > 1.3 μg/ml for ≥70% of serotypes, but Humoral immune status in adult lung transplant recipients 89

≥2-fold increase between pre and postvaccination antibody titers for < 70% of serotypes), (3) moderately impaired (antibody levels > 1.3 μg/ml for < 70% of serotypes) or (4) severely impaired (antibody levels > 1.3 μg/ml for ≤ 2 serotypes).

BOS is defined as an FEV1 less than or equal to 80% of the mean of the 2 best post transplantation values taken at least 3 weeks apart (baseline value) in the ab- 21, 22 sence of other causes. BOS-stages are defined as follows: stage 1, FEV1

66-80% of baseline; stage 2, FEV1 51-65% of baseline; and stage 3, FEV1 < 50% of baseline. Pulmonary function testing was carried out according to the standards of the European Respiratory Society.23

Immunosuppressive therapy given after lung transplantation consisted of tacrolimus, mycophenolate mofetil and prednisone. In addition, on day 0 and day 4 posttransplantation 20 mg basiliximab was administered as induction therapy. The dosage of tacrolimus was based on serum drug levels; the target serum level was 10-15 μg in the first year and 7-10 μg/ml thereafter. The dosage of mycophenolate mofetil was 1000 mg twice daily in the first three months, then 750 mg twice daily for the remainder of the first year and then 500 mg twice daily thereafter. The dosage of prednisone was initially 100 mg daily, which was tapered to 15 mg daily in the first 6 months, and then 10 mg daily thereafter.

For statistical analyses the Fisher’s exact test, Mann-Whitney U test were used where appropriate. For data management and statistical analyses Microsoft Excel 2010 and IBM SPSS Statistics for Windows (version 22.0) were used. Graphs were made with GraphPad Prism version 2.0, and SPSS Statistics for Windows (version 22.0). Differences were considered to be significant at p-values < 0.05.

The local medical ethics committee approved the study. All patients gave formal written informed consent.

Results

Fifty-five patients were included in the study. The baseline characteristics are shown in Table 1. Thirty-two patients were female and median age at screening was 52 years. Thirty-one patients (56%) were transplanted because of COPD/ emphysema and 8 patients (15%) suffered from pulmonary fibrosis. Eight patients (15%) had an IgG level below 7.0 g/l at time of screening for lung transplantation. Twenty-two patients (49%) had one or more IgG subclasses below normal values. Forty patients (73%) had a normal response to the first vaccination with 23vPPV (before transplantation). 90 Chapter 6

Table 1. Patient characteristics at time of screening for lung transplantation.

Patient Characteristics Total 55 Female 32 Median age at screening (years) 52 Age Range (years) 23-60 Eurotransplant Diagnosis Category COPD/Emphysema 31 Alpha-1 Antitrypsin Deficiency 6 Idiopathic Pulmonary Fibrosis 5 Other Pulmonary Fibrosis 3 Hypersensitivity Pneumonitis 2 Sarcoidosis 1 Bronchiectasis 1 Histiocytosis X 2 Bronchiolitis Obliterans 1 Lymphangioleiomyomatosis 2 Cystic Fibrosis 1 Immunoglobulins IgM < 0.4 g/l 1 IgA < 0.7 g/l 1 IgG < 7.0 g/l 8 IgG1 < 4.9 g/l 19 IgG2 < 1.5 g/l 7 IgG3 < 0.2 g/l 1 IgG4 < 0.08 g/l 7 Response to Pneumococcal Vaccination Normal Response (3-plex) 24 Impaired Response (3-plex) 6 Normal Response (8-/14-plex) 16 Impaired Response (8-/14-plex) 9

Immunoglobulin G subclass measurements were available in 53 patients. Response to pneumococcal vaccination was categorized in accordance with the 2015 AAAAI/ACAAI “Practice Parameter for the Diagnosis and Management of Primary Immunodeficiency” for 8- and 14-plex pneumococcal antibody panels. For 3-plex pneumococcal antibody panels a normal response was defined as having a postvaccination titer > 20 U/ml and at least a 2-fold increase for 2 of the 3 pneumococcal serotypes tested. Humoral immune status in adult lung transplant recipients 91

Median follow-up after transplantation was 6.6 years (interquartile range 4.6-9.2, 379 patient-years). The median levels of all immunoglobulin isotypes significantly decreased within 6 months after lung transplantation. After 4 years the median levels of the serum immunoglobulins were still significantly lower than before lung transplantation (p < 0.001 for IgA, p < 0.001 for IgM and p=0.004 for IgG). After the first year post transplantation there was a significant increase of median serum IgG levels (p=0.003) but serum levels were still significantly lower than before the lung transplantation (p < 0.001). All IgG subclasses were significantly higher pretransplant compared to all posttransplant periods (p < 0.05), except for IgG3 levels in the period 38-48 months and the period after 48 months.

Prior to lung transplantation 7 patients (13%) had an IgG level < 7 g/l, including 1 patient with an IgG level < 5 g/l. Seven to twelve months after lung transplantation 35 patients (65%) had an IgG level < 7 g/l (p < 0.0001) including 10 patients (19%) with an IgG level < 5g/l (p=0.004) (Table 2). After 4 years posttransplantation, 18 patients (43%) had median IgG levels < 7.0 g/l and 5 patients (12%) had median IgG levels < 5.0 g/l (p=0.04 and p=0.40, respectively compared to 7-12 months posttransplantation; p=0.001 and p=0.08, respectively compared to pretransplantation).

Follow up of antibodies against pneumococcal serotypes showed a significant, progressive decline of protective titers in the posttransplant period up to the moment of the second vaccination with 23vPPV (Figure 1). The percentage of pneumococcal antibodies above cutoff (> 20 U/ml for 3-plex; ≥1.3 μg/l for 8-, 13- or 14-plex) pretransplant was significantly higher compared to all posttransplant periods (p < 0.0001). Within the first year after transplantation 86% of the patients had protective antibodies levels against less than 70% of the tested serotypes compared to 31% before transplantation (p < 0.0001, Table 2).

The pneumococcal antibody response to the second vaccination with 23vPPV (after transplantation) was measured on a 13- or 14-plex antibody panel (n=49 and n=4, respectively). Serotype-specific antibody titers before and after vaccination are shown in Figure 2. The median interval between the first and second vaccination was 6.1 years (IQR 5.2-7.9) and between the moment of transplantation and the second vaccination 4.4 years (IQR 2.8-6.5). The polysaccharide antibody response was normal in 6 patients, mildly impaired in 7 patients, moderately impaired in 32 patients and severely impaired in 8 patients. The response was abnormal in 87% of the patients. Of the forty patients who showed a normal antibody response to the first vaccination (before transplantation), 33 had an impaired response to the second vaccination (after transplantation; 6 patients had a normal response; 1 patient was receiving IVIG). All patients 92 Chapter 6 18 (43) 5 (12) after 48 mo after posttransplant 42 42 4.87 (4.24-5.85) 1.24 (1.73) 1.84 (1.19-2.30) 0.70 (0.45) 0.35 (0.25-0.58) 7.41 (3.40) 0.14 (0.06-0.22) - 37-48 mo post 22 (48) 5 (11) transplant 44 46 4.76 (4.15-5.73) 1.31 (1.20) 1.75 (1.16-2.35) 0.64 (0.62) 0.35 (0.24-0.59) 7.42 (3.27) 0.17 (0.08-0.25) - 25-36 mo post transplant 29 (54) 4 (8) 52 54 4.68 (3.90-5.55) 1.43 (1.07) 1.54 (1.15-2.25) 0.71 (0.61) 0.30 (0.20-0.50) 6.75 (3.03) 0.19 (0.08-0.25) - 13-24 mo post transplant 26 (51) 8 (16) 47 51 4.60 (3.70-6.10) 1.36 (0.99) 1.50 (1.20-2.15) 0.72 (0.55) 0.30 (0.20-0.40) 6.76 (2.77) 0.18 (0.08-0.30) - 7-12 mo post transplant 35 (65) 10 (19) 47 54 4.30 (3.50-5.27) 1.41 (1.04) 1.53 (1.30-2.35) 0.60 (0.51) 0.30 (0.20-0.40) 6.31 (2.26) 0.20 (0.10-0.31) - 0-6 mo post transplant 33 (70) 10 (21) 40 47 4.13 (3.26-4.98) 1.70 (1.23) 1.48 (1.30-2.25) 0.48 (0.34) 0.28 (0.20-0.34) 6.23 (2.40) 0.30 (0.15-0.49) Pretransplant 7 (13) 1 (2) 53 55 5.70 (4.55-6.80) 2.46 (1.63) 2.20 (1.85-2.80) 1.00 (0.67) 0.40 (0.30-0.60) 8.50 (2.41) 0.30 (0.20-0.55) N IgG < 7.0 g/l (%) N IgG < 5.0 g/l (%) IgG subclasses N patients Immunoglobulins N patients IgG1 g/l (IQR) IgA g/l (IQR) IgG2 g/l (IQR) IgM g/l (IQR) IgG3 g/l (IQR) IgG g/l (IQR) IgG4 g/l (IQR) Immunoglobulin, IgG subclass and pneumococcal antibody levels before and after transplantation. before and after antibody levels 2. Immunoglobulin, IgG subclass and pneumococcal Table Table 2. Continued Humoral immunestatusinadultlungtransplantrecipients

Pretransplant 0-6 mo post- 7-12 mo post- 13-24 mo post- 25-36 mo post- 37-48 mo post- after 48 mo transplant transplant transplant transplant transplant posttransplant Anti-PPS antibodies N patients 54 28 22 21 17 31 %serotypes 93 (56-100) 37 (21-62) 43 (25-61) 31 (15-48) 32 (15-53) 31 (15-51) above cut-off % patients 31 86 86 100 100 94 < 70% protective titers % patients 22 54 59 76 76 71 < 50% protective titers

Mean values for the pretransplant period were calculated per patient from all available measurements before transplantation. Mean values for the posttransplant period were calculated per patient from available measurements during each specific time-interval. The mean values of all pooled individual means are stated in the Table. IgA pretransplant was significantly higher compared to all posttransplant periods (Wilcoxon signed ranks test; p < 0.001). IgM pretransplant significantly higher compared to all posttransplant periods (Wilcoxon signed ranks test; p < 0.001). IgG pretransplant significantly higher compared to all posttransplant periods -(Wil coxon signed ranks test; p < 0.005). All IgG subclasses pretransplant significantly higher compared to all posttransplant periods (Wilcoxon signed ranks test; p < 0.05), except for IgG3 levels in period 38-48 months and period after 48 months. The percentage of anti-PPS antibodies above cutoff (> 20 U/ml for 3-plex; ≥1.3 μg/l for 8-, 13- or 14-plex) pretransplant was significantly higher compared to all posttransplant periods (Wilcoxon signed ranks test; p < 0.0001). 93 94 Chapter 6

Figure 1. Follow-up of anti-pneumococcal antibody titers after lung transplantation. The median percentage of pneumococcal serotypes with anti-PPS antibody levels above cutoff is shown on the Y-axis for various time points. Brackets indicate the interquartile range. Cutoff values were > 20 U/ml for 3-plex and ≥1.3 μg/l for 8-, 13- or 14-plex.

who showed an abnormal antibody response to the first vaccination (before transplantation) also had an impaired response to the second vaccination (after transplantation). Sixteen patients showed a normal antibody response to the first vaccination, measured on an 8- or 13-plex antibody panel. Fourteen of these 16 (87.5%) had an abnormal antibody response to the second vaccination.

Twenty-two patients (40%) developed BOS during follow-up. Median time until BOS was 4.9 years (IQR 3.6-6.5) after lung transplantation. Eleven patients were classified as BOS1, 5 patients as BOS2 and 6 patients as BOS3 at the end of follow up. Eight patients died during follow up, after a median of 6.5 years posttransplantation (IQR 5.5-8.1). Four out of 8 patients died from BOS and 3 of the remaining patients had BOS but died due to a nonrelated cause. BOS was significantly associated with mortality (p=0.005). There was no significant correlation between vaccination responses or IgG levels before and after transplantation and BOS or death. Humoral immune status in adult lung transplant recipients 95

Figure 2. Response to pneumococcal vaccination after lung transplantation. Serotype specific antibody concentrations before (purple dots) and 3-6 weeks after (orange dots) vaccination with the 23-valent pneumococcal polysaccharide vaccine. Geometric means are indicated with black bars. The horizontal black line indicates the threshold antibody concentration of 1.3 μg/ml.

There were 3 patients with one or more confirmed pneumococcal infections during follow up, including 1 patient with invasive pneumococcal disease 3 months after lung transplantation. All 3 patients had a moderately impaired response to the second pneumococcal vaccination (after transplantation). Two of 3 patients had a normal response to the first pneumococcal vaccination (before transplantation); the other patient had a severely impaired response to the first vaccination, as well as low IgG levels pretransplantation (IgG level 5.92 g/l). Three other patients received antibody replacement therapy with intravenous immunoglobulins (IVIG). Two of these 3 patients had an impaired pneumococcal antibody response before transplantation. The indication to start IVIG was a rapid decline of serum IgG level within 3 months after transplantation (< 3.5 g/l). During follow up none of these 3 patients had severe or recurrent infections under IVIG, but all 3 patients did develop BOS.

Discussion

Long-term follow up of this large cohort of lung transplant recipients revealed a significant impairment of the humoral immune system. This impairment 96 Chapter 6

becomes evident in the first year after lung transplantation. There is a significant decrease of all immunoglobulins and IgG subclasses and pneumococcal polysaccharide antibodies. After the first year there is partial recovery of all immunoglobulins and subclasses reflecting the tapering of the immune suppressive therapy. However, immune status is still significantly lower compared to the pretransplant period. Several years posttransplantation, the pneumococcal polysaccharide antibody response after vaccination with 23vPPV was still impaired in 87% of all patients.

As far as we know this is the first study that reports on the detailed response to pneumococcal polysaccharide vaccination after lung transplantation. The response to 23vPPV was clearly lower posttransplantation compared to pretransplantation and almost all patients fulfilled the criteria of an impaired vaccination response according to the 2015 AAAAI/ACAAI classification.20 There was no relation between the interval of the vaccination with 23vPPV before transplantation and the pneumococcal antibody levels 1 year after transplantation. It would be interesting to correlate the pneumococcal vaccination response with infection frequency. In our study, because of the retrospective character, this was not possible for all infections.

It is recommended to observe an interval of 5 years between vaccination and revaccination (13vPCV plus 23vPPV). 3, 10-12 These recommendations are based on data from immunocompromised populations in general and the optimal vaccination schedule might be different in lung transplant patients. Our results show that immune status improves after the first year of transplantation when immunosuppressive therapy is tapered. In this light, it would seem logical to administer the pneumococcal vaccinations at this moment, ie, 1 year after transplantation. We think this timing would provide optimal protection against pneumococcal infections. Another strategy might be measuring the serum levels of pneumococcal antibody titers regularly and vaccinate when titers are considered to be unprotective. However, the definition of a protective level is subject of discussion and could also be different for various serotypes.20, 24 When using the commonly used cutoff values of 1.3 µg/ml for protective antibody titers for > 70% of measured serotypes,20 we found that the majority of our cohort (86%) had unprotective antibody titers within the first year after transplantation. Therefore, we would advise to revaccinate all patients 1 year after transplantation. Although they did not meet the criteria for an adequate response, nearly all patients were capable of producing a partial antibody response. This indicates that pneumococcal vaccination can still be beneficial in these patients. The vaccination response might improve by adding 13vPCV to the vaccination regime, but this requires further study. Humoral immune status in adult lung transplant recipients 97

Forty percent of the patients in our cohort developed BOS during follow up. BOS was significantly associated with mortality during the study period. We did not find association between BOS and the vaccination response, but our study was likely underpowered to detect any existing association, as the vast majority of patients (87%) had an impaired vaccination response. We also did not find an association between BOS and hypogammaglobulinemia. In comparison with previously published studies4-8 a similar rate of hypogammaglobulinemia was found within the first year. In addition to the existing studies we found that serum IgG and IgG subclass levels increase after the first year of transplantation but were still significantly lower compared to pretransplant values. Hypogammaglobulinemia has been associated with a shorter survival in other studies.25, 26 However, a relation between hypogammaglobulinemia and mortality was not found. This could be explained by the relatively low mortality (15%) at the end of the follow up compared to other studies.

The indication to start IVIG is a combination of recurrent infections and an abnormal immune status investigation,27 also after lung transplantation. Start- ing IVIG should not only be based on serum IgG levels, unless the levels are extremely low. This was the case in the 3 patients who were put on IVIG in the present study. These patients did not develop any further recurrent or severe infections under IVIG. In addition to the patients included in this study, we have had several lung transplant recipients who presented with recurrent infections and severe hypogammaglobulinemia or low anti-pneumococcal antibodies. They were treated with IVIG which led to increased IgG levels and clinically relevant reduction of infectious episodes. The results from this study show that most patients become humoral immunodeficient after lung transplantation. Therefore, to our opinion IVIG should always be considered in case of recurrent infections in combination with hypogammaglobulinemia and/or and impaired pneumococcal antibody response.

A possible limitation of this study is selection bias since only patients that sur- vived until the second vaccination with 23vPPV (ie, at least 1 year after transplantation) were included. This contributes to the relatively low mortality in our cohort. It is not clear whether the patients who died prior to the second pneumococcal vaccination were immunologically different from the included patients. Another limitation of our study is the use of different assays to measure anti-pneumococcal antibodies, reflecting changing diagnostic methods over time. This makes it difficult to directly compare anti-pneumococcal antibody levels at different time points. However, we have corrected for this by using the percentage of antibody titers above internationally acknowledged cutoff values. The interpretation of the response to the first pneumococcal vaccination (before transplantation) is complicated by the use of the 3-plex antibody panel to meas- 98 Chapter 6

ure pneumococcal antibody levels in a part of the patients. This measurement might overestimate the proportion of normal responders compared to 8-, 13- and 14-plex platforms. In a recent study in lung transplant candidates (in the pretransplantation setting) we found a slightly lower rate of impaired response to 23vPPV measured on a 14-plex platform compared to the pretransplantation vaccination response in this study (including 3-plex measurements).28

In conclusion we found a significantly suppressed humoral immunity that was most outspoken in the first year after lung transplantation. The response to pneumococcal polysaccharide vaccination posttransplantation was clearly lower than pretransplantation. However, most patients did show a partial response to vaccination. Therefore, revaccination with pneumococcal vaccines should be considered 1 year after transplantation.

References

1. Fishman JA. Infection in solid-organ transplant recipients. N Engl J Med. 2007;357(25):2601-2614. 2. Weigt SS, DerHovanessian A, Wallace WD, Lynch JP,3rd, Belperio JA. Bronchiolitis obliterans syndrome: The achilles’ heel of lung transplantation. Semin Respir Crit Care Med. 2013;34(3):336- 351. 3. L’Huillier AG, Kumar D. Immunizations in solid organ and hematopoeitic stem cell transplant patients: A comprehensive review. Hum Vaccin Immunother. 2015;11(12):2852-2863. 4. Goldfarb NS, Avery RK, Goormastic M, et al. Hypogammaglobulinemia in lung transplant recipients. Transplantation. 2001;71(2):242-246. 5. Kawut SM, Shah L, Wilt JS, et al. Risk factors and outcomes of hypogammaglobulinemia after lung transplantation. Transplantation. 2005;79(12):1723-1726. 6. Yip NH, Lederer DJ, Kawut SM, et al. Immunoglobulin G levels before and after lung transplantation. Am J Respir Crit Care Med. 2006;173(8):917-921. 7. Noell BC, Dawson KL, Seethamraju H. Effect of hypogammaglobulinemia on the incidence of community-acquired respiratory viral infections after lung transplantation. Transplant Proc. 2013;45(6):2371-2374. 8. Ohsumi A, Chen F, Yamada T, et al. Effect of hypogammaglobulinemia after lung transplantation: A single-institution study. Eur J Cardiothorac Surg. 2014. 9. Chambers DC, Davies B, Mathews A, Yerkovich ST, Hopkins PM. Bronchiolitis obliterans syndrome, hypogammaglobulinemia, and infectious complications of lung transplantation. J Heart Lung Transplant. 2013;32(1):36-43. 10. Centers for Disease Control and Prevention (CDC). Use of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine for adults with immunocompromising conditions: Recommendations of the advisory committee on immunization practices (ACIP). MMWR Morb Mortal Wkly Rep. 2012;61(40):816-819. 11. Danziger-Isakov L, Kumar D, AST Infectious Diseases Community of Practice. Vaccination in solid organ transplantation. Am J Transplant. 2013;13 Suppl 4:311-317. 12. Kumar D. Immunizations following solid-organ transplantation. Curr Opin Infect Dis. 2014;27(4):329-335. 13. Duchini A, Goss JA, Karpen S, Pockros PJ. Vaccinations for adult solid-organ transplant recipients: Current recommendations and protocols. Clin Microbiol Rev. 2003;16(3):357-364. 14. Eckerle I, Rosenberger KD, Zwahlen M, Junghanss T. Serologic vaccination response after solid organ transplantation: A systematic review. PLoS One. 2013;8(2):e56974. Humoral immune status in adult lung transplant recipients 99

15. Vlug A, Nieuwenhuys EJ, van Eijk RV, Geertzen HG, van Houte AJ. Nephelometric measurements of human IgG subclasses and their reference ranges. Ann Biol Clin (Paris). 1994;52(7- 8):561-567. 16. Van Kessel DA, Horikx PE, Van Houte AJ, De Graaff CS, Van Velzen-Blad H, Rijkers GT. Clin- ical and immunological evaluation of patients with mild IgG1 deficiency. Clin Exp Immunol. 1999;118(1):102-107. 17. Meerveld-Eggink A, van der Velden AM, Ossenkoppele GJ, van de Loosdrecht AA, Biesma DH, Rijkers GT. Antibody response to polysaccharide conjugate vaccines after nonmyeloablative allogeneic stem cell transplantation. Biol Blood Marrow Transplant. 2009;15(12):1523-1530. 18. Elberse KE, Tcherniaeva I, Berbers GA, Schouls LM. Optimization and application of a multiplex bead-based assay to quantify serotype-specific IgG against streptococcus pneumoniae polysaccharides: Response to the booster vaccine after immunization with the pneumococcal 7-valent conjugate vaccine. Clin Vaccine Immunol. 2010;17(4):674-682. 19. van Kessel DA, Hoffman TW, van Velzen-Blad H, Zanen P, Rijkers GT, Grutters JC. Response to pneumococcal vaccination in mannose-binding lectin-deficient adults with recurrent respiratory tract infections. Clin Exp Immunol. 2014;177(1):272-279. 20. Bonilla FA, Khan DA, Ballas ZK, et al. Practice parameter for the diagnosis and management of primary immunodeficiency. J Allergy Clin Immunol. 2015;136(5):1186-205.e1-78. 21. Estenne M, Maurer JR, Boehler A, et al. Bronchiolitis obliterans syndrome 2001: An update of the diagnostic criteria. J Heart Lung Transplant. 2002;21(3):297-310. 22. Meyer KC, Raghu G, Verleden GM, et al. An international ISHLT/ATS/ERS clinical practice guideline: Diagnosis and management of bronchiolitis obliterans syndrome. Eur Respir J. 2014;44(6):1479-1503. 23. Quanjer PH, Tammeling GJ, Cotes JE, Pedersen OF, Peslin R, Yernault JC. Lung volumes and forced ventilatory flows. report working party standardization of lung function tests, european community for steel and coal. official statement of the european respiratory society. Eur Respir J Suppl. 1993;16:5-40. 24. Borgers H, Moens L, Picard C, et al. Laboratory diagnosis of specific antibody deficiency to pneumococcal capsular polysaccharide antigens by multiplexed bead assay. Clin Immunol. 2010;134(2):198-205. 25. Florescu DF, Kalil AC, Qiu F, Schmidt CM, Sandkovsky U. What is the impact of hypogammaglobulinemia on the rate of infections and survival in solid organ transplantation? A meta-analysis. Am J Transplant. 2013;13(10):2601-2610. 26. Lichvar AB, Ensor CR, Morrell MR, et al. On-demand immunoglobulin-G replacement is not associated with benefits in lung transplant recipients with hypogammaglobulinemia. The Journal of Heart and Lung Transplantation. ;35(4):S69. 27. Orange JS. Hossny EM. Weiler CR. Ballow M. Berger M. Bonilla FA. Buckley R. Chinen J. El-Gamal Y. Mazer BD. Nelson RP Jr. Patel DD. Secord E. Sorensen RU. Wasserman RL. Cunningham-Rundles C. Primary Immunodeficiency Committee of the American Academy of Allergy, Asthma and Immunology. Use of intravenous immunoglobulin in human disease: A review of evidence by members of the primary immunodeficiency committee of the american academy of allergy, asthma and immunology. J Allergy Clin Immunol. 2006;117(4 Suppl):S525-53. 28. van Kessel DA, Hoffman TW, van Velzen-Blad H, van de Graaf EA, Grutters JC, Rijkers GT. Immune status assessment in adult lung transplant candidates. Transpl Immunol. 2017 ;40:31-34.

PART III

ANTIBODY REPLACEMENT THERAPY IN PATIENTS WITH HUMORAL IMMUNODEFICIENCY

CHAPTER 7

Antibody replacement therapy in primary antibody deficiencies and iatrogenic hypogammaglobulinemia

T.W. Hoffman1 D.A. van Kessel1,2 H. van Velzen-Blad3 J.C. Grutters1,2 G.T. Rijkers3,4

1Department of Pulmonology, St. Antonius Hospital, Nieuwegein, The Netherlands 2Division of Heart and Lungs, University Medical Center Utrecht, Utrecht, The Netherlands 3Department of Medical Microbiology and Immunology, St. Antonius Hospital, Nieuwegein, The Netherlands 4Department of Science, University College Roosevelt, Middelburg, The Netherlands

Exp Rev Clinical Immunol 2015;11(8):921-33 104 Chapter 7

Abstract

Antibody replacement therapy has been used in the treatment of primary antibody deficiencies (PADs) for several decades, and an evidence-based guideline for its treatment is currently available. By contrast, the use of antibody replacement therapy in iatrogenic hypogammaglobulinemia (IHG), a condition that is associated with immunosuppressive medication, has hardly any evidence base and no guidelines. As IHG can be equally as severe as PAD and is much more prevalent, evidence-based guidelines are urgently needed. This review will focus on the differences and similarities between PAD and IHG and the use of antibody replacement therapy in both conditions. Suggestions for the development of evidence-based guidelines and future research are given. Antibody replacement therapy 105

Introduction

Agammaglobulinemia and hypogammaglobulinemia are conditions characterized by low or absent circulating antibodies that predispose to severe recurrent infections. These conditions are the hallmark of the so-called primary antibody deficiencies (PADs). The reported prevalence for PADs ranges from 1.3 to 2.9/ 100,000 persons in Europe.1, 2 The actual prevalence is probably higher due to underdiagnosis of PADs in general and underrepresentation of less severe forms of PAD in these estimates.1, 3 In accordance with this presump- tion, a telephone survey in the US estimated the prevalence of primary immunodeficiencies (of which PADs represent the largest part) to be 1in 1200 individuals.4

Apart from the intrinsic impairment of the immune system that is the basis of PADs, hypogammaglobulinemia can also be caused by various exogenous factors. These factors include radiation therapy and immunosuppressive medications such as corticosteroids, B-cell targeting biologicals and cytotoxic drugs.5 This condition develops in a subgroup of the patients under immunosuppressive treatment and is called iatrogenic hypogammaglobulinemia (IHG). As in hypogammaglobulinemia due to PADs, these patients can present with recurrent infections, with possibly severe consequences. Hard epidemiological data on IHG are lacking, but most probably, IHG is significantly more prevalent than PAD. 6, 7 Furthermore, the prevalence of IHG is likely to increase because of the increased use of immunosuppressive therapies, especially biologicals.8, 9

The mainstay of treatment in PAD has not changed since its first recognition more than half a century ago by Col. Ogden C Bruton10 and is still based on the repeated infusion of antibodies from pooled donor plasma, which is also known as antibody, immunoglobulin or gammaglobulin replacement therapy. The use of antibody replacement therapy in PAD is defined in international guide lines.11-13 By contrast, the use of antibody replacement therapy in IHG misses clear international guidelines, and efforts have only recently been made to provide clear treatment indications.6

Several aspects of the use of antibody replacement therapy in PADs and in IHG will be reviewed here. First, the pathogenesis of both conditions will be discussed. Thereafter, an overview of the clinical and laboratory manifestations of both conditions will be given. Then, the evidence base for the use of antibody replacement therapy in PAD and IHG is presented. Finally, the similarities and differences between both conditions will be discussed, as well as future prospects for the use of antibody replacement therapy in both conditions. 106 Chapter 7

Throughout this review, the IHGs caused by immunosuppressive therapy with systemic corticosteroids, rituximab (antiCD20) and immunosuppressive drug regimens used after solid organ transplantation are used to illustrate the group of IHGs. The use of immunoglobulin products for their immunomodulatory properties or hypogammaglobulinemia in the context of (immunosuppressive treatment for) hematological malignancies will not be reviewed here.

Pathogenesis of PADs & IHG

PADs are caused by genetic defects that affect B-cell development at various stages, as is illustrated in Figure 1. The abnormal B-cell development mechanisms that underlie PAD pathogenesis are beyond the scope of this review and are discussed elsewhere.14 In Table 1, some of the known genetic mutations in several common PADS are shown. PADs that derive from mutations affecting the early stages of B-cell development lead to severe impairments in antibody production (agammaglobulinemia), whereas mutations affecting the later stages of B-cell development generally lead to milder impairments in antibody production (hypogammaglobulinemia). Hypogammaglobulinemia is classified as mild (IgG level 5–6.9 g/l), moderate (IgG level 3–4.9 g/l) or severe (IgG level < 3 g/l).15 Serum IgG levels < 1 g/l are termed agammaglobulinemia. Several aspects of PAD pathogenesis are still unknown. For example, a mutation that normally leads to severe immunodeficiency (i.e., X-linked agammaglobulinemia) has also been reported in patients with a less severe immunodeficiency (specific antibody deficiency).16 In common variable immunodeficiency (CVID), only 10–15% of the patients have mutations in known genes,17 some of which only seem to be associated with disease risk.18 Moreover, the age range in which a certain type of PAD can become manifest is broad.19 Other, still unknown genetic and environmental factors are thought to play a role both in onset as well as clinical severity.18 In mild antibody deficiencies, no causative genetic factors have been identified. T-cell function is generally intact in PAD, except in CVID.20 Antibody replacement therapy 107

Figure 1. B-lymphocyte development and differentiation: genetic defects and targets for immunosuppressive medication.14 In the bone marrow, B-lymphocyte development starts with differentiation of progenitor B cells into pro-B cells. These cells then differentiate into pre-B cells which can be characterized by a rearranged heavy chain which is expressed with a surrogate light chain and the Ig-α and -ß proteins in the form of a pre-BCR. Upon successful rearrangement of the light chain, pre-B cells differentiate into immature B-cells, which express the complete surface IgM molecule in the context of the BCR. Immature B-cells leave the bone marrow compartment and via transitional B-cells differentiate into naïve mature B-cells, which present both IgM and IgD on their surface. Naïve mature B-cells migrate to secondary lymphoid tissues. Upon encountering specific antigen, the B-cell can be activated, in most cases with the help of T-cells. Activated B-cells either differentiate into short-lived plasma cells that produce IgM antibodies or the activated B-cells migrate to the follicular zone of lymphoid follicles. There they proliferate rapidly and undergo somatic hypermutation. The resulting B-cell clones with the highest antigen specificity are stimulated by follicular dendritic cells and follicular T-cells to proliferate and undergo immunoglobulin class switching. These cells differentiate into either long-lived plasma cells or circulating memory B-cells. Long-lived plasma cells home in the bone marrow and produce antibodies of any class over prolonged periods. Circulating memory B-cells either reside in the circulation or home to the bone marrow. They become active upon renewed antigen stimulation. Mutations in genes that are involved in the transition from pro-B cells to pre-B cells can lead to XLA or ARA, for example, genes encoding the different components of the pre-BCR. Mutations in genes involved in the development of naïve mature B-cells out of transitional B-cells or in the B-cell migration to secondary lymphoid tissues lead to CVID. Also, mutations in genes encoding B-cell activating factor receptors lead to CVID, as the B-cell activating factor stimulates B-cell proliferation and differentiation. Mutations in genes involved in the immunoglobulin class switching can lead to HIgM, SIgD or SAD. The blue bulbs that are present on B-cells at various stages of differentiation represent CD20 expression, thus indicating which B-cell stages are targeted by rituximab. The C-icons indicate what immune cells are mainly suppressed by corticosteroid treatment. ARA: Autosomal recessive agammaglobuline- mias; BCR: B-cell receptor; CVID: Common variable immunodeficiency; HIgM: Hyper-IgM syndrome; SAD: Specific polysaccharide antibody deficiencies; SIgD: Specific immunoglobulin deficiencies; XLA: X-linked agammaglobulinemia. 108 Chapter 7 11 No, only in case of severe No, only in case of severe recurrent infections and impaired vaccination response No, only in case of severe No, only in case of severe recurrent infections and impaired vaccination response Indication for antibody antibody for Indication therapy replacement Always Always Always Only in case of severe Only in case of severe recurrent infections and impaired vaccination response Always Low IgG1, IgG2, IgG3 and/ Low or IgG4 Low IgA Low Low IgM Low Laboratory characteristics Low or absent IgM, IgG and Low IgA Impaired response to vaccination or absent IgM, IgG and Low IgA Impaired response to vaccination IgG, IgA and IgE levels Low with normal or high IgM levels Impaired response to vaccination Low IgG, as well as low IgA, IgG, as well low Low and/or IgM Impaired response to vaccination Bacterial infections Bacterial Bacterial infections, Bacterial disease autoimmune Bacterial infections, viral Bacterial infections, autoimmune disease Clinical manifestations Clinical Bacterial infections, Bacterial disease, autoimmune malignancy infections, Bacterial disease, autoimmune malignancy infections, Bacterial disease, autoimmune malignancy Bacterial infections, viral Bacterial infections, autoimmune disease, malignancy Primary Deficiencies Antibody

14 98 14 14 Unknown Unknown Mutations in IGHM, CD79A, B29, IGLL1, PI3K, LRBA or BLNK Mutations in TNFRSF13B, CD19, TNFRSF13C, ICOS, MS4A1, CR2, or CD81 Unknown Mutations in CD40, CD40L, UNG, or NEMO AID, Mutations in BTK Underlying cause Underlying Selective immunoglobulin Selective subclass deficiency Selective IgA deficiency Selective Autosomal recessive recessive Autosomal agammaglobulinemia variable Common immunodeficiency (CVID) IgM deficiency Selective Hyper IgM syndrome X-linked X-linked agammaglobulinemia Disorder Clinical and laboratory manifestations of the most common PADs and three immunosuppressive therapies. and three immunosuppressive PADs 1. Clinical and laboratory manifestations of the most common Table Table 1. Continued Antibody replacementtherapy

Primary Antibody Deficiencies Disorder Underlying cause Clinical manifestations Laboratory characteristics Indication for antibody replacement therapy11

Selective polysaccharide Unknown Bacterial infections Impaired specific antibody Only in case of severe antibody deficiency production in response to recurrent infections (SAD) polysaccharide vaccination Iatrogenic Hypogammaglobulinemias Corticosteroid Suppression of T-cell Bacterial, viral and fungal Low IgM, IgG and/or IgA No formal guidelines hypogammaglobulinemia* mediated immunity and B-cell infections Possible impaired antibody functioning 21 response to polysaccharide vaccination Rituximab Depletion of CD20-positive Bacterial, viral and fungal Low IgM, IgG and/or IgA No formal guidelines hypogammaglobulinemia** B-cell lineages24 infections Possible impaired antibody response to polysaccharide vaccination Hypogammaglobulinemia Suppression of T-cell Bacterial, CMV and fungal Low IgM, IgG and/or IgA No formal guidelines after solid organ mediated immunity and infections Possible impaired antibody transplantation B-cell functioning to various response to polysaccharide degrees31 vaccination Genes that are affected in primary antibody deficiencies are indicated, as well as the immunosuppressive mechanism for three immunosuppressive therapies. The use of antibody replacement therapy according to the current guidelines is summarized for the individual conditions. †Corticosteroid maintenance therapy is sometimes indicated for the management of chronic obstructive pulmonary disease, asthma and rheumatoid arthritis. ‡Rituximab maintenance therapy can be indicated for the management of rheumatoid arthritis, systemic lupus erythematosus, various other autoimmune diseases and hematological malignancies. AID: Gene encoding the activation-induced cytidine deaminase protein; B29: Ig-ß protein of the B-cell receptor complex; BLNK: B-cell linker gene; BTK: Bruton’s tyrosine kinase; CD19: Gene encoding the B-lymphocyte antigen CD19; CD20: B-cell differentiation antigen;CD40 : Gene encoding the CD40 molecule; CD40L: Gene encoding the CD40 ligand molecule; CD79A: Gene encoding the Ig-α protein of the B-cell receptor complex; CD81: Gene encoding the CD81 protein; CR2: Gene encoding the complement receptor type 2 (CD21) protein; CVID: Common variable immunodeficiency;ICOS : Inducible T-cell co-stimulator gene; IGHM: Gene enco- 109 ding the C region of the heavy chain of the pre-B cell receptor; IGLL1: Immunoglobulin lambda-like polypeptide 1 gene (surrogate light chain); LRBA: Gene encoding the lipopolysaccharide and beige-like anchor (LRBA) protein; MS4A1: Gene encoding the CD20 protein; NEMO: Gene encoding the NF kappa-B essential modulator protein; PI3K: Gene encoding phosphatidylinositol 3-kinase; SAD: Selective polysaccharide antibody deficiency; TNFRSF13B: Tumor necrosis factor receptor superfamily, member 13B gene; TNFRSF13C: Tumor necrosis factor receptor superfamily, member 13C gene; UNG: Gene encoding the uracil N-glycosylase protein. 110 Chapter 7

Table 2. Overview of common immunosuppressive therapies that can lead to iatrogenic hypogammaglobulinemia

Immunosuppressive agents that can cause hypogammaglobulinemia Strong immunosuppressants Adalimumab99 Ciclosporin5 Cyclophosphamide5 Etanercept99 Imatinib5 Infliximab99 Mycophenolate mofetil32 Rituximab5 Tocilizumab100 Mild immunosuppressants Azathioprine5 Corticosteroids5 Gold-based drugs5 Sulfalazine5

Table 2 presents an overview of immunosuppressive therapies that can lead to IHG. The effector mechanisms of two immunosuppressive therapies are also illustrated in Figure 1. Corticosteroids exert a myriad of anti-inflammatory and immunosuppressive effects. Corticosteroids are widely used in the treatment of various inflammatory and autoimmune conditions. Apart from suppressing pro-inflammatory T-cells and increasing antibody catabolism, corticosteroids also suppress B-cell proliferation and survival.21 Hypogammaglobulinemia has been reported after both short-term high-dose treatment and in long-term low-dose treatment with systemic corticosteroids (> 5 mg/day for more than 2 years).5, 22 It has been demonstrated that corticosteroid doses > 12.5 mg/day were associated with hypogammaglobulinemia.23 The immunoglobulin class that is most affected is IgG.

Rituximab is a monoclonal antibody directed against the CD20 molecule, which is present on immature and mature B-cells, but not on hematopoietic stem cells, pro-B cells, plasma cells or other cell lines. It is used in the treatment of several malignant and autoimmune conditions. Rituximab induces complement- and antibody-mediated cytotoxic reactions, resulting in a 90% decrease in the number of circulating CD20-positive B-cells.24 Also, rituximab is suggested to modulate regulatory T-cells to a limited extent.24 Rituximab therapy induced low IgM and IgG levels for a period of > 4 months in 22.4 and 3.5% of over 3000 rheumatoid arthritis (RA) patients, respectively.25 In a cohort of 243 patients with systemic lupus erythematosus (SLE) and various vasculitides treated with Antibody replacement therapy 111

rituximab, moderate or severe hypogammaglobulinemia (IgG < 5 g/l) was observed in 56% of the patients. It was transient and improved spontaneously in approximately half of them before the end of follow-up (median 42 months). IgM levels < 0.4 g/l were found in 58% of the 243 patients.15 A large review of the literature identified 21 cases of hypogammaglobulinemia after rituximab treatment out of 351 patients with idiopathic thrombocytopenic purpura (6%).26 The prevalence of hypogammaglobulinemia in patients receiving rituximab treatment is related to the number of treatment cycles and the cumulative rituximab dose,27, 28 as well as to the immunoglobulin levels before initiation of rituximab therapy.15 Rituximab-induced hypogammaglobulinemia is mostly transient, particularly when it develops within 6 months of the first rituximab dose.15 More rarely, rituximab can lead to hypogammaglobulinemia that persists for years after cessation of treatment.29 Rituximab-induced hypogammaglobulinemia is the form of IHG that likely resembles PADs the most. This is illustrated by human CD20 deficiency, which causes a CVID-like phenotype.30

The treatment schemes used after solid organ transplantation include immunosuppressive medication that collectively affects B-cells directly and also suppresses T-cell function.31 Variation between treatment schemes does exist, depending on the individual transplanted organs, and individual immuno suppressants affect the B-cell compartment to different degrees (e.g., mycophenolate mofetil is a particularly strong B-cell suppressant).32 A recent meta-analysis estimated the percentage of patients that develop hypogammaglobulinemia after solid organ transplantation to be 45% in the first year post-transplantation.33 Lower IgG levels pre-transplantation are associated with an increased probability of post-transplantation hypogammaglobulinemia in lung transplantation recipients.34

In general, the cumulative dose of immunosuppressive therapy appears to be related to the risk of IHG. An important contributing factor is the underlying disease. In RA and idiopathic thrombocytopenic purpura, the incidence of post-rituximab hypogammaglobulinemia is relatively low, while it is higher in SLE and vasculitides. These differences could be due to differences in the mechanisms of disease, or the underlying disease is rather a marker for the severity and duration of the total amount of immunosuppression given to a patient. Several genetic and environmental factors are probably of influence as well.8 112 Chapter 7

Manifestations of PAD & IHG

The clinical presentation and laboratory manifestations in PAD and IHG comprise a broad spectrum. Generally speaking, the severity of the immunological abnormalities found in laboratory investigations correlates with the severity of the clinical presentation. It is the current opinion that the clinical phenotype of a given PAD is determined by a combination of genetic and environmental factors.19 In IHG, these factors, plus the underlying disease, are likely to influence the clinical phenotype. Despite this heterogeneity, the individual disorders in both PAD and IHG have a number of distinctive features in common. The most prevalent PADs and three IHG-causing immunosuppressive therapies, as well as the underlying causes and clinical and laboratory manifestations are summarized in Table 1.

The most common clinical presentation of PAD and IHG is recurrent respiratory tract infections. Because antibodies are of vital importance in antibacterial immune responses, PADs are mostly characterized by recurrent bacterial infections. The role of antibodies in the protection against viral pathogens, in general, is of less importance. PAD patients do not commonly present with fungal or recurrent viral infections, apart from enteroviral infections.14 Furthermore, PADs have been associated with the development of autoimmune disease and malignancy.14 In addition, because of an oftentimes-significant diagnostic delay in PAD patients, organ damage can be present at the time of diagnosis.35, 36 Also, non-infectious pulmonary complications can be seen in CVID, which are termed granulomatous-lymphocytic interstitial lung disease. The pathogenetic background of this type of pulmonary disease is not yet known.37 Common laboratory manifestations of PADs include low immunoglobulin isotype or subclass levels, low isohemagglutinin antibody titers, low antibody titers to protein and polysaccharide antigens, and an impaired response to pneumococcal and other vaccinations. In some PADs, mature B-cells are absent in blood and bone marrow.

IHGs appear to be associated with recurrent bacterial infections, but to a lesser extent than PADs. Based on available data, it is suggested that the most common infections under systemic corticosteroids are bacterial, but the risk of viral and fungal infections is also increased.38 Low IgG levels at baseline, as well as sustained low IgG levels for a period ≥4 months under rituximab treatment are associated with an increased risk of serious infections in RA patients.39 Other studies did not find an increased general infection risk in RA patients.25 In patients with vasculitides that have low IgG levels after treatment with rituximab, an increased infection risk has been reported. 40, 41 This is not confirmed by other studies, although antibiotic prophylaxis may have reduced the infection Antibody replacement therapy 113

frequency in these hypogammaglobulinemic patients.42 In RA patients treated with immunosuppressants, there seems to be no increased risk for malignancies, except for a higher risk of certain cutaneous malignancies.43

Patients who develop moderate or severe IHG (IgG < 4g/l) after solid organ transplantation have an increased risk for respiratory tract infections in general, and also specifically for cytomegalovirus, Aspergillus and other fungal infections.33 Hypogammaglobulinemia (defined as IgG < 6.5 g/l) was associated with a significantly increased risk for post-transplantation bacterial infections and sepsis in living donor liver transplant recipients.44 Hypogammaglobulinemia after lung transplantation is not associated with an increased risk for community-acquired respiratory viral infections.45 Whether the viral and fungal infections are really attributable to IHG or IHG is a biomarker for severe immunosuppression in these patients receiving immunosuppressive combination therapy is still a matter of debate.33 Apart from a higher risk of infections, hypogammaglobulinemia after solid organ transplantation has been associated with decreased survival.33, 46-48 Furthermore, solid organ transplantation recipients, in general, have an increased risk for malignancy.49 Whether IHG further increases this risk is not known yet.

Laboratory manifestations of IHG can be the same as those seen in PADs, but with great variation between patients. Corticosteroid treatment, in general, does not seem to cause an impaired response to pneumococcal vaccination,50-52 and also, patients with corticosteroid-induced IHG mostly have a normal vaccination response.53 RA patients treated with rituximab and methotrexate respond less well to pneumococcal vaccination compared to RA patients treated with methotrexate alone.54 A systematic review by Eckerle et al.55 suggests that most solid organ transplant recipients can adequately respond to various vaccinations, but the evidence base for this is poor. Specifically, response to pneumococcal vaccination was evaluated in nine trials with substantial heterogeneity and was estimated to be 83%. The definition of a sufficient response was very loose compared to that in PAD guidelines, so the response rate was likely overestimated.

It should be noted that the majority of patients with IHG do not have recurrent infections.15 Also, not in all patients with infections related to immunosuppression, hypogammaglobulinemia will be found. It remains to be seen what factors influence the clinical presentation of patients with IHG. Environmental factors, as well as genetic predisposition, the effect of other immunosuppressants and immunosuppressive effects outside of the B-cell compartment certainly play a role. 114 Chapter 7

Antibody replacement therapy in PADs

International guidelines recommend the use of antibody replacement therapy in all patients with severe forms of PAD.11, 56 Antibody replacement therapy is also indicated in patients with milder forms of PAD and having severe recurrent infections.11, 56 Assessment of the response to vaccination is a standard parameter for determining the immune status of PAD patients, but there is still some controversy about the use of vaccination response to determine eligibility for antibody replacement therapy.57 The indications for antibody replacement therapy in the most common PADs according to the current guidelines are summarized in Table 1.

Antibody replacement therapy is given either in the form of intravenous immunoglobulins (IVIG) or in the form of subcutaneous immunoglobulins (SCIG). These delivery modes have different pharmacokinetic properties, each with their distinct advantages.58 Higher quantities of IgG can be injected, and therefore, higher serum levels can be achieved via IVIG. However, SCIG therapy can give more constant IgG levels between infusions, and can be self-administered more easily. IVIG therapy is mostly given once per month, while SCIG therapy is given weekly or daily. A recent evidence-based review did not establish the superiority of one form over the other regarding effectiveness or safety, but did note that the potential for self-administration of SCIG generally leads to a higher treatment satisfaction.59 In some situations, IVIG can still be preferable, for example, when subcutaneous access is lacking, when immediate correction of IgG levels is needed, or when a patient prefers monthly infusions to weekly or daily infusions.58

Although double-blinded placebo-controlled studies of antibody replacement therapy in PAD have never been performed, the cumulative evidence of existing studies is sufficiently compelling to establish the efficacy of both SCIG and IVIG in the prevention of infections in patients with severe PAD.11 The evidence base for the use of antibody replacement therapy in mild PAD is more limited and these patients should have a history of documented severe infections and impaired antibody response to a pure polysaccharide vaccine.57 Moreover, the necessity and effectiveness of antibody replacement therapy can be evaluated by monitoring the infection frequency and sometimes by means of periodic cessation.11, 56 Antibody replacement therapy has become very safe over the decades, and the risk for pathogen transmission or severe adverse events is very low. 60 The most common infusion-related adverse events of IVIG therapy include fever, chills, headache and myalgia. These are usually related to infusion speed. Less frequent, but more severe adverse effects include anaphylactic reactions, acute renal tubular necrosis, aseptic meningitis and thromboembolic Antibody replacement therapy 115

events. These are more likely to occur with the use of higher immunoglobulin doses.61, 62 SCIG therapy is commonly associated with local swelling and redness, but systemic reactions are rarer than with IVIG.61, 62 When adverse events occur, (temporarily) switching from one mode of administration to another may be helpful.

Commonly used immunoglobulin dosages for PAD patients vary between 200 and 800 mg immunoglobulin per kilogram bodyweight per month. Although a strong correlation has been reported between the amount of immunoglobulin substituted and serum IgG levels obtained, a standard substitution dose can lead to quite variable serum IgG levels.63 In general, higher dosages of immunoglobulins lead to a better therapeutic response,64 but this is not always so, and the use of higher-than-normal immunoglobulin doses has not been investigated thoroughly.65 Furthermore, it has been proposed that each patient may have his or her own ‘biological IgG level’, meaning the serum IgG level is associated with the best therapeutic response.58 This concept bears relation to the variable IgG levels that are found in healthy individuals. An individual’s biological IgG level is thought to depend on immune physiologic factors and comorbid conditions.58 This supports the individualization of immunoglobulin dosage based on both clinical and laboratory parameters. Some authors suggest that this can be achieved by increasing the immunoglobulin dose by 10–15% every month, until symptoms of infections are controlled.58 In this case, the doses of immunoglobulins are solely based on the patient’s clinical status.63

Antibody replacement therapy in IHG

In comparison with PAD, the use of antibody replacement therapy for IHG is much less well documented. In patients under corticosteroid immunosuppression, there are scarce reports of antibody replacement therapy. Most hypogammaglobulinemic steroid-dependent patients have a sufficient antibody response to vaccination, and antibody replacement therapy would thus not be warranted.53 In addition, when the vaccination response is impaired, this is mostly not associated with infections that can be attributed to the immunological defect that is found.66 Only when these patients have an impaired vaccination response and present with recurrent infections, antibody replacement therapy should be considered.

For rituximab-associated hypogammaglobulinemia, some authors recommend that only in case of IgG levels < 2 g/l and recurrent infections, antibody replacement therapy should be initiated.67 Others use a less strict cutoff value and recommend antibody replacement therapy in hypogammaglobulinemic 116 Chapter 7

patients who present with recurrent infections and do not respond sufficiently to prophylactic antibiotics.68 Some authors recommend IVIG as infection pro phylaxis in all infants and young children treated with rituximab,67 but others warn that the need for this remains questionable.69 Reports on the use of antibody replacement therapy in rituximab-associated hypogammaglobulinemia are scarce outside the domain of malignancy. All available reports only represent a handful of patients and IVIG, rather than SCIG, was used in most cases. These patients include adults and children with various autoimmune diseases, including SLE, autoimmune hemolytic anemia and autoimmune vasculitis. Most reports on symptomatic hypogammaglobulinemia do not elaborate on the effect of antibody replacement therapy on infection frequency or IgG levels. 40, 41, 70, 71

In a case report that does give information on these parameters, antibody replacement therapy led to increased IgG levels and decreased infection frequency.72 In another report, a patient who was treated with two cycles of rituximab for dermatomyositis developed severe symptomatic hypogammaglobulinemia. Subsequent initiation of IVIG maintenance therapy did not restore the immunoglobulin levels and infections were eventually managed with antimicrobial therapy.73 In one case series, six patients with autoimmune disease (RA [n = 3], SLE [n = 2] and autoimmune hemolytic anemia [n = 1]) developed symptomatic hypogammaglobulinemia (defined as IgG < 5.8 g/l) that could not be adequately managed with antibiotic treatment. These patients were started on IVIG replacement therapy and were reported to have a reduced rate of infections and hospital admissions, although quantitative data are lacking in the report.74 Another case series reported on 12 patients with autoimmune disease who received antibody replacement therapy for moderate or severe hypogammaglobulinemia (defined as IgG < 5 and < 3 g/l, respectively) and recurrent or persistent infections despite antibiotic prophylaxis. All the 12 patients were started on IVIG therapy and 6 patients subsequently switched to SCIG once stabilized. A sustained overall reduction in frequency and severity of infections was observed in 10/12 patients after initiation of antibody replacement therapy. The infection frequency was not quantified in the report, and therefore, statistical evaluation of the results was not performed.68 A recent report of three patients with idiopathic thrombo cytopenic purpura who were treated with rituximab and subsequently developed symptomatic hypogammaglobulinemia also reported that IVIG maintenance therapy reduced the number of infections.26

The use of antibody replacement therapy in asymptomatic hypogammaglobulinemia was judged to be successful in the available reports, based on the absence of infections and normalization of IgG levels.75 Other reports do not mention the efficacy of antibody replacement therapy for asymptomatic hypogammaglobu- Antibody replacement therapy 117

linemia.41, 76 Antibody replacement therapy has been used effectively in children with symptomatic and asymptomatic hypogammaglobulinemia under rituximab.67, 77, 78

There are guidelines on the use of antibody replacement therapy in solid organ transplantation recipients, but these mostly focus on the immunomodulatory effect of immunoglobulins.79 A review by Jordan et al.80 briefly mentions antibody replacement maintenance therapy, but found that there were few reports on antibody replacement therapy in post-transplantation hypogammaglobulinemia. The authors, nevertheless, concluded that monthly IVIG replacement therapy was effective in correcting post-transplantation hypogammaglobulinemia. A study of special interest is a double-blinded randomized controlled trial of anti-CMV–enriched immunoglobulins in heart transplantation recipients with moderate post-transplantation hypogammaglobulinemia (defined as IgG 3.5–5 g/l). This study comprised a small patient population (n = 23), which was followed over a period of 6 months. The group of patients that received antibody replacement therapy every time they had low IgG levels had a significantly lower number of CMV infections, compared to controls.81 A retrospective cohort study reported on 55 heart transplant recipients with post-transplant hypogammaglobulinemia (defined as IgG < 6 g/l) and at least one severe infection which were treated with three or more monthly infusions of IVIG until normalization of IgG levels (> 7.5 g/l). After IVIG therapy, humoral immune status in these patients was comparable with 55 non-hypogammaglobulinemic controls. Furthermore, the mean number of infections in the 6 months after IVIG therapy was significantly lower than that before IVIG therapy.82 A double-blinded randomized controlled crossover trial in which 11 lung transplant recipients with hypogammaglobulinemia (defined as IgG < 5 g/l) received IVIG and placebo for 3 months each did not find a reduced risk of bacterial infections under IVIG.83 A recent cohort study reported findings on 54 lung transplant recipients with hypogammaglobulinemia (defined as IgG level < 6 g/l) who were treated with monthly IVIG until the IgG levels normal- ized (> 7 g/l). Both overall survival and chronic lung allograft dysfunction-free survival were similar in the hypogammaglobulinemic patients compared to 30 non-hypogammaglobulinemic patients.84 Also, the number of infections did not significantly differ between both groups. Despite the obvious limitations of this study (most importantly, the retrospective character), the authors suggest that antibody replacement therapy equalizes survival in hypogammaglobulinemic lung transplant recipients compared to non-hypogammaglobulinemic controls, in part by reducing the risk of opportunistic infections. Some authors recommend that IVIG therapy should be started in all post-transplantation patients with severe hypogammaglobulinemia, as well as in patients with recurrent infections associated with low IgG levels.6 118 Chapter 7

In summary, the evidence base for the use of antibody replacement therapy in IHG is very small compared to the evidence base in PAD. Most available reports present positive findings, namely, that antibody replacement therapy reduces infection frequency in patients with IHG and recurrent infections. However, multiple patients who continued to have recurrent infections despite antibody replacement therapy have been reported. Almost all available reports describe the use of IVIG therapy, and data on the use of SCIG for IHG are virtually absent. Adverse effects of antibody replacement therapy are not discussed in most reports, but in three reports on post-solid organ transplantation hypogammaglobulinemic patients (combined n = 79, of whom 67 received ≥3 IVIG infusions), no moderate or severe adverse reactions were reported.81-83 Discontinu- ation of antibody replacement therapy due to adverse effects is not mentioned in any of the reports. Based on available evidence, patients who do not present with infectious manifestations and are prophylactically started on antibody replacement therapy remain infection free. Of course, it is unknown whether these patients would have had any infections without prophylactic antibody replacement therapy. Well-designed studies on the effect of antibody replacement therapy on the quality of life in IHG patients are lacking.

Discussion

In view of the similarities between PAD and IHG, it is remarkable that the use of antibody replacement therapy is so well established in one group (PAD) but is still poorly defined in the other (IHG). Guidelines for the treatment of IHG will have to include similar indications for the use of antibody replacement therapy, such as a proven susceptibility to infections, combined with insufficient response to a polysaccharide vaccine. Other management options, such as pro phylactic antibiotics and vaccinations,74 and importantly, cessation of immuno suppressive therapy will also have to be considered in IHG. Some issues remain that are relevant to the use of antibody replacement therapy in both PAD and IHG. Because there is more experience with antibody replacement therapy in the context of PAD, the situation in PAD will be used to exemplify these issues. It should be kept in mind, however, that these points are equally applicable to the use of antibody replacement therapy in IHG.

An important discussion point is the inherent imperfection related to any form of substitution therapy, which is that it only relieves the consequences of the disease but does not restore the causative defect. This is especially problematic in the substitution of antibodies because the production of antibodies in a normally functioning immune system is driven by pathogen exposure. Using antibodies from a pool of individual donors who have been exposed to the same pathogens as Antibody replacement therapy 119

the patient in question can protect the PAD or IHG patient against those pathogens. Inevitably, immunodeficient or immunosuppressed patients will remain vulnerable to novel or mutated pathogens. This becomes evident if we examine the infection frequency of immunodeficient patients under antibody replacement maintenance therapy. Analyses of multiple studies on PAD patients who receive antibody replacement therapy report infection rates ranging from 2.0 to 5.2 infections per patient per year.64, 85 Furthermore, organ damage still develops or progresses in patients under adequate antibody replacement therapy,36 possibly due to subclinical infections. It has also been suggested that because only IgG is substituted, an important part of the physiological airway defense (i.e., IgA and IgM) can still be missing, leaving patients with IgA or IgM immune defects prone to mucosal infections.86 Nevertheless, despite the above shortcomings, antibody replacement therapy has improved the quality of life of a large number of PAD patients and is still the most effective therapy available for these patients.

Immunoglobulins are a very costly product. The cost for antibody replacement therapy in an average adult PAD patient varies approximately between US$20,000 and US$40,000 per year, depending on the country and the immunoglobulin form and dosage.87, 88 Also, because immunoglobulin products are produced from donor plasma, they have a limited availability, and periodic shortages of immunoglobulin products have already occurred.89 The extended use of immunoglobulin products to IHG would lead to higher demand and, therefore, could be viewed as undesirable. Concerns about uncontrolled prescription of immunoglobulins for iatrogenic immune deficiencies have been expressed.90 In the future, prioritization algorithms might be necessary to distrib- ute the limited amount of immunoglobulin product that is available.91, 92 However, while most PAD patients are treated with lifelong antibody replacement therapy, for most IHGs, the use of antibody replacement therapy can be limited to periods of B-cell dysfunction. In addition, PAD and IHG both comprise a broad spectrum of clinical severity that overlaps for a great part. In designing guidelines for both PAD and IHG, the cost and availability aspects of immunoglobulin preparations should be kept in mind, but the guidelines should also acknowledge the conditions in which antibody replacement therapy can be most effective.

In conclusion, PAD and IHG are conditions characterized by an increased susceptibility for infections due to an impaired capacity to produce antibodies. While the treatment of PAD is guided by international guidelines and has a substantial evidence base, the treatment of IHG does not have it. As IHG is far more prevalent then PAD, likely to become even more prevalent in the future and can be a condition equally severe as PAD, the establishment of evidence-based guidelines is urgently required. These guidelines cannot be fully identical to those for the PADs, but should be tailored specifically. 120 Chapter 7

Expert commentary

Based on the available reports, antibody replacement therapy seems to be effective in reducing infection frequency in most patients with IHG and recurrent infections. There is no consensus on the precise management of these patients and when antibody replacement therapy should be started. Multiple authors agree that regular monitoring of IgG levels is advised both before the start71, 74 and during strong immunosuppressive medication.6, 8, 26, 42, 74 Other authors recommend that immunoglobulin levels should only be checked in case of recurrent, serious or unusual infections. 43, 67 As the risk factors for developing IHG are not yet known, we recommend monitoring of immunoglobulin levels before initiation of and every 3 months during strong immunosuppressive therapy (e.g., rituximab or post-solid organ transplantation immunosuppression). For milder immunosuppressive therapies (e.g., systemic corticosteroids > 5 mg/day for longer than 2 years), we recommend measuring the immunoglobulin levels before initiation. Follow-up measurements should only be performed for prolonged periods of immunosuppression in accordance with the treating physician’s clinical judgment. In addition, immunoglobulin levels should be measured in case of recurrent, serious or unusual infections in patients receiving any kind of immunosuppressive therapy.

We propose that the spectrum of iatrogenic immune deficiencies should also include the iatrogenic equivalent of specific antibody deficiency (characterized by infections, normal immunoglobulin levels and an impaired response to vaccination), and thus should not be limited to immune deficiencies that present with hypogammaglobulinemia. Therefore, response to pneumococcal poly saccharide vaccination should be measured in case of recurrent infections, even when IgG levels are within the normal range. Moreover, the response to pneumococcal polysaccharide vaccination should be measured in all patients who present with low IgG levels prior to initiation of or during immunosuppressive therapy. Measurement of this response includes a baseline measurement of anti-pneumococcal polysaccharide antibodies, as well as a follow-up measurement 4–8 weeks after vaccination.93 Pneumococcal polysaccharide vaccination is preferable as a functional immune status investigation because there is a large body of evidence for this vaccination in PAD diagnostics. Also, the large number of pneumococcal serotypes that can be tested allows for greater precision in determining functional immune status.93 Whether response to pneumococcal polysaccharide vaccination should be measured in all patients before they receive immunosuppressive therapy can be debated. Vaccination of patients with a pneumococcal polysaccharide vaccine prior to immunosuppressive therapy can also be considered in a preventive setting, rather than as diagnostic, in which case a pneumococcal conjugate vaccine could also be used. Antibody replacement therapy 121

When should antibody replacement therapy be started in patients with IHG then? Despite the similarities between PAD and IHG, the use of antibody substitution therapy in both conditions differs in some aspects. PAD is mostly recognized in patients who present with recurrent infections and is subject to significant diagnostic delay. Organ damage, especially bronchiectasis, may already be present in a significant part of PAD patients before treatment is started.36 By contrast, the development of IHG can be monitored by regular determination of serum immunoglobulin levels. It would be important to identify patients who are at a high risk of developing symptomatic hypogammaglobulinemia, so that therapy can be started before any serious infections develop.8 This poses a dilemma: should patients with hypogammaglobulinemia and who do not have recurrent or severe infections be prophylactically started on antibody replacement therapy?

There is a narrow line between adequately preventing severe infections and overtreatment. We propose that antibody replacement therapy should only be given prophylactically in patients with severe immune hypogammaglobulinemia and immune dysfunction due to multiple immunosuppressants or the underlying disease. Which patients qualify for prophylactic antibody replacement therapy is subject to their physician’s clinical judgment. In patients with hypogammaglobulinemia and recurrent or severe infections, we recommend preventive vaccination with pneumococcal and influenza vaccines, possibly combined with prophylactic antibiotics as a first line of treatment. There should be a low threshold for a trial of antibody replacement therapy if antibiotics are inadequate, which is in line with what other authors have suggested and prac- ticed.8, 68 For patients with recurrent infections and an impaired vaccination response, but no hypogammaglobulinemia, we recommend that the clinical presentation should be leading. When recurrent or severe infections persist despite preventive vaccinations and antibiotic prophylaxis, a trial of antibody replacement therapy for 6 months can be considered. Antibody replacement therapy can initially be given in the form of IVIG, but switching to SCIG therapy could be considered when the patient is clinically stable.68 Starting IVIG doses that have been used are 400 mg/kg/month, which were then titrated to effect on infection rate and a minimal serum IgG concentration of 8 g/l.68

Also, the moment of antibody replacement therapy cessation differs between PAD and IHG. Antibody replacement therapy can be useful in IHG as long as patients are at an increased risk for infectious complications. In principle, this is as long as immunosuppressive therapy is given, as immunoglobulin levels usually normalize after discontinuation of immunosuppressive therapy.15, 21, 27 In case of a substitution trial, antibody replacement therapy should be given for at least 6 months. When the clinical benefit is not evident, antibody replacement 122 Chapter 7

therapy can be stopped after 6 months and the clinical response to cessation should be evaluated. Depending on the outcome, antibody replacement therapy should then either be reinitiated or other management options should be sought. When the clinical benefit is evident during the trial, antibody replacement therapy should be continued until immunosuppressive therapy is stopped and can then be tapered off. In some cases, tapering off will cause the return of hypogammaglobulinemia and infections. Prolonged antibody replacement therapy may then be warranted.68

An area of special interest is the possible overlap of PAD and IHG. It has been suggested in some reports that asymptomatic, mild PADs will become manifest upon immunosuppression.24, 71, 94 In RA patients treated with repeated cycles of rituximab, borderline immunoglobulin levels before treatment were associated with hypogammaglobulinemia during treatment.27 The concept of mild PADs underlying IHG could be one of the factors that determine the clinical phenotype in these patients. When the use of immunosuppressants can trigger an underlying PAD to become manifest, antibody replacement therapy will be needed even after discontinuation of immunosuppressive therapy. When hypogammaglobulinemia is detected prior to the start of immuno suppressive therapy, closer monitoring of immunoglobulin levels and clinical status is recommended. A schematic overview of our recommendations for the management of IHG is provided in Figure 2.

Five-year view

As the prevalence of IHG will very likely increase due to the growing use of strong immunosuppressive therapies, international guidelines for the treatment of IHG should be established in the near future. The use of antibody replacement therapy in IHG can then be properly evaluated. Studies on the risk factors for the development of symptomatic IHG as well as the factors that influence the response to antibody replacement therapy are necessary. Another promising field of study is that of latent PADs that become manifest after immunosuppressive therapy. In this context, it would be of interest to study the response to pneumococcal vaccination prior to immunosuppressive therapy. Furthermore, home-based therapy with SCIG warrants further investigation in patients with IHG. SCIG has already proven to be beneficial in post-transplantation lung recipients and some patients with rituximab-associated IHG.68, 95 Another study on the use of SCIG in IHG has recently been announced.96 Antibody replacement therapy 123

Figure 2. Recommendations for follow-up and treatment of patients receiving immunosuppressive medications.

Future developments in the treatment of immune deficiency can benefit patients with PAD as well as patients with IHG. Current guidelines for PAD are not carved in stone and can be, and are still being, improved upon.57 For example, more re search is needed to evaluate optimal doses and IgG trough levels for the treatment of PAD patients.97 Developments in this area will most likely focus on individualization of treatment in accordance with the concept of ‘Biological IgG Levels’. An improvement in patient’s quality of life could be achieved by the implementation of home-based therapy wherever this is possible. 124 Chapter 7

Key issues • The pathogenesis of primary antibody deficiency (PAD) is based on defects at various stages of B-cell development. Immunosuppressive therapies can suppress B-cell development at various stages and can thereby cause iatrogenic hypogammaglobulinemia (IHG). • The clinical and laboratory manifestations of both PAD and IHG comprise a broad spectrum and vary between the type of PAD or IHG and even between patients with the same type of immune deficiency. • The most common clinical features are recurrent bacterial respiratory tract infections, but also viral and fungal infections. • Substitution treatment with intravenous or subcutaneous immunoglobulins can decrease the infection frequency and improve the quality of life in PAD patients. • The evidence base for the use of antibody replacement therapy in IHG is limited compared to that in PAD. Based on that limited evidence, antibody replacement therapy seems to be effective in reducing the infection frequency in IHG patients. • Routine measurement of immunoglobulin levels is advised before and during immunosuppressive therapy. • Antibody replacement therapy in IHG differs from that in PAD in that the development of IHG can be monitored and that antibody replacement therapy can be started before a patient presents with severe infections and possibly organ damage. • The best time of cessation of antibody replacement therapy in IHG is not always clear. This should be after the immunosuppressive therapy is stopped, but in some cases, prolonged substitution treatment may be warranted. • Before the start of antibody replacement therapy, prophylactic antibiotics, vaccination and cessation of immunosuppressive therapy should be considered in the treatment of IHG. • Some forms of PAD and IHG present with low immunoglobulin levels and an impaired response to vaccination, but no clinical signs of immune deficiency. In these cases, restraint should be exercised in starting antibody replacement therapy.

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CHAPTER 8

Long-term clinical outcome of antibody replacement therapy in humoral immunodeficient adults with respiratory tract infections

D.A. van Kessel1,2 T.W. Hoffman1 H. van Velzen-Blad3 P. Zanen1,2 J.C. Grutters1,2 G.T. Rijkers3,4

1Department of Pulmonology, St. Antonius Hospital, Nieuwegein, The Netherlands 2Division of Heart and Lungs, University Medical Center Utrecht, Utrecht, The Netherlands 3Department of Medical Microbiology and Immunology, St. Antonius Hospital, Nieuwegein, The Netherlands 4Department of Science, University College Roosevelt, Middelburg, The Netherlands

EBioMedicine 2017 Apr;18:254-60 132 Chapter 8

Abstract

In severe humoral immunodeficiency the indication for antibody replacement therapy (ART) is clear, and supported by several large studies. However, for milder forms of humoral immunodeficiency, the indication for ART is less clear. This is a retrospective cohort study of 87 adults with recurrent respiratory tract infections who received ART. The patients had severe or mild humoral immunodeficiency, and were followed up for a median of 62 months. Infection frequency, pharmacy-registered antibiotics use and hospital admissions significantly decreased under ART compared to the year prior to starting ART (median 5.50(anamnestically)-0.82(physician-confirmed) infections/year, p < 0.001; median 4.00-2.05 antibiotics courses/year, p < 0.001; mean 0.75-0.44 hospital admissions/year, p=0.009). These beneficial effects of ART were seen in both severe and mild immunodeficiency. Bronchiectasis was present in 27 patients when ART was started, but was not associated with clinical outcomes. An increase in hospital admissions under ART, observed in some patients, was significantly associated with pulmonary emphysema and current smoking. In conclusion, this study shows that ART is a long-term effective therapy in adults with recurrent respiratory tract infections with severe as well as with milder forms of humoral immunodeficiency. Long-term clinical outcome of antibody replacement therapy 133

Introduction

In 1952 Colonel Ogden C. Bruton described the first case of agammaglobulinemia in a child with recurrent respiratory tract infections (RTI). This patient was successfully treated with antibody replacement therapy (ART).1 Agamma globulinemia and hypogammaglobulinemia are states of absent or low levels of circulating antibodies. These conditions are the hallmark of primary antibody deficiencies (PADs). PADs vary in severity: from agammaglobulinemia to mild hypogammaglobulinemia to normogammaglobulinemia and impaired specific antibody production to vaccination. Various gene mutations have been found to cause PADs, but in most patients the precise cause remains unknown.2 The estimated prevalence for PADs ranges from 1.3 to 2.9/100,000 persons in Europe,3 although the actual prevalence is likely higher due to underdiagnosis of PADs and underrepresentation of mild PADs in these estimates.3 The most notable clinical manifestations of PADs are recurrent RTI,4 and the treatment of choice is still ART, as introduced by Bruton 64 years ago. Prognosis is mainly determined by organ damage due to infections and the development of autoimmune or malignant disease.

The indication for ART is evident in case of hypo- or agammaglobulinemia. 4, 5 Despite the lack of randomized placebo-controlled trials, there is a broad consensus to provide ART to these patients. Non-randomized studies demon- strating the benefit of ART in hypo- or agammaglobulinemic patients have been published decades ago, as well as more recently.6-12 The Primary Immunodefi- ciency Committee of the American Academy of Allergy, Asthma and Immunology performed a systematic literature review of the use of antibody replacement therapy (ART) in human disease in 2006.5 Despite the absence of double-blind placebo-controlled studies the committee found the existing studies compelling enough to indicate ART in patients with severe antibody deficiency (agammaglobulinemia or hypogammaglobulinemia), such as common variable immunodeficiency (CVID) or X-lined agammaglobulinemia (XLA).

In milder forms of humoral immunodeficiencies e.g. IgG subclass deficiency (IgGSD) or specific antibody deficiency (SAD) there is less evidence supporting ART. There are few studies that thoroughly compare clinical and immunological characteristics before and during ART in these patients. The most extensive study is a retrospective study of 132 adult patients with IgGSD and ≥ 4 RTI per year.13 Treatment with ART in the form of subcutaneous immunoglobulins (SCIG) resulted in a significant reduction of antibiotic-treated RTI in the most recent year of ART. Other studies have much smaller patient populations or were unable to obtain objective measures of infection frequency or baseline measurement with which to compare infection frequency under ART.14-16 134 Chapter 8

There have been no studies of ART with long-term follow up and robust outcome parameters in patients with mild immunodeficiency. As ART is an expensive therapy (20,000-30,000 euros per patient per year) it is important to establish clinical efficacy in patients with mild immunodeficiency.

The outpatient clinic of the Department of Pulmonology at the St. Antonius Hospital is a referral center for adult patients with recurrent RTI. Clinical and immunological characteristics of the patients are systematically evaluated, and then a multidisciplinary decision is made whether or not to initiate ART. This patient group comprises both severe adult-onset immunodeficiency such as CVID and mild adult-onset immunodeficiency such as SAD. Here we present the clinical and immunological data from adult patients with recurrent RTI who were treated with ART at our center. The objective of this study was to determine the efficacy of ART in this population. Clinical outcome parameters were compared between the period prior to ART and long-term follow-up.

Patients and methods

In this retrospective cohort study we included 87 patients referred for analysis of recurrent RTI in the period 1992 until 2014. The clinical and immunological screening before starting ART comprised infection history and immune status investigation according to the immune status protocol of that time (the Dutch Na- tional Working Group for Immunodeficiencies (WID) and European Society for Immunodeficiencies (ESID) protocols).17-19 Data were retrieved from patient records. For every patient, the history and laboratory results, including response to pneumococcal vaccination, at time of immunological screening were collected, as well as clinical and laboratory follow up data after that time. Data were collected up until July 2014, or until a patient was lost to follow up or died. Thus, there was no fixed length of the follow-up period. If patients gave written consent their home pharmacy was contacted with a request for providing the patient’s antibiotics use. Antibiotics use was only available for patients who started with ART after 2002. The local medical ethics committee approved of the study and allowed con- tacting the pharmacies of patients that had passed away. The study was conducted in accordance with the principles of the Declaration of Helsinki (2013 version).

Antibody Replacement Therapy

ART was started in patients with a diagnosed immune deficiency and recurrent infections. Intravenous immunoglobulins (IVIG) were given monthly at the outpatient department of the hospital or at home, whereas SCIG was (self-)administered daily or weekly at home. The choice between these two administration Long-term clinical outcome of antibody replacement therapy 135

routes was made on an individual basis. The preferred administration route for ART was IVIG in case of CVID. The starting dose depended on serum IgG levels. In patients with IgG levels < 5 g/l the dose was 400mg/kg/ every 4 weeks. In case of SAD or IgGSD the preferred route was SCIG with a starting dose of 2 ml (160mg/ml) daily or 15 ml (160mg/ml) every week by a subcutaneous infusion pump, depending on patient preference. Dose adjustments and changes in administration route were made based on IgG trough levels and infection frequency. The target IgG trough level was 7 g/l. ART with SCIG was usually discontinued after six months to evaluate infection frequency. ART was restarted if infection frequency and/or antibiotics use increased.

Immune Status Investigations

Standard laboratory investigations included blood leucocyte count with differentiation, complement status, serum immunoglobulins and IgG subclass-level measurement and pneumococcal antibody response after vaccination. All patients were vaccinated intramuscularly with a single dose of 23-valent pneumococcal polysaccharide vaccine (Pneumovax 23; Merck, Rahway, NJ, USA). Blood samples were routinely drawn before and 3–6 weeks after vaccination. Serum samples were stored at −80°C until use. Until 2008 total antibodies against capsular polysaccharides of two or three Streptococcus pneumoniae serotypes (3, 4 and 9) were measured by ELISA as previously described.20 After 2008 IgG antibodies against 8, 13 or 14 different pneumococcal polysaccharides were measured as described previously.21,22 For categorization of the antibody response to pneumococcal polysaccharide vaccination, the 2005 AAAAI/ ACAAI classification criteria were used.23

Diagnostic Classification of Immunodeficiencies

Patients were classified in different categories of primary immunodeficiency according to recent definitions. A patient was classified as CVID when fulfilling the following criteria: (1) IgG level < 7 g/l; (2) IgM level < 0.4 g/l and/or IgA level < 0.7 g/l; (3) onset of immunodeficiency at > 2 years of age; (4) absent isohemag- glutinins and/or poor response to vaccinations; and (5) exclusion of other defined causes of hypogammaglobulinemia.24 When patients fulfilled criteria 1, 3 and 5, but not criteria 2 and/or 4, they were classified as Idiopathic Primary Hypogammaglobulinemia (IPH).25 Patients with IgA levels < 0.07 g/l and normal serum IgG and IgM levels in whom other causes of low IgA levels had been excluded, were categorized as IgA deficiency. 4 When patients had normal levels of IgG, but IgG subclass levels below cut-off value they were classified as IgGSD.4 Patients with normal levels of immunoglobulins and IgG subclasses, but an impaired response to pneumococcal polysaccharide vaccination, were classified as 136 Chapter 8

SAD.4 Patients with a serum monoclonal protein were classified as MGUS when: (1) serum M-protein < 30g/l; (2) bone marrow clonal plasma cells < 10%; (3) no evidence of other B-cell proliferative disorders; and (4) no related organ or tissue impairment.26 For the purpose of this study, we refer to CVID and IPH as severe immunodeficiency, and to IgGSD and SAD as mild immunodeficiency.

Clinical Outcome Parameters

Recurrent RTI were defined as having three or more infectious periods per year. Infection frequency prior to starting ART was based on patient reporting. Infection frequency after ART was based on confirmation of infectious episodes by a physician. Infectious episodes were categorized as sinusitis, bronchitis or pneumonia. Data on antibiotics use was provided by patient’s home pharmacies. In the Netherlands antibiotics are only available with a prescription from a physician and all antibiotics use is registered. Hospital admissions were scored in case of infection and/or exacerbation of underlying pulmonary disease.

Data Management and Statistics

Data collection and management were performed using Microsoft Office Excel 2011. Statistical analyses were performed using IBM SPSS Statistics version 24. Fisher’s exact test was used for comparing dichotomous variables where appropriate. Mann-Whitney U test and Wilcoxon Signed Rank test were used for comparing continuous variables where appropriate. Graphs were created in IBM SPSS Statistics version 24.

Results

Eighty-seven patients were included in the study. Patient characteristics prior to ART are shown in Table 1. Fifty-five patients were female. The median age was 61 years at start of ART. In the majority of the patients comorbid conditions were present. Thirty-six patients were diagnosed with chronic obstructive pulmonary disease (COPD). COPD patients were significantly more likely to smoke or have smoked (32/36 COPD patients versus 20/51 non-COPD patients; p = 0.0001) to use corticosteroid maintenance therapy (11/36 COPD patients versus 6/51 non-COPD patients; p = 0.02), and to have been admitted to the hospital in the previous year (23/36 COPD patients versus 18/51 non-COPD patients; p = 0.001). HRCT-scan of the thorax showed bronchiectasis in 27 patients and pulmonary fibrosis in 3 patients. Infectious episodes prior to ART included sinusitis in 62 patients, bronchitis in 80 patients and pneumonia in 62 patients. Twenty-eight patients had undergone sinus surgery. Patients Long-term clinical outcome of antibody replacement therapy 137

reported to have had infections for a median of 5 years (interquartile range 2 – 18.5 years) at time of immunological screening. The results of the immune status investigations are shown in Supplementary Table 1. The group of mild immunodeficiency consists of 37 patients with IgGSD (n = 27) or SAD (n = 10). The group of severe immunodeficiency consists of 43 patients with CVID (n = 15) or IPH (n = 28). The total group includes these two groups, as well as patients with MGUS (n = 5), IgA deficiency (n = 1) and no immunological defect (n = 1).

Seventeen patients were initially treated with IVIG and 70 patients with SCIG. In two patients with anti-IgA antibodies, pre-treatment with SCIG was given prior to starting IVIG. Twenty patients switched from SCIG to IVIG and one patient from IVIG to SCIG. Seventeen types of adverse effects were reported in 15 patients during IVIG. Adverse effects were reported in 23 patients during SCIG (Table 2). Adverse effects were mild except in one patient who developed a moderately severe systemic reaction after changing to another brand of IVIG. In 11 patients the therapy was stopped because of mild adverse reactions or because the therapy itself was perceived as too burdensome.

The total study group comprised 543 patient-years including 383 patient-years under ART. The median follow-up time was 62 months (interquartile range 34.5 – 98.5). Thirty-seven patients were treated with IVIG during a median time of 23 months (interquartile range 6 – 63) and 71 patients with SCIG during a median of 11 months (interquartile range 7 – 50). The follow up of 10 patients, who were referred for immunological evaluation and initiation of ART, took place in the referring hospital. In 35 patients therapy with SCIG was discontinued after 4 – 8 months to evaluate the effect. In 19 of these patients it was necessary to restart ART after median of 12 months (interquartile range 2.5 – 15) due to increasing infection frequency. Details on comorbidities during the follow up, as well as details on mortality and the causes of death are shown in Supplementary Table 2. Mortality was associated with male gender (p = 0.005), higher age at start of ART (p = 0.007), COPD Gold 4 (p = 0.02), and development of malignancy during follow up (p = 0.0008).

The outcome measures of efficacy of antibody replacement therapy are summarized in Table 3 and Figure 1. Anamnestically patients had a median number of 5.50 infections per year (interquartile range 4.00 – 8.00; available in 50 patients). In the year prior to ART the median number of antibiotic courses was 4.00 (interquartile range 2.00 – 6.25; available in 46 patients). The mean number of hospital admissions was 0.75. Thirty of 76 patients were admitted at least one time in the year prior to ART. 138 Chapter 8

Table 1. Clinical characteristics prior to the start of antibody replacement therapy

Demographic characteristics Total 87 Female 55 Median age at start of antibody replacement therapy (IQR) 61 (50.5 – 67) Comorbid conditions Allergic disease 22 Chronic obstructive pulmonary disease 36 Asthma 21 Auto-immune disease 8 Previous malignancy 8 Previous sinus surgery 28 Smoking status Never smoker 35 Ex-smoker 28 Current smoker 24 Lung function [1] Normal 25 GOLD I 10 GOLD II 20 GOLD III 11 GOLD IV 3 Chest HRCT scan [2] Bronchopathy 19 Emphysema 25 Bronchiectasis 27 Pulmonary fibrosis 3 Medication Corticosteroid maintenance therapy 17 Other immunosuppressive maintenance therapy 1 Infectious episodes Sinusitis 62 Bronchitis 80 Pneumonia 62 Encapsulated bacteria in sputum culture 62 Median number of years since onset of infections (IQR) [3] 5 (2 – 18.5)

IQR: interquartile range. [1] Data available in 69 patients. [2] Data available in 75 patients. [3] Data available in 83 patients. Long-term clinical outcome of antibody replacement therapy 139

Table 2. Adverse events during antibody replacement therapy

Adverse events IVIG Mild systemic reaction - Self-limiting cardiac arrhythmia 1 - Fever after infusion 3 - Hypertension after infusion 1 - Urticaria 1 - Multiple symptoms 10 Moderately severe systemic reaction - Dyspnea and hypotension. 1 SCIG Mild local reaction - Pain at infusion site 7 - Hematoma at infusion site 7

- Hardening of skin at infusion site 8 - Rash at infusion site 1

Each type of adverse event is scored only one time per patient.

The yearly frequency of physician confirmed infections under ART was significantly lower than the anamnestically obtained infection frequency prior to therapy (p < 0.001). The yearly frequency of antibiotics use was significantly lower under ART compared to the year prior to ART (p < 0.001). The antibiotics use decreased under ART in 33 out of 46 patients and increased in 10 patients. Clinical and outcome parameters were not significantly different between the two groups. There were no significant differences in age, gender, time to diagnosis, type of PAD, or comorbid conditions.

The yearly number of hospital admissions was significantly lower under ART in the whole cohort compared to the year prior to ART (p = 0.009). The number of hospital admissions decreased in 26 out of 76 patients, increased in 19 patients and 31 patients had no admissions before or under ART. In the group with an increase in hospital admissions 15 out of 19 patients were not admitted to the hospital in the year before ART. In these 19 patients, there were more current smokers (p = 0.08) and significantly less ex-smokers (p = 0.02). Nine patients showed radiologically confirmed emphysema on thorax HRCT (p = 0.01 compared to patients with no increase in hospital admissions). Six of these 9 patients were current smokers. The combination of HRCT-confirmed emphysema and 140 Chapter 8 - - - - 0.009 < 0.001 < 0.001 p-value 0.82 2.05 Total 0.44 (0.81) Under ART (0.00 – 1.38) (1.49 – 4.28) 46 50 76 5.50 4.00 Base line 0.75 (1.36) (4.00 – 8.00) (2.00 – 6.25) - - - 0.17 0.002 < 0.001 p-value 0.73 1.64 0.39 (0.81) Under ART (0.00 – 1.25) (0.28 – 2.62) 21 25 36 Severe immunodeficiency Severe 5.00 3.00 Infections Base line 0.64 (1.29) (4.00 – 7.50) (3.00 – 5.50) - - - 0.03 0.02 < 0.001 p-value 0.96 3.73 0.51 (0.86) Under ART (0.00 – 1.66) (1.67 – 5.63) 22 21 35 Mild immunodeficiency 6.00 4.50 Base line 0.80 (1.32) (4.00 – 8.00) (2.00 – 7.00) - - Data available available Data in N patients Median number of infec tions/year (IQR) Mean number of hospi - tal admissions/year (SD) Data available available Data in N patients Data available available Data in N patients Median courses of anti Median courses (IQR) biotic therapy/year IQR: interquartile range. SD: standard deviation. The group of mild immunodeficiency consists of 37 patients with IgGSD (n = 27) or SAD (n = 10). The group of severe severe of group The 10). = (n SAD or 27) = (n IgGSD with patients 37 of consists immunodeficiencymild of group The deviation. standard SD: range. interquartile IQR: IgA 5), = (n MGUS with patients as well as groups, two above the includes group total The 28). = (n IPH or 15) with = patients (n 43 CVID of consists immunodeficiency minimum The ART. under and before both available are data whom in patients only using calculated are p-values 1). = (n defect immunological no and 1) = (n deficiency of non-nor in spite the minimum period under ART is six months. In case of hospital admissions, mean values are indicated period before start of ART is one year, statistical significance (p-value < 0.05). mal distributions, because median values are all 0. Boldface in dicates Infectious outcome parameters under antibody replacement therapy parameters 3. Infectious outcome Table Long-term clinical outcome of antibody replacement therapy 141

Figure 1. Infectious outcomes during antibody replacement therapy [a] Number of antibiotics courses per patient per year prior to antibody replacement therapy (blue bars) and under antibody replacement therapy (green bars). [b] Number of hospital admissions per patient per year prior to antibody replacement therapy (blue bars) and under antibody replacement therapy (green bars). [c] Net change in antibiotics use per patient per year, when comparing the year prior to antibody replacement therapy with antibiotics use under antibody replacement therapy. Negative values on the Y-axis represent a decrease in antibiotics use under antibody replacement therapy. [d] Net change in hospital admissions per patient per year, when comparing the year prior to antibody replacement therapy with hospital admissions under antibody replacement therapy. Nega- tive values on the Y-axis represent a decrease in hospital admissions under antibody replacement therapy. N.B. patients were sorted on the X-axis for clarity and patient order is not the same in all graphs.

being a current smoker at the start of ART was significantly more present in patients with increased hospital admissions under ART (6/14 patients versus 2/57 patients; p = 0.0005). Five of 19 patients had a malignancy before ART (p = 0.02 compared to patients with no increase in hospital admissions). All 5 patients were considered disease free at the start of ART. There were no significant differences in age, gender, and time to diagnosis between the patients with an increase in hospital admissions compared to those with no increase in hospital admissions. 142 Chapter 8

Subgroup analysis, with CVID and IPH patients combined in one group (n = 43) and SAD and IgGSD patients combined in another group (n = 37) showed a significant decrease under ART of infection frequency (p < 0.001 in both groups) and antibiotics use (p = 0.002 in CVID/IPH and p = 0.02 in SAD/IgGSD). The number of hospital admissions decreased in both groups as well, but not significantly in the severe immunodeficiency group (p = 0.17 in CVID/IPH and p = 0.03 in SAD/IgGSD). Before the start of ART, there were no significant differences in infection frequency, antibiotics use and number of hospital admissions.

Outcome measures for the different administration routes are shown in Supplementary Table 3. All three outcome parameters decreased under both IVIG and SCIG, but not always significantly. Immunoglobulin levels at baseline and mean immunoglobulin levels under IVIG and SCIG are shown in Table 4. IgG levels increased significantly under antibody replacement therapy (p = 0.001 for IVIG and p < 0.001 for SCIG respectively).

There were 27 patients with bronchiectasis. No significant correlation was found between bronchiectasis and the time since start of respiratory tract infections and start ART. Numbers of infections and hospital admissions prior to ART were not significantly different between patients with and without bronchiectasis. IgG and IgG1 levels prior to ART were significantly higher in the group of bronchiectasis patients (p = 0.04 and p = 0.02).

Discussion

This single center cohort study shows the long-term efficacy of ART severe but also in mild immunodeficiency. We report 383 patient-years under ART with a long follow up for most of the patients. In patients with severe immunodeficiency (CVID/IPH) and patients with mild immunodeficiency (IgGSD/SAD) treatment with ART resulted in a significant decrease of the use of antibiotics, hospital admissions and infection frequency. Only the decrease in hospital admissions in the severe immunodeficiency group was not significant. Our results confirm and extend earlier studies where the beneficial effect of ART was established mainly with IVIG. In contrast to other studies, our patient group is homogeneous in clinical symptomatology but heterogeneous in underlying humoral immunodeficiency. Analysis was carried out with the patients serving as their own controls using robust outcome parameters such as hospital admissions and pharmacy-registered antibiotics use. There is no over- the-counter availability of antibiotics in the Netherlands, which permitted us to register and analyze all antibiotics use of a patient. Treatment with ART Table 4. Serum immunoglobulin levels during antibody replacement therapy Long-term clinicaloutcome ofantibodyreplacementtherapy

IVIG SCIG

N patients Baseline level Level under p-value N patients Baseline level Level under p-value IVIG SCIG IgM median (IQR) 25 0.60 (0.35 – 1.30) 0.45 (0.30 – 1.23) 0.08 33 0.70 (0.45 – 0.90) 0.55 (0.41 – 0.96) 0.009 IgA median (IQR) 21 1.00 (0.15 – 1.65) 0.88 (0.18 – 1.65) 0.73 33 1.10 (0.50 – 2.35) 1.41 (0.48 – 2.34) 0.92 IgG median (IQR) 29 5.40 (3.35 – 8.70) 7.48 (6.44 – 8.55) 0.001 45 6.70 (5.25 – 8.45) 7.58 (6.15 – 9.57) < 0.001 IgG1 median (IQR) 21 3.20 (2.10 – 5.75) 5.45 (4.14 – 6.27) < 0.001 59 4.60 (3.70 – 6.60) 5.30 (4.20 – 6.65) 0.04 IgG2 median (IQR) 21 0.90 (0.40 – 1.25) 2.30 (1.75 – 2.79) < 0.001 59 1.40 (1.00 – 2.20) 1.88 (1.50 – 2.45) < 0.001 IgG3 median (IQR) 21 0.30 (0.15 – 0.50) 0.30 (0.16 – 0.43) 0.22 59 0.40 (0.20 – 0.60) 0.37 (0.20 – 0.56) 0.03 IgG4 median (IQR) 19 0.10 (0.10 – 0.30) 0.17 (0.06 – 0.38) 0.28 59 0.20 (0.10 – 0.30) 0.20 (0.10 – 0.37) 0.02

IQR: interquartile range. p-values were calculated by comparing baseline levels with patient-specific median levels under (a specific form of) antibody replacement therapy. Boldface indicates statistical significance (p-value < 0.05). 143 144 Chapter 8

was generally well tolerated. The therapy was discontinued in eleven patients because of mild adverse reactions or because it was considered too burdensome.

The number of hospital admissions increased under ART in 19 patients. This was significantly associated with smoking status and radiologically confirmed emphysema and even more strongly with both factors combined. Smoking and chronic respiratory diseases are both known risk factors for severe RTI.27 Obviously, ART will not overcome structural lung damage. This raises the question whether there is an indication for ART in patients with progressive underlying respiratory disease and immunodeficiency. Nevertheless, it is not known whether the number of hospital admissions would have been even higher without ART. ART might slow disease progression, as infectious episodes can cause worsening of underlying respiratory disease. Despite existing comorbidity, most patients in our study showed a significant response to ART.

In clinical practice there should be a high index of suspicion for an immuno deficiency in patients with lung disease and recurrent RTI not responding to conventional therapies. Immunological screening may reveal an immuno deficiency that contributes to the susceptibility to infections. ART can be given intravenously or subcutaneously depending on IgG levels and patient preference. In general, for patients with severe immunodeficiency the intravenous route is preferred. These patients need higher doses of immunoglobulins to achieve normal trough levels, and it is burdensome to give these quantities subcutaneously. All CVID patients in our cohort received immunoglobulins via the intravenous route. Therapy effect can be evaluated by monitoring infection frequency, use of antibiotics and hospital admissions in combination with IgG trough levels. Temporary discontinuation of therapy, followed by careful evaluation can also be helpful to select patients who benefit from ART.

In our cohort bronchiectasis was not associated with a higher mortality or a longer diagnostic delay. Bronchiectasis has been associated with a diagnostic delay in other studies28 and is considered to be an important predictor of the prognosis of the disease. However an association with mortality has not always been found. We did not find an association with mortality or diagnostic delay possibly because we did not differentiate between mild and severe bronchiectasis. In our study 36% (27 of 75 patients) of the patients had bronchiectasis. The reported prevalence in other cohorts, consisting largely of CVID patients, varied from 11.2 – 47%.11, 28, 29

Limitations of our study are mainly related to the retrospective character. Because of this not all data were complete. Infection frequency before ART was determined anamnestically, while during ART all infections were physician- Long-term clinical outcome of antibody replacement therapy 145

confirmed. The latter is a stricter interpretation. The decrease of infection frequency during ART is very significant but should be interpreted with caution. In observational studies without control groups the phenomenon of regression to the mean can be inadvertently classified as a therapeutic effect. We find this unlikely in our study because the decision to start ART was based on an increase of infection frequency and spontaneous decrease of infection frequency in immunodeficient patients is not often seen. Another limitation is that the diagnoses of the patients receiving IVIG were different from the patients receiving SCIG. Patients receiving IVIG had more severe forms of immunodeficiency. However, in our study we did not intend to directly compare IVIG with SCIG, but rather to investigate the efficacy of ART as a whole. During the long follow up period there have inevitably been changes in diagnostic protocols and definition of antibody response. The IgG pneumococcal antibody response was categorized using the diagnostic criteria of 2005, 23 because these formed the basis for the decision to start ART in most patients. In 2015 the criteria were updated. 4 The new criteria have a stricter definition of an adequate antibody response.

In conclusion, ART is an effective and well-tolerated therapy. Measurement of IgG levels and assessment of pneumococcal antibody response after pneumococcal polysaccharide vaccination leads to selection of patients that benefit from ART. In this study we confirm that adults with recurrent RTI and an underlying mild or severe humoral immunodeficiency show a decrease in infection frequency, antibiotics use and hospital admissions upon treatment with ART.

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1. Bruton OC. Agammaglobulinemia. Pediatrics. 1952;9(6):722-728. 2. Durandy A, Kracker S, Fischer A. Primary antibody deficiencies.Nat Rev Immunol. 2013;13(7):519- 533. 3. Edgar JD, Buckland M, Guzman D, et al. The united kingdom primary immune deficiency (UK- PID) registry: Report of the first 4 years’ activity 2008-2012. Clin Exp Immunol. 2014;175(1):68- 78. 4. Bonilla FA, Khan DA, Ballas ZK, et al. Practice parameter for the diagnosis and management of primary immunodeficiency.J Allergy Clin Immunol. 2015;136(5):1186-205.e1-78. 5. Orange JS. Hossny EM. Weiler CR. Ballow M. Berger M. Bonilla FA. Buckley R. Chinen J. El-Gamal Y. Mazer BD. Nelson RP Jr. Patel DD. Secord E. Sorensen RU. Wasserman RL. Cun- ningham-Rundles C. Primary Immunodeficiency Committee of the American Academy of Al- lergy,Asthma and Immunology. Use of intravenous immunoglobulin in human disease: A review of evidence by members of the primary immunodeficiency committee of the american academy of allergy, asthma and immunology. J Allergy Clin Immunol. 2006;117(4 Suppl):S525-53. 6. Nolte MT, Pirofsky B, Gerritz GA, Golding B. Intravenous immunoglobulin therapy for antibody deficiency. Clin Exp Immunol. 1979;36(2):237-243. 146 Chapter 8

7. Ammann AJ, Ashman RF, Buckley RH, et al. Use of intravenous gamma-globulin in antibody immunodeficiency: Results of a multicenter controlled trial. Clin Immunol Immunopathol. 1982;22(1):60-67. 8. Cunningham-Rundles C, Siegal FP, Smithwick EM, et al. Efficacy of intravenous immunoglobulin in primary humoral immunodeficiency disease. Ann Intern Med. 1984;101(4):435-439. 9. Roifman CM, Lederman HM, Lavi S, Stein LD, Levison H, Gelfand EW. Benefit of intravenous IgG replacement in hypogammaglobulinemic patients with chronic sinopulmonary disease. Am J Med. 1985;79(2):171-174. 10. Quartier P, Debre M, De Blic J, et al. Early and prolonged intravenous immunoglobulin replacement therapy in childhood agammaglobulinemia: A retrospective survey of 31 patients. J Pedi- atr. 1999;134(5):589-596. 11. Quinti I, Soresina A, Guerra A, et al. Effectiveness of immunoglobulin replacement therapy on clinical outcome in patients with primary antibody deficiencies: Results from a multicenter prospective cohort study. J Clin Immunol. 2011;31(3):315-322. 12. Lucas M, Lee M, Lortan J, Lopez-Granados E, Misbah S, Chapel H. Infection outcomes in patients with common variable immunodeficiency disorders: Relationship to immunoglobulin therapy over 22 years. J Allergy Clin Immunol. 2010;125(6):1354-1360.e4. 13. Olinder-Nielsen AM, Granert C, Forsberg P, Friman V, Vietorisz A, Bjorkander J. Immunoglob- ulin prophylaxis in 350 adults with IgG subclass deficiency and recurrent respiratory tract infections: A long-term follow-up. Scand J Infect Dis. 2007;39(1):44-50. 14. Bjorkander J, Bengtsson U, Oxelius VA, Hanson LA. Symptoms in patients with lowered levels of IgG subclasses, with or without IgA deficiency, and effects of immunoglobulin prophylaxis. Monogr Allergy. 1986;20:157-163. 15. Abdou NI, Greenwell CA, Mehta R, Narra M, Hester JD, Halsey JF. Efficacy of intravenous gammaglobulin for immunoglobulin G subclass and/or antibody deficiency in adults. Int Arch Allergy Immunol. 2009;149(3):267-274. 16. Abrahamian F, Agrawal S, Gupta S. Immunological and clinical profile of adult patients with selective immunoglobulin subclass deficiency: Response to intravenous immunoglobulin therapy. Clin Exp Immunol. 2010;159(3):344-350. 17. Vossen JM, Zegers BJ. Diagnostic studies in patients suspected of having an immunodeficiency disorder. Tijdschr Kindergeneeskd. 1988;56(5):174-184. 18. de Vries E, Kuijpers TW, van Tol MJ, van der Meer JW, Weemaes CM, van Dongen JJ. Immu- nology in medical practice. XXXV. screening of suspected immunodeficiency: Diagnostic protocols for patients with opportunistic or recurrent severe infections, wasting and failure to thrive. Ned Tijdschr Geneeskd. 2000;144(46):2197-2203. 19. de Vries E. Clinical Working Party of the European Society for Immunodeficiencies (ESID). Patient-centred screening for primary immunodeficiency: A multi-stage diagnostic protocol designed for non-immunologists. Clinical & Experimental Immunology. 2006;145(2):204-214. 20. Van Kessel DA, Horikx PE, Van Houte AJ, De Graaff CS, Van Velzen-Blad H, Rijkers GT. Clin- ical and immunological evaluation of patients with mild IgG1 deficiency. Clin Exp Immunol. 1999;118(1):102-107. 21. Meerveld-Eggink A, van der Velden AM, Ossenkoppele GJ, van de Loosdrecht AA, Biesma DH, Rijkers GT. Antibody response to polysaccharide conjugate vaccines after nonmyeloablative allogeneic stem cell transplantation. Biol Blood Marrow Transplant. 2009;15(12):1523-1530. 22. Elberse KE, Tcherniaeva I, Berbers GA, Schouls LM. Optimization and application of a multiplex bead-based assay to quantify serotype-specific IgG against streptococcus pneumoniae polysaccharides: Response to the booster vaccine after immunization with the pneumococcal 7-valent conjugate vaccine. Clin Vaccine Immunol. 2010;17(4):674-682. 23. Bonilla FA, Bernstein IL, Khan DA, et al. Practice parameter for the diagnosis and management of primary immunodeficiency. Ann Allergy Asthma Immunol. 2005;94(5 Suppl 1):S1-63. 24. Conley ME, Notarangelo LD, Etzioni A. Diagnostic criteria for primary immunodeficiencies. representing PAGID (pan-american group for immunodeficiency) and ESID (european society for immunodeficiencies).Clin Immunol. 1999;93(3):190-197. Long-term clinical outcome of antibody replacement therapy 147

25. Driessen GJ, Dalm VA, van Hagen PM, et al. Common variable immunodeficiency and idiopathic primary hypogammaglobulinemia: Two different conditions within the same disease spectrum. Haematologica. 2013;98(10):1617-1623. 26. International Myeloma Working Group. Criteria for the classification of monoclonal gammo- pathies, multiple myeloma and related disorders: A report of the international myeloma working group. Br J Haematol. 2003;121(5):749-757. 27. Torres A, Peetermans WE, Viegi G, Blasi F. Risk factors for community-acquired pneumonia in adults in europe: A literature review. Thorax. 2013;68(11):1057-1065. 28. Brent J, Guzman D, Bangs C, et al. Clinical and laboratory correlates of lung disease and cancer in adults with idiopathic hypogammaglobulinaemia. Clin Exp Immunol. 2016;184(1):73-82. 29. Resnick ES, Moshier EL, Godbold JH, Cunningham-Rundles C. Morbidity and mortality in common variable immune deficiency over 4 decades.Blood . 2012;119(7):1650-1657.

148 Chapter 8 4 43 83 20 84 13 84 87 Total 6·88 (5·00-8·90) 1·35 (0·70-2·30) 0·70 (0·48-1·10) 0 1 0 0 1 0 1 1 8·6 (-) 2·40 (-) 1·50 (-) Normal 0 1 0 1 1 0 1 1 8·4 (-) Other 0·01 (-) 0·60 (-) IgAdef 1 3 0 1 5 2 4 5 13·60) MGUS 8·20 (5·55- 1·00 (0·70-1·30) 0·45 (0·24-1·65) 0 1 0 27 27 27 27 28 IPH 5·61 (5·10-6·15) 1·66 (1·10-2·20) 0·80 (0·55-0·92) Severe Severe 3 7 Immunodeficiency 15 15 12 15 15 15 CVID 3·70 (3·00-4·25) 0·30 (0·20-0·50) 0·40 (0·29-0·85) 0 0 0 0 10 10 10 10 SAD 10·98) 9·35 (9·00- 2·05 (1·53-3·08) 0·70 (0·63-0·85) Mild 0 1 5 4 Immunodeficiency 26 25 26 27 IgGSD 8·70 (7·80-9·78) 2·00 (0·90-2·70) 0·80 (0·50-1·70) IgG IgA IgM (IQR) Total < 7·0 g/l Anti-IgA < 0·70 g/l < 0·40 g/l Median (g/l) Median (g/l) Median (g/l) antibodies [1] Web extra materialWeb respiratory tract with infections adults immunodeficient humoral in therapy replacement antibody of Long-termoutcome clinical Diana A. Pieter van Hoffman, van Zanen, Heleen Velzen-Blad, Kessel, Thijs Jan C. W. Grutters Rijkers Ger and T. at time of start antibody replacement therapy 1. Results of immune status investigations Supplementary Table Long-term clinicaloutcome ofantibodyreplacementtherapy IgG1 27 10 14 27 5 1 1 85 < 4·90 g/l 11 0 14 25 1 0 0 51 Median (g/l) 5·50 (4·20-7·40) 6·50 (5·58-6·98) 2·90 (2·40-3·35) 3·90 (3·60-4·45) 6·90 (5·00-9·20) 6·0 (-) 5·6 (-) 4·40 (3·60-6·20)

IgG2 27 10 14 27 5 1 1 85 < 1·50 g/l 14 0 12 19 4 0 0 49 Median (g/l) 1·40 (1·10-2·50) 2·35 (2·13-2·75) 0·60 (0·13-0·80) 1·20 (1·00-1·50) 1·00 (0·90-1·00) 3·7 (-) 1·9 (-) 1·30 (0·90-2·10)

IgG3 27 10 14 27 5 1 1 85 < 0·20 g/l 5 0 3 3 2 0 0 13 Median (g/l) 0·50 (0·20-0·60) 0·40 (0·20-0·88) 0·25 (0·20-0·30) 0·30 (0·20-0·40) 0·44 (0·10-0·50) 0·60 (-) 0·70 (-) 0·40 (0·20-0·60)

IgG4 27 10 12 27 5 1 1 83 < 0·08 g/l 7 0 5 3 2 0 0 17

Isohemagglu- 18 8 12 21 4 1 1 65 tinins ≤ 1:8 3 2 9 2 0 0 0 16 149 150 Chapter 8 - 17 17 13 14 45 52 75 83 Total - - - - 0 1 0 1 Normal - - - - 1 1 1 1 Other IgAdef - - 2 2 3 3 5 5 MGUS 8 8 3 3 10 14 21 25 IPH Severe Severe 2 2 2 2 Immunodeficiency 10 11 14 15 CVID - - 4 4 6 6 10 10 SAD Mild 5 5 4 5 Immunodeficiency 15 16 24 26 IgGSD

8-plex 23vPPV Impaired Impaired Impaired Impaired 2-/3-plex response response response response 13-/14-plex Response to to Response Table 1. Continued Table IQR: interquartile range. Lower limits g/l,0·40 are normal of g/l,0·70 g/l,7·0 Lower range. g/l,4·9 interquartile IQR: g/l,1·5 0·2 g/l g/l0·08 and IgA,IgM, for respectively. IgG4, and IgG3 IgG2, IgG1, IgG, For 2- and 3-plex measurements patients were classified asresponder or non-responder on the basis of theirtotal pneumococcal polysaccharide antibodyresponse after vaccination. A responder was defined as having a post-vaccination titer > 20 U/ml and atleast a two-fold increase for two of two or two of the three sero pneumococcal - polysaccha pneumococcal IgG post-vaccination their on based responder low or responder as classified were patientsmeasurements, 14-plex or 13- 8-, For 20. tested types ride antibody response profile. 24 A positive antibodyresponse to a given serotype was defined as IgGa post-vaccination antibody concentration responses ≥ 1·3 μg/mlantibody or a the > 4-fold of 70% least at if responder a be to considered was patient A samples. serum post-vaccination and pre- the between increase concentration antibody < 0·025 g/l. of IgA levels in case measured positive. [1] anti-IgA antibodies were were serotypes) 6 of 8, 10 14 and 13 tested (i.e. tested the serotypes to Long-term clinical outcome of antibody replacement therapy 151

Supplementary Table 2. Follow up outcomes of 87 patients started on antibody replacement therapy

Follow up time Median follow up time (IQR) 62 months (34·5-98·5) Median time on IVIG (IQR) – 37 patients 23 months (6-62) Median time on SCIG (IQR) – 71 patients 11 months (7-50) Clinical outcomes [1] Died during study period 20 Malignancy 5 Pneumonia 4 Sepsis 2 End-stage COPD 4 Myocardial infarction 1 Unknown 4 Diagnosed with malignancy during follow up 9 Breast cancer 2 Lung cancer 4 Oropharynx cancer 1 Gastric cancer 1 Rectal cancer 1 Diagnosed with autoimmune disease during 4 follow up Primary biliary cirrhosis 1 Sjögren’s syndrome 1 Rheumatoid arthritis 1 Ulcerative Colitis 1 Sinus surgery during follow up 16 First-time sinus surgery 8

IQR: interquartile range. [1]: Ten patients were referred to our clinic for immunological screening and initiation of ART, but subsequently followed in the referring hospital. These patients are not included in these follow up outcome measures. 152 Chapter 8 - - - 0·009 < 0·001 < 0·001 p-value 1·38) 4·28) Total 0·82 (0·00- 2·05 (1·49- 0·44 (0·81) Under ART 50 46 76 8·00) 6·25) Baseline 5·50 (4·00- 4·00 (2·00- 0·75 (1·36) - - - 0·26 < 0·001 < 0·001 p-value SCIG 1·67) 4·36) 0·96 (0·00- 2·00 (1·41- 0·42 (0·79) Under SCIG 39 40 60 6·75) 6 (4-8) Baseline 4·00 (3·00- 0·55 (1·03) - - - 0·23 0·10 < 0·001 p-value IVIG 1·27) 4·80) 0·29 (0·00- 2·40 (1·57- 0·69 (1·22) Under IVIG 20 16 31 8·00) 6·75) Baseline 6·00 (4·25- 4·00 (1·00- 1·00 (1·61) Infections in N available Data patients Median number of infections/year (IQR) Antibiotics in N available Data patients Hospital Admissions Hospital in N available Data patients Median courses of Median courses antibiotic thera - (IQR) py/year Mean number of hospital admissi - ons/year (SD) Infectious outcome parameters during antibody replacement therapy parameters 3. Infectious outcome Supplementary Table IQR: interquartile range. SD: standard deviation. p-values are routes, with calculated Signed Wilcoxon administration Rank test different using for only patientsp-values in calculating whom data When are months. available both six before and is ART under period minimum the year, one is ART of start before period minimum The ART. under patientsonly via that who administration ART for route received at least six months are included. In the case of hospital admissions, mean values in are indicated spite of 0. all are median values non-normal distributions, because PART IV

REMARKABLE CLINICAL CASES

CHAPTER 9

Defective antibody response against pneumococcal serotype 9V in a patient with a single episode of pneumonia

D.A. van Kessel1,2 T.W. Hoffman1 H. van Velzen-Blad3 B. Meek3 S. van Mens3 J.C. Grutters1,2 G.T. Rijkers3,4

1Department of Pulmonology, St. Antonius Hospital, Nieuwegein, The Netherlands 2Division of Heart and Lungs, University Medical Center Utrecht, Utrecht, The Netherlands 3Department of Medical Microbiology and Immunology, St. Antonius Hospital, Nieuwegein, The Netherlands 4Department of Science, University College Roosevelt, Middelburg, The Netherlands

Submitted to Pneumonia after revision 156 Chapter 9

Abstract

Patients with recurrent respiratory tract infections and an impaired response to pneumococcal polysaccharide vaccination are diagnosed as a specific antibody deficiency. In adult patients with pneumococcal pneumonia an impaired antibody response to the infecting pneumococcal serotype can sometimes be found. It is unknown whether these patients are unable to produce an adequate anti-polysaccharide antibody response to pneumococcal vaccination after recovery. We describe a case of invasive pneumonia caused by Streptococcus pneumoniae serotype 9V in a previously healthy 35-year-old female. This patient did not produce serotype specific antibodies against the infecting serotype during disease. After pneumococcal polysaccharide vaccination 3 months after recovery, she responded adequately to most other pneumococcal serotypes, but still had no response to the infecting serotype 9V. After 9 years however, prior to pneumococcal conjugate vaccination, normal antibody levels against 9V were found. These antibody levels further increased after pneumococcal conjugate vaccination. In our opinion this is a first description of a temporary deficient response to the infecting pneumococcal serotype in adults, while other reports with similar observations all concerned children. Defective antibody response in a patient with pneumonia 157

Background

Community acquired pneumonia (CAP) is a serious disease, most frequently caused by Streptococcus pneumoniae.1 Invasive and non-invasive pneumococcal disease have a high mortality risk especially in the elderly patient with comorbidities.2 Vaccination with a 23-valent pneumococcal polysaccharide vaccine (23vPPV) induces antibody production against the external polysaccharide capsule of the pneumococcus.3 An impaired response to pneumococcal polysaccharide vaccination can be risk factor for recurrent respiratory tract infections.4 Vaccination with 23vPPV is part of the immunological screening in patients with recurrent respiratory tract infections.5 Those patients with an impaired response to pneumococcal polysaccharides are diagnosed with a specific antibody deficiency.4

Previously it was shown that a substantial proportion of patients with pneumococcal pneumonia did not show an antibody response to the infecting pneumococcal serotype either during the clinical course of the disease or during convalescence.6,7 This raises the question whether these patients are able to mount a serotype-specific antibody response after vaccination with 23vPPV.

We describe a patient with pneumococcal pneumonia in whom the infecting serotype was identified. After recovery, an assessment of the humoral immune status was made, including analysis of the antibody response to pneumococcal polysaccharide vaccination.

Case Presentation

A 35-year-old female was seen at the emergency department of our hospital. She presented with fever up to 40 degrees Celsius, shaking chills, dry cough, nausea, headache and right-sided chest pain. These symptoms were present for one week. Her medical history was unremarkable; she didn’t use any medication and was a non-smoker. The diagnosis pneumonia was made by physical and laboratory examination. The chest radiography showed a large right-sided lobar infiltrate (Figure 1). Because of impending respiratory insufficiency and hypotension she was admitted to the intensive care unit and immediately intubated. Treatment with penicillin and erythromycin was started. Streptococcus pneumoniae was detected as the causative microorganism by a blood and sputum culture and positive urine antigen testing, and identified as serotype 9V. A pharyngeal swab showed no signs of respiratory viruses on PCR testing. Antibiotic treatment was converted to penicillin only. 158 Chapter 9

Figure 1. (A) Chest X-ray at time of presentation at the emergency department showing a large right-sided lobar infiltrate. (B) Chest X-ray at the outpatient department taken 42 days after initial presentation showing almost complete resolution of the infiltrate.

Following 9 days of mechanical ventilation she was transferred to the ward. She made a good recovery and was discharged after 16 days.

Antibody titers against the capsular polysaccharides of 14 pneumococcal serotypes were measured on a Luminex platform as previously described (serotypes 1, 3, 4, 6B, 7F, 8, 9N, 9V, 12F, 14, 18C, 19A, 19F, and, 23F; Danish nomenclature).6 Samples were taken at the day of admission and 42 days later. In the recovery sample the patient showed a low antibody titer and no titer rise against pneumococcal serotype 9V. She had not been vaccinated with a pneumococcal vaccine in the past.

Three months after admission to the hospital she was seen at the outpatient department for a work-up according to the diagnostic protocol developed by the European Society for Immunodeficiency.5 In that context, 23vPPV was administered. Pneumococcal polysaccharide antibodies against 8 pneumococcal serotypes were measured on a Luminex platform before and 4 weeks after vaccination (serotypes 3, 4, 6B, 9V, 14, 18C, 19F, and 23F; Danish nomenclature). The results showed that the patient had a sufficient antibody response to 5 of the 8 pneumococcal serotypes tested (Table 1), but no response whatsoever against her infecting serotype 9V. The patient therefore did not have an adequate antibody response specifically to pneumococcal serotype 9V either after natural exposition or after vaccination with 23vPPV. Immunological work up showed no other abnormalities, i.e. serum immunoglobulins, IgG-subclasses and complement were all normal (Table 2). After this first episode of pneumonia, for nine years, the patient has had no severe or recurrent infections. Defective antibody response in a patient with pneumonia 159

Table 1. Pneumococcal antibodies (µg IgG/ml) during pneumonia and after pneumococcal polysaccharide (conjugate) vaccination. A sufficient serotype-specific response is defined as having titers higher than 1.3 µg/ml and at least a two-fold increase between pre- and post-vaccination titers.4

23V Pneumococcal 13V Pneumococcal Community-acquired polysaccharide vacci conjugate vaccination pneumonia nation (+ 3 months) (+10 years) Jan-Feb May-June July-Au- Timeline 2005 2005 gust 2014 Hospital Pneumococcal Recovery Pre-vacci- Post-vacci- Pre-vacci- Post-vacci- admission serotype (day 42) nation nation nation nation (day 1) 3 0.12* 0.14 0.05 2.43 6.27 13.09 4 0.06 0.03 0.15 0.17 0.02 0.42 6B 0.34 0.33 0.05 0.28 2.63 42.76 9V 0.43 0.25 0.08 0.06 4.06 15.35 14 2.25 2.67 3.21 7.28 6.91 9.55 18C 3.41 4.23 3.76 49.84 39.80 37.41 19F 1.03 1.08 0.60 6.85 31.96 77.10 23F 0.47 0.55 0.47 2.13 1.55 17.29

Table 2. Laboratory results of immunological work-up, performed 3 months after recovery from the episode of pneumonia.

Immunoglobulins IgM (g/l) 1.01 IgG (g/l) 11.6 IgA (g/l) 2.19

IgG1 (g/l) 6.9 IgG2 (g/l) 3.4 IgG3 (g/l) 0.4 IgG4 (g/l) 0.5 Specific IgG antibodies EBV VCA pos CMV neg Toxoplasma neg Rubella pos Isohaemagglutinins Anti-A 1/32 Complement CH50 (%) 95 AP50 (%) 75 MP (%) 90 160 Chapter 9

Recently the patient was included in the so-called CAPolista study (patient #6).8 In this study, patients who have experienced an episode of community acquired pneumonia were vaccinated with 13 valent pneumococcal conjugate vaccine and antibodies were measured before and after vaccination. Antibodies were measured on a Luminex platform as previously described (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F; Danish nomenclature).8 Quite unexpectedly, in the pre (conjugate) vaccination serum now a high anti-9V titer of 4 µg IgG/ml was found, which increased 3.8 fold after vaccination (Table 1). Also for serotype 6B the antibody titers had increased substantially in the 10 years after pneumococcal polysaccharide vaccination. For serotype 4 the antibody levels were and remained low (Table 1).

Discussion and conclusions

We describe a case of severe pneumonia in a previously healthy 35-year-old female. Immunological investigation showed an absent pneumococcal antibody response to the infecting serotype during the course of the disease. Upon vaccination with the 23vPPV three months after recovery, 9V antibodies remained low. Nine years later, a protective (i.e. > 1.3 μg/ml) and even high level of 9V antibodies was found already before revaccination with the 13-valent conjugate vaccine, and increased further after conjugate vaccination. According to the 2015 AAAAI/ACAAI criteria4 this patient has an impaired response to pneumococcal capsular polysaccharides as she had an adequate response to less than 70% of the tested serotypes (5 out of 8). In our opinion however, this patient does not meet the diagnostic criteria (inadequate response to pneumococcal polysaccharide vaccination, in combination with clinical characteristics suggestive of an immunodeficiency, such as recurrent infections),4 because she had no recurrent respiratory tract infections before or after the episode of the pneumococcal pneumonia.

The high antibody titer against 9V (and 6B for that matter) 9 years after the invasive pneumonia caused by serotype 9V and 23vPPV vaccination indicates a temporary hyporesponsiveness to selective serotypes including the infecting serotype that recovered and high antibody levels were observed after 9 years that further increased upon revaccination with the 13-valent conjugate vaccine. We repeated all serotype specific antibody measurements with essentially the same results (not shown). We have no additional blood samples during the 9-year period; the patient was in good clinical condition and didn’t report back at the hospital. We therefore do not know when during this period the 9V antibodies increased or whether there has been renewed colonization or a subclinical infection with 9V during that period. At any rate, there has been a temporary defect in the ability to respond to serotype 9V pneumococci. The cellular and/or molecular causes of this temporary hyporesponsiveness are unknown. Defective antibody response in a patient with pneumonia 161

Serotype-specific hyporesponsiveness towards the infecting serotype has been described in literature in children. In children who had been vaccinated with PCV prior to developing invasive pneumococcal disease, antibody levels directly after the infection were lower against the infecting serotype compared to other vaccine serotypes.9 Furthermore, it has been observed that some children with invasive pneumococcal disease remained hyporesponsive to the infecting pneumococcal serotype, even after recovery.9,10 A possible explanation for this phenomenon is that hyporesponsiveness to PCV can be caused by pneumococcal carriership at time of vaccination. It has been shown that nasopharyngeal colonization with a specific pneumococcal serotype at the time of vaccination is associated with a lower response to that serotype, even after subsequent booster vaccinations.11,12 Another possible explanation is that during the pneumococcal infection, a high load of circulating polysaccharide antigens can cause a temporary immune paralysis.13 Above considerations have been made based on observations in children, and data in adults are lacking.

In this case immune investigations were performed because the patient partic- ipated in a study on pneumococcal pneumonia in our center. This specific immunological defect would normally not have been found, as she did not meet the criteria for immunological screening.5 This case gives rise to the debate whether all hospitalized patients with a first episode of pneumococcal pneumonia should undergo an immune status assessment or at least pneumococcal vaccination and measurement of antibodies to the most common pneumococcal serotypes. The CAPolista study has shown that the vast majority of CAP patients that were vaccinated with a conjugated polysaccharide vaccine do show an adequate antibody response.8

In conclusion, these findings suggest that temporary hyporesponsiveness towards the causative pneumococcal serotype in CAP patients may occur but does not necessarily indicate a selective immunodeficiency since recovery of the impairment occurred over time. We do not know whether the temporary impairment was the cause of pneumonia or occurred due to a high load of capsular polysaccharides because of this pneumonia. However, this case report illustrates that next to children, also in adults pneumococcal infections may be associated with temporary hyporesponsiveness to infecting serotypes.

References

1. Drijkoningen JJ, Rohde GG. Pneumococcal infection in adults: Burden of disease. Clin Micro- biol Infect. 2014;20 Suppl 5:45-51. 2. Torres A, Peetermans WE, Viegi G, Blasi F. Risk factors for community-acquired pneumonia in adults in europe: A literature review. Thorax. 2013;68(11):1057-1065. 162 Chapter 9

3. Tomczyk S, Bennett NM, Stoecker C, et al. Use of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine among adults aged > /=65 years: Recom- mendations of the advisory committee on immunization practices (ACIP). MMWR Morb Mortal Wkly Rep. 2014;63(37):822-825. 4. Bonilla FA, Khan DA, Ballas ZK, et al. Practice parameter for the diagnosis and management of primary immunodeficiency. J Allergy Clin Immunol. 2015;136(5):1186-205.e1-78. 5. de Vries E, Clinical Working Party of the European Society for Immunodeficiencies (ESID). Patient-centred screening for primary immunodeficiency: A multi-stage diagnostic protocol designed for non-immunologists. Clin Exp Immunol. 2006;145(2):204-214. 6. van Mens SP, Meijvis SC, Endeman H, et al. Longitudinal analysis of pneumococcal antibodies during community-acquired pneumonia reveals a much higher involvement of streptococcus pneumoniae than estimated by conventional methods alone. Clin Vaccine Immunol. 2011;18(5):796-801. 7. Cremers AJ, Lut J, Hermans PW, Meis JF, de Jonge MI, Ferwerda G. Avidity of antibodies against infecting pneumococcal serotypes increases with age and severity of disease. Clin Vaccine Immunol. 2014;21(6):904-907. 8. Wagenvoort GHJ, Vlaminckx BJM, van Kessel DA, et al. Pneumococcal conjugate vaccination response in patients after community-acquired pneumonia, differences in patients with S. pneumoniae versus other pathogens. Vaccine. 2017;35(37):4886-4895. 9. Brousseau N, Andrews N, Waight P, et al. Antibody concentrations against the infecting serotype in vaccinated and unvaccinated children with invasive pneumococcal disease in the united kingdom, 2006-2013. Clin Infect Dis. 2015;60(12):1793-1801. 10. Tamura K, Matsubara K, Ishiwada N, et al. Hyporesponsiveness to the infecting serotype after vaccination of children with seven-valent pneumococcal conjugate vaccine following invasive pneumococcal disease. Vaccine. 2014;32(13):1444-1450. 11. Dagan R, Givon-Lavi N, Greenberg D, Fritzell B, Siegrist CA. Nasopharyngeal carriage of streptococcus pneumoniae shortly before vaccination with a pneumococcal conjugate vaccine causes serotype-specific hyporesponsiveness in early infancy. J Infect Dis. 2010;201(10):1570- 1579. 12. Rodenburg GD, van Gils EJ, Veenhoven RH, et al. Lower immunoglobulin G antibody responses to pneumococcal conjugate vaccination at the age of 2 years after previous nasopharyngeal carriage of streptococcus pneumoniae. J Pediatr. 2011;159(6):965-70.e1. 13. Borrow R, Stanford E, Waight P, et al. Serotype-specific immune unresponsiveness to pneumococcal conjugate vaccine following invasive pneumococcal disease. Infect Immun. 2008;76(11):5305-5309. CHAPTER 10

An unusual presentation of a patient with severe hypogammaglobulinemia

T.W. Hoffman1* D.A. van Kessel1,2* M.J.D. van Tol3 G. Vidarsson4 C.M. Jol-van der Zijde3 G.T. Rijkers5,6 H. van Velzen-Blad5

*Contributed equally to this article

1Department of Pulmonology, St. Antonius Hospital, Nieuwegein, The Netherlands 2Division of Heart and Lungs, University Medical Center Utrecht, Utrecht, The Netherlands 3Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands 4Department of Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory, University of Amsterdam Academic Medical Center, Amsterdam, The Netherlands 5Department of Medical Microbiology and Immunology, St. Antonius Hospital, Nieuwegein, The Netherlands 6Department of Science, University College Roosevelt, Middelburg, The Netherlands

Submitted 164 Chapter 10

Abstract

A full-term infant presented with two episodes of bacterial meningitis within the first two months of life. Serum analysis revealed a virtual absence of IgG. As infants largely depend on the prenatal transfer of maternal IgG in the first months of life, the mother was also investigated. She was a healthy 37-year-old female with a relatively uncomplicated medical history. She reported no severe or recurrent infections. Serum analysis revealed a very low IgG level (0.57 g/l) and normal levels of IgM and IgA. The mother was vaccinated with pneumococcal polysaccharide and diphtheria-tetanus-poliomyelitis vaccines. Surpris- ingly, she initially mounted a normal antibody response to both vaccines. However, follow up of anti-tetanus IgG antibodies showed a rapid decline. Af- ter exclusion of secondary causes of hypogammaglobulinemia, we diagnosed the patient with a primary antibody deficiency. The patient has received intravenous immunoglobulins for sixteen years, and is still in excellent health. Over the years, numerous additional immunological investigations were performed. None revealed an underlying immunological defect. In conclusion, this patient was coincidentally found to have severe hypogammaglobulinemia, and is doing very well on immunoglobulin replacement therapy for over sixteen years. Until now, no specific molecular or cellular defect has been found that explains this immunodeficiency. An unusual presentation of a patient with severe hypogammaglobulinemia 165

Introduction

Primary antibody deficiencies (PADs) are conditions characterized by hypo- or agammaglobulinemia and/or an impaired antibody response upon vaccination. The various types of PADs can range in severity from the virtual absence of immunoglobulins at a young age to adult-onset low immunoglobulin levels to normal immunoglobulin levels with an impaired specific antibody response after vaccination.1 Patients with PADs most often present with recurrent or severe infections, or infections with unusual pathogens. Infections are commonly of the respiratory tract. The diagnosis of a PAD can be made after taking an infection history, performing immunological laboratory investigations, including measurement of the response to vaccinations, and excluding other causes of the observed immunological defects.2 Treatment for hypogammaglobulinemia most often is immunoglobulin replacement therapy with Intravenous Immu- noglobulin (IVIG), prepared from pooled plasma of healthy adults. This therapy, with IVIG mostly containing IgG, effectively restores humoral immunity and has been shown to reduce infection frequency.3 Here we present a patient with normal IgM and IgA serum levels who was coincidentally found to be IgG deficient because of serious infections in her newborn child. The patient was followed for more than sixteen years, and over the years various diagnostic investigations have been performed. The patient provided written informed consent for the case to be published.

Case Presentation

A 37-year-old female was diagnosed with hypogammaglobulinemia after the birth of her third child. She had previously had two pregnancies, resulting in two healthy children. The first pregnancy had been complicated by HELLP (hemolysis, elevated liver enzymes and low platelet count) syndrome and the second by gestational diabetes. Also, the third pregnancy was complicated by gestational diabetes. The delivery after 38 weeks of pregnancy was uncomplicated. The child was male, had normal length and weight for his gestational age and had a good start. However, within two months, the child suffered from two episodes of bacterial meningitis. Retrospective analysis of the infant’s blood, taken at three weeks of age (during the first meningitis episode), showed a complete absence of IgG. As infants in the first months of life depend on the active transfer of maternal IgG across the placenta by the neonatal Fc-receptor (FcRn),4 the mother was evaluated. 166 Chapter 10

Original presentation

The mother reported a relatively uncomplicated medical history. She had been diagnosed with mild psoriasis at 14 years of age and had undergone an appen- dectomy at 15 years of age. The psoriasis has been in remission since early adulthood. She did not smoke, had no known allergies, used no medications and did not report any symptoms or complaints at the time of evaluation. She reported having one episode of tonsillitis per year since more than ten years, one episode of severe cystitis requiring hospitalization at 10 years of age and one episode of pyelonephritis at 28 years of age. She worked as an elementary school teacher. Physical examination showed a healthy female, and no abnormalities were found. Specifically, there was no lymphadenopathy or splenomegaly and her palatine tonsils were relatively large but otherwise unremarkable. Laboratory analysis revealed severe hypogammaglobulinemia. Serum IgA level was 3.8 g/l, IgM was 0.4 g/l and IgG was 0.57 g/l. All IgG subclass levels were low. The results of other immunological and additional laboratory investigations are shown in Table 1. Of note, there were no or very low circulating antibodies against specific antigens including diphtheria-tetanus-polio, despite a normal vaccination history. No other laboratory or immunological abnormalities were found. An HRCT-scan of the thorax showed no abnormalities. At diagnosis, the patient was vaccinated with a 23-valent unconjugated pneumococcal polysaccharide vaccine and diphtheria-tetanus-polio vaccine. Pre-vaccination antibody levels were very low against all tested antigens (Table 2). Because of the very low circulating IgG levels, the patient was started on IVIG at a dosage of 400 mg/kg/4 weeks immediately after obtaining post-vaccination blood samples. Surprisingly, she mounted normal antibody responses against both polysaccharide and protein antigens. IgG anti-tetanus antibodies showed a normal relative avidity for recognition of the antigen (data not shown). Isotype analysis of anti-pneumococcal polysaccharide antibodies showed relatively low levels of IgG antibodies five weeks after vaccination.

Follow up

After start of immunoglobulin replacement, IgG anti-tetanus antibodies were monitored for a period of 6 months. Compared to healthy controls, the patient showed an unusually fast decline of anti-tetanus IgG antibody levels. After six months, the IgG antibody level was reduced to less than 20% of the original post-vaccination antibody titer (Figure 1). Four years after start of immunoglobulin replacement the patient was doing well, she reported no infections in the past years and had steady IgG trough levels within the normal range under an IVIG dosage of 600 mg/kg/month (Figure 2). At the patients’ request, immunoglobulin replacement therapy was discontinued. An unusual presentation of a patient with severe hypogammaglobulinemia 167

Table 1. Laboratory results at time of diagnosis Reference ranges are based on the reference values used at the time of diagnosis. Absolute counts/ µl were derived from leukocyte count and differential (dual platform).

Test Result Reference range Erythrocytes (x10^12/L) 4.4 3.8 – 4.9 Ht (%) 41 36 – 44 Hb (mmol/l) 8.6 7.7 – 9.6 Leukocytes (x10^9/L) 4.8 3.0 – 10.0 Lymphocytes (%, absolute counts/μL) 35, 1700 20 – 35, 1000 – 2800 Thrombocytes (x10^9/L) 152 150 – 300 CRP (mg/l) < 5 < 5 ESR (mm/h) 9 2 – 12 Serum protein level (g/l) 65 66 – 79 Albumin (g/l) 40.4 35 – 45 Serum protein electrophoresis Normal Normal Blood type A positive Anti-B isohemagglutinin titer 1:16 ≥1:8 Immunoglobulins IgM (g/l) 0.4 0.4 – 2.5 IgG (g/l) 0.57 7.0 – 17.0 IgG1 (g/l) 0.5 4.9 – 11.4 IgG2 (g/l) 0.1 1.5 – 6.4 IgG3 (g/l) < 0.1 0.2 – 1.1 IgG4 (g/l) < 0.1 0.1 – 1.4 IgA (g/l) 3.8 0.5 – 3.7 IgA1 (g/l) 2.9 0.6 – 2.4 IgA2 (g/l) 0.37 0.1 – 1.6 Salivary IgA (mg/l) > 100 > 60 Specific IgG antibodies Sero-positive cutoff EBV VCA (titer) < 1:100 > 1:100 CMV (AU/ml) < 0.5 ≥ 15 Rubella (IU/ml) < 0.5 > 10 Toxoplasma (IU/ml) < 0.5 > 3 Autoimmune serology Sero-negative cutoff ANA titer < 1:40 < 1:40 RA Negative Negative 168 Chapter 10

Table 1. Continued Complement Reference range C3 (mg/dL) 133 90 – 180 C4 (mg/dL) 30 15 – 40 CH50 (%) 114 75 – 125 AP50 (%) 129 78 – 128 Lymphocyte subsets CD3+ (%, absolute counts/ µL) 71, 1200 55 – 83, 500 – 2300 CD4+ (%, absolute counts/µL) 42 28 – 57 CD8+ (%, absolute counts/µL) 29 10 – 39 CD4/CD8 ratio 1.4 1.0 – 3.5 NK-cells (%, absolute counts/µL) 15, 250 7 – 31, 90 – 600 B-cells (%, absolute counts/µL) 16, 275 3 -19, 100 – 500

The reason was that she wanted to know whether her IgG level would drop again when not receiving IVIG. Discontinuation of IVIG led to a marked decline of IgG levels (from 7.1 to 3.2 g/l). Therefore, after two months, IVIG-therapy was reinstated and IgG levels returned to the normal range. Until the present time, the patient has consistently received IVIG in a dosage of 400-600 mg/kg/month (at present 400 mg/kg/month). During the follow up period, which currently spans more than 16 years, she has not reported serious or frequent infections and has not developed any comorbidity. She is presently doing well. She is satisfied with the immunoglobulin replacement therapy, and although she did not have serious infections before IVIG, she has not had any episode of tonsillitis during IVIG, as opposed to yearly episodes before. In addition, the patient reported feeling less tired while on IVIG compared to both the time before IVIG was started and the brief period IVIG was stopped.

Family

There was no family history of autoimmune disorders, confirmed immunodeficiency and recurrent or severe infections. All three children of the patient are healthy. The third child has had no severe infectious episodes since the two episodes of meningitis. The immunoglobulin levels of all children were measured one and sixteen years after diagnosis of their mother. IgA, IgM and IgG levels were normal on both occasions. Furthermore, the patient has two living parents and one brother. All three are healthy and do not report a high infection frequency or serious infections. IgM, IgG and IgA levels of both parents and the brother were measured sixteen years after diagnosis of the patient and were normal. Table 2. Antibody response to vaccinations An unusualpresentationofapatientwithsevere hypogammaglobulinemia Pneumococcal vaccination was performed with 23-valent pneumococcal polysaccharide vaccine. Rabies vaccination was performed with a purified chicken embryo cell vaccine. Protective antibody level values are based on Plotkin 30 for DTP-vaccine, on van Kessel et al. 31 for 3-plex total anti-pneumococcal antibody measurement, on Bonilla et al. 2 for 14-plex IgG anti-pneumococcal antibody measurements and on Brinkman et al. 32 for median concentrations of Rabies antibodies after vaccination of healthy adults. The pre-vaccination samples taken six and eleven years after diagnosis, respectively, were taken just prior to a scheduled gift of IVIG, the post-vaccination samples were taken respectively one week and just prior to the next scheduled gift of IVIG. A normal response to pneumococcal vaccination is defined as a two-fold increase over baseline and values ≥20 U/ml for at least 2/3 serotypes when using 3-plex total anti-pneumococcal antibody measurement and as a two-fold increase over baseline and values ≥1.3 µg/ml for at least 70% of serotypes (i.e., 10 out of 14 serotypes) when using 14-plex IgG anti-pneumococcal antibody measurement.

Vaccine/antibodies Before IVIG Under IVIG Protective levels Time of diagnosis +6 years +11 years Prevaccina- +3 weeks +5 weeks Prevaccina- 3 weeks post- Prevaccina- 5 weeks post- tion tion vaccination tion vaccination DTP Diphteria (Ig; AU/ml) < 0.01 1.42 0.21 0.30 0.24 0.25 > 0.1 Tetanus (Ig; AU/ml) 0.02 > 16 2.93 7.26 2.23 4.00 > 0.1 Poliomyelitis type l (Ig; titer) < 1/2 ≥1/4098 ≥ 1:8 Poliomyelitis type II (Ig; titer) < 1/2 1/128 ≥ 1:8 Poliomyelitis type III (Ig; titer) < 1/2 1/256 ≥ 1:8 Pneumococcal vaccination PPS3 (Ig; (U/ml) 6 107 64 ≥ 20 PPS3 IgM:IgG:IgA 199:28:1203 100:100:100 PPS4 (Ig; U/ml) 3 19 18 ≥ 20 PPS4 IgM:IgG:IgA 52:14:121 100:100:100 PPS9 (Ig; U/ml) 1 20 12 ≥ 20 169 Table 2. Continued 170 Vaccine/antibodies Before IVIG Under IVIG Protective levels Time of diagnosis +6 years +11 years Prevaccina- +3 weeks +5 weeks Prevaccina- 3 weeks post- Prevaccina- 5 weeks post- tion tion vaccination tion vaccination PPS9 IgM:IgG:IgA 109:49:359 100:100:100 PPS1 (IgG; µg/ml) 1.3 1.1 ≥1.3 PPS3 (IgG; µg/ml) 0.82 0.79 ≥1.3 PPS4 (IgG; µg/ml) 0.55 0.57 ≥1.3 PPS6B (IgG; µg/ml) 2.5 2.9 ≥1.3 PPS7F (IgG; µg/ml) 2.4 2.6 ≥1.3 (PPS8 (IgG; µg/ml) 1.9 1.8 ≥1.3 PPS9N (IgG; µg/ml) 0.94 0.94 ≥1.3 PPS9V (IgG; µg/ml) 1.2 1.4 ≥1.3 PPS12F (IgG; µg/ml) 1.4 0.98 ≥1.3 PPS14 (IgG; µg/ml) 4.5 5.6 ≥1.3 PPS18C (IgG; µg/ml) 1.9 2.2 ≥1.3 PPS19A (IgG; µg/ml) 2.9 3.2 ≥1.3 PPS19F (IgG; µg/ml) 2.5 3.0 ≥1.3 PPS23F (IgG; µg/ml) 1.4 1.9 ≥1.3 Rabies 4 weeks postvaccination Median response controls

Rabies (IgG; IU/ml) 0.13 0.19 1.9 Chapter 10 primary vaccination Rabies (IgG; IU/ml) 0.24 0.34 18.1 secondary vaccination An unusual presentation of a patient with severe hypogammaglobulinemia 171

Figure 1. IgG anti-tetanus antibodies before and after tetanus vaccination at time of diagnosis Blue dots represent antibody measurements in patient’s serum samples. The grey lines represent the expected decline of IgG anti-tetanus antibodies at a normal rate, based on the geometric mean of values measured in 20 healthy adult donors. Samples were taken prior to the start of immunoglobulin replacement (samples at 3 and 5 weeks post-vaccination), and after initiation of immunoglobulin replacement (6 and 8 months post-vaccination). TT = tetanus

Additional immunological investigations

Over the years, many additional immunological investigations have been performed with the aim to delineate the cellular and/or molecular cause of the immunodeficiency. Several additional vaccinations under immunoglobulin replacement therapy were given. Six years after diagnosis, the patient was vaccinated with a rabies vaccine (purified chicken embryo cell vaccine) to investigate the response to a T-cell dependent neo-antigen. She showed an impaired antibody response to this vaccine upon primary vaccination as well as secondary vaccination 3.5 months later (Table 2). At the same time, she was booster vaccinated with the diphtheria-tetanus vaccine, showing lower antibody responses than to the vaccination at time of diagnosis. Eleven years after diagnosis she was revaccinated with pneumococcal polysaccharide vaccine, as well as with diphtheria-tetanus vaccine. She showed almost no change in antibody levels in response to both vaccines (Table 2). In interpreting these responses, it should be kept in mind that the patient was receiving IVIG and therefore most if not all of the pre-vaccination antibodies were passively administered. 172 Chapter 10

Figure 2. Follow up of IgG levels since diagnosis The IgG dosage is indicated by shade of grey. Periods without IVIG are colored white.

IgG (except IgG3) has a long half-life of 3 weeks, as compared to the one week for similarly sized proteins. The long half-life of IgG is due to FcRn-mediated recycling. FcRn is also responsible for transport of IgG across the placenta.4 To test if a defect in FcRn-mediated functions could potentially explain our patient’s phenotype, we sequenced the FcRn gene as well as the B2m gene. No genetic defects were found in coding sequences. FcRn expression was measured in monocytes and this analysis indicated normal levels (Figure 3A). In addition, the turnover of IgG from the gammaglobulin preparations was measured. This was done by measuring the elimination of H435-IgG3, a rare IgG3 allotype present in gammaglobulin preparations, but not endogenously produced by most people of Western-European origin. People of Western-European origin (including our patient) normally produce R435-IgG3. R435-IgG3 has a half-life of one week due to diminished binding to FcRn, while H435-IgG3 has an extended half-life of three weeks like the other IgG subclasses, due to increased binding to FcRn.5,6 The results were compatible with a normal IgG turnover, also suggesting a normal expression and function of the FcRn in vivo (Figure 3B). This is supported by the observation of a normal serum albumin concentration (Table 1), as FcRn is also involved in rescuing albumin from degradation. 4

The development of B-cells is influenced by expression of the Fcgamma IIb receptor (FcγRIIb). This is one of the Fc-gamma receptors, a class of receptors that bind to antibodies and regulate the immune response. FcγRIIb is the only inhib- itory Fc-gamma receptor and is the only Fc-gamma receptor that is present on B-cells. It controls the magnitude and persistence of antibody responses through effects on mature B-cells, memory B-cells and plasma cells.7 In mice, over-expression of FcγRIIb has been found to lead to reduced serum IgG levels and suppression of late IgG antibody responses.8 Therefore, we examined the expression of FcγRIIb on monocytes, as previously described.9 This was found to be normal. Furthermore, multiplex ligation-dependent probe amplification analysis of several common single nucleotide polymorphisms and investigation An unusual presentation of a patient with severe hypogammaglobulinemia 173

Figure 3. FcRn expression and function (A) FcRn expression on blood monocytes as measured by flow cytometry through staining with anti-FcRn monoclonal antibody (DVN22). Blue shade represents DVN22 positive cells; grey shade represents negative isotype control. FcRn expression was measured on monocytes because of their high FcRn expression.33 (B) Measurement of H435-IgG3 levels after a gift of IVIG 16 years after diagnosis. Data points represent means and standard deviations of multiple measurements (four dilutions in duplo per time point).

of copy number variations in all Fc-gamma receptors was performed, as previously described.10 Also this revealed no abnormalities. Finally, long-range PCR of the FcgammaR2b gene10 was performed, which was also normal (data not shown).

To explore the possibility of a defect in B-cell or T-cell development, analysis of lymphocyte populations and B- and T-cell differentiation stages was performed with flow cytometry on a blood sample obtained at 16 years after diagnosis (Table 3). The numbers of T-, NK- and B-cells and the distribution of T-cell differentiation stages in the CD4+ and CD8+ T-cell subsets were normal. Also, B-cell distribution amongst the various differentiation stages was normal, including the numbers of circulating IgG isotype switched memory B-cells and natural effector (marginal zone) B-cells. In addition, after in vitro stimulation of peripheral blood mononuclear cells (PBMC) in the presence of IL-21 and anti-CD40 +/- anti-IgM or CpG, IgM, IgG and IgA were produced (Figure 4).

Further genetic evaluation of the TNFRSF13B gene was performed. This gene encodes the TACI protein, a transmembrane protein involved in B-cell development, survival and antibody production.11 A great variety of mutations in TNFRSF13B have been reported in patients with common variable immunodeficiency (CVID), but also in patients with milder types of PADs.11 One report describes a patient with a homozygous C104R mutation who presented with very low IgG levels, low IgA levels, normal IgM levels, and an initially normal antibody response to vaccination that had returned to baseline after 14 months.12 174 Chapter 10

Table 3. Lymphocyte and B-cell subset analysis 16 years after diagnosis Percentages of B-cell differentiation stages are within CD19-positive cells (=B-cells). Percentages of CD4+ and CD8+ T-lymphocytes are within CD3-positive cells (=T-cells). Percentages of CD4+ and CD8+ T-cell differentiation stages are within the respective T-cell subsets (CD4+ or CD8+). Markers used were as follows: CD3+ for T-lymphocytes; CD16/56+ CD3- for NK-cells; CD19+ for B-lymphocytes; CD- 38high CD24high for transitional B-cells; CD38dim CD24dim IGD+ CD27- for naïve mature B-cells; CD- 38dim IgD+ CD27+ for Marginal zone/Natural effector B-cells; CD38dim IgD- CD27+ for memory B-cells; CD45RO- CCR7+ CD27+ CD28+ for naïve T-lymphocytes; CD45RO+ CCR7+ CD27+ CD28+ for central memory T-lymphocytes; CD45RO+/- CCR7- for effector memory T-lymphocytes.

Percentage Absolute counts /µL Reference range (absolute counts/µL) White blood cells 5.4 4.0 – 10.0 (x10^9/L) Lymphocytes 1200 1000 – 2800 T-lymphocytes 1110 700 – 2100 NK-cells 130 90 – 600 B-lymphocytes 230 100 – 500 B-cell differentiation % of B-cells stages Transitional B-cells 6 13 3 – 50 Naïve mature B-cells 35.4 81 57 – 447 Natural effector B-cells 36.5 84 9 – 88 Memory B-cells 20.1 45 13 – 122 IgM memory B-cells 5.9 13 1 – 33 IgG memory B-cells 4.7 11 5 – 59 IgA memory B-cells 8.7 20 2 – 35 Plasmablasts 0.2 T-cell differentiation % of T-cells/subset stages CD4+ T-lymphocytes 59.7 700 300 – 1400 Naïve 41.6 Central memory 41.3 Effector memory 16.3 CD8+ T-lymphocytes 38.1 400 200 – 1200 Naïve 20.0 Central Memory 13.1 Effector Memory 66.3 CD4/CD8 ratio 1.75 1.0 – 3.5 An unusual presentation of a patient with severe hypogammaglobulinemia 175

Figure 4. Immunoglobulin levels in culture supernatant after in vitro stimulation of PBMC acquired 16 years after diagnosis PBMC were cultured for 7 days in the presence of the indicated (combination of) stimuli. IgG, IgA and IgM in the culture supernatant were quantified by sandwich ELISA. Stimuli: anti-CD40: MAB89, 0.5 µg/ ml; anti-IgM: 1 µg/ml; CpG: ODN2006, 1 µg/ml, IL-21: 20 ng/ml. Triangles: patient (freshly isolated PBMC); filled circles day control (viably frozen PBMC); open circles: historic controls.

This patient did not have any infectious manifestations, similar to our patient. Because of the somewhat similar laboratory findings and clinical presentation, we sequenced exon 3 of the TNFRSF13B gene in our patient. No abnormalities were found, and therefore a C104R mutation can be excluded.

Discussion

Here we describe a 37-year-old female patient with a virtual absence of IgG in serum. This was a coincidental finding, because the patient was otherwise healthy and had no recurrent or unusual infections. There was no evidence for a defect in specific IgG synthesis or catabolism and no general protein loss. However, we did find a rapid decline of circulating specific IgG antibodies in response to tetanus vaccination. Further booster vaccinations given over the course of follow up for more than sixteen years showed a diminishing response against tetanus and diphtheria. Additional investigations revealed that all differentiation stages of the B-cell lineage were present in the blood in normal numbers and an intact functional IgG recycling system was demonstrated. IgM, IgG and IgA were produced upon in vitro stimulation of B-cells. 176 Chapter 10

As other causes of hypogammaglobulinemia have been excluded, we have to conclude that our patient has a PAD with an unknown cause. She did not fulfill the diagnostic criteria for CVID,13 as she had normal IgM and IgA levels. Appar- ently, the patient does fit within the diagnostic category of Idiopathic Primary Hypogammaglobulinemia,14 but our patient had nearly absent circulating IgG levels and initially an overall normal antibody response to vaccination with T-cell dependent as well as T-cell independent antigens, although the titer of IgG anti-tetanus antibodies rapidly declined and the IgG response to PPS3, PPS4 and PPS9 was low. Therefore, our patient is not comparable with other patients in this category. PADs are caused by B-cell developmental defects, sometimes in combination with defects in other hematopoietic cell lines.15 In some cases, specific gene mutations have been found to underlie PADs. These mutations can lead to the impairment of B-cell differentiation at various stages.15 In the presence or absence of a known genetic defect, functional deficiencies in B-cell development can be detected, such as a low number of class-switched memory B-cells16,17 or marginal zone (natural effector) B-cells.18 Furthermore, patients have been described with no clear B-cell development or follicular T helper cell defects, despite clear hypogammaglobulinemia.14 Rare cases of low IgG levels caused by mutations in the β2-microglobulin gene (the protein product of which associates with the α-chain of MHC-class I family proteins, including FcRn) have been reported resulting in impaired FcRn-function (i.e., increased IgG and albumin turnover).4,19,20 Because of the clinical resemblance of our patient with FcRn deficient patients we have performed extensive molecular, cellular and functional studies on FcRn. We have to conclude that our patient is not FcRn deficient.

It is unknown how long this patient had been hypogammaglobulinemic prior to the first immunological evaluation. She had no remarkable history of infections, except for a yearly episode of tonsillitis since more than 10 years prior to the start of IVIG. One would expect that such a low IgG level would have led to more outspoken infectious manifestations. However, the patient did have a normal IgM and a high IgA (i.e., high IgA1 and normal IgA2) serum level and initially showed an adequate antibody response after vaccinations. Therefore, it could be argued that she was relatively well protected against (mucosal) infections despite such a low circulating IgG level. It has been shown for various forms of PAD that normal numbers of class- switched memory B-cells are associated with less severe infectious manifestations and less organ complications.16,21-23 In contrast to most CVID patients, our patient had normal numbers of circulating germinal center derived memory B-cells as well as marginal zone B-cells. Furthermore, NK cells and all differentiation stages in the CD4+ and CD8+ T-cell subsets and in the B-cells, including IgG+ switched memory B-cells, were normal, indicating a normal development. An unusual presentation of a patient with severe hypogammaglobulinemia 177

Also, B-cell function in vitro was not impaired. This all points to a post germinal center defect in B-cell development.

To the best of our knowledge, there are no other cases reported in the literature that have a similar clinical presentation with no severe, persistent or frequent infections, but with a profound IgG deficiency and normal IgA and IgM levels, an initially adequate IgG response to protein vaccines that rapidly declines and no abnormalities in B-cell differentiation stages or B-cell function in vitro. As the hypogammaglobulinemia in our patient was a coincidental finding, and there was no clinical presentation suggestive of an immunodeficiency, there might be other apparently healthy humans with comparable immunological characteristics that have not yet been recognized.

It cannot be excluded that the clinical presentation and laboratory findings in our patient represent a precursor stage of CVID. In contrast to the response to initial vaccinations (i.e., adequate IgG anti-tetanus antibody levels directly after vaccination, but with a fast decline, and a relatively low IgG anti-polysaccharide response), there was a lower response to later vaccinations. However, total IgM and IgA did not decrease at all during the sixteen years follow up (data not shown). A possible explanation for the presentation and laboratory findings in our patient might be a defect in the homing of IgG antibody secreting plasma cells to the bone marrow. In recent years, various factors that influence B-cell differentiation to plasma cells, plasma cell homing to the bone marrow and plasma cell survival in the bone marrow have been discovered.24,25 Interest- ingly, in mouse models of defective plasma cell homing/survival, a vaccination response comparable to that of our patient is seen. In mice deficient in the known plasma cell survival factors Aiolos, CD93 or Zbtb20, the antibody response to vaccination is initially adequate, but rapidly declines.26-29 As far as we know, the human phenotype of a plasma cell homing/survival defect has never been described.

In conclusion, we present a patient that showed a profound hypogammaglobulinemia after her (third) newborn child presented with two episodes of meningitis and was found to have absent IgG in blood. The patient had no history of severe or recurrent infections. Immunological analysis revealed very low serum IgG levels, normal IgA and IgM levels and an initially adequate antibody response to vaccination with T-cell dependent antigens that rapidly declined. There were no abnormalities in T-cell and B-cell differentiation stages and production of IgG after in vitro stimulation of PBMC was normal. The patient has been doing very well under immunoglobulin replacement therapy for more than sixteen years. Thus far, we have not been able to find a cellular or molecular explanation for this remarkable clinical presentation. 178 Chapter 10

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20. Ardeniz O, Unger S, Onay H, et al. Beta2-microglobulin deficiency causes a complex immunodeficiency of the innate and adaptive immune system. J Allergy Clin Immunol. 2015;136(2):392-401. 21. Alachkar H, Taubenheim N, Haeney MR, Durandy A, Arkwright PD. Memory switched B cell percentage and not serum immunoglobulin concentration is associated with clinical complications in children and adults with specific antibody deficiency and common variable immunodeficiency. Clin Immunol. 2006;120(3):310-318. 22. Vodjgani M, Aghamohammadi A, Samadi M, et al. Analysis of class-switched memory B cells in patients with common variable immunodeficiency and its clinical implications. J Investig Allergol Clin Immunol. 2007;17(5):321-328. 23. Aghamohammadi A, Abolhassani H, Biglari M, et al. Analysis of switched memory B cells in patients with IgA deficiency. Int Arch Allergy Immunol. 2011;156(4):462-468. 24. Nutt SL, Hodgkin PD, Tarlinton DM, Corcoran LM. The generation of antibody-secreting plasma cells. Nat Rev Immunol. 2015;15(3):160-171. 25. Recaldin T, Fear DJ. Transcription factors regulating B cell fate in the germinal centre. Clin Exp Immunol. 2016;183(1):65-75. 26. Cortes M, Georgopoulos K. Aiolos is required for the generation of high affinity bone marrow plasma cells responsible for long-term immunity. J Exp Med. 2004;199(2):209-219. 27. Chevrier S, Genton C, Kallies A, et al. CD93 is required for maintenance of antibody secretion and persistence of plasma cells in the bone marrow niche. Proc Natl Acad Sci U S A. 2009;106(10):3895-3900. 28. Wang Y, Bhattacharya D. Adjuvant-specific regulation of long-term antibody responses by ZBTB20. J Exp Med. 2014;211(5):841-856. 29. Chevrier S, Emslie D, Shi W, et al. The BTB-ZF transcription factor Zbtb20 is driven by Irf4 to promote plasma cell differentiation and longevity. J Exp Med. 2014;211(5):827-840. 30. Plotkin SA. Correlates of protection induced by vaccination. Clin Vaccine Immunol. 2010;17(7):1055-1065. 31. Van Kessel DA, Horikx PE, Van Houte AJ, De Graaff CS, Van Velzen-Blad H, Rijkers GT. Clinical and immunological evaluation of patients with mild IgG1 deficiency. Clin Exp Immu- nol. 1999;118(1):102-107. 32. Brinkman DM, Jol-van der Zijde CM, ten Dam MM, et al. Vaccination with rabies to study the humoral and cellular immune response to a T-cell dependent neoantigen in man. J Clin Immu- nol. 2003;23(6):528-538. 33. Vidarsson G, Stemerding AM, Stapleton NM, et al. FcRn: An IgG receptor on phagocytes with a novel role in phagocytosis. Blood. 2006;108(10):3573-3579.

CHAPTER 11

Summary and general discussion

Summary and general discussion 183

Summary and general discussion

Recurrent respiratory tract infections are a common problem, and can be due to a great variety of causes, including an underlying immunodeficiency. This thesis investigates the value of humoral immune status assessment in patients with respiratory tract infections, lung transplant candidates/recipients and the long-term treatment results of primary antibody deficiency with antibody replacement therapy.

Immune status assessment in patients with recurrent respiratory tract infections and suspected primary antibody deficiency

Immune status assessment is currently recommended for patients that present with recurrent respiratory tract infections, when other causes are unlikely.1 The European Society for Immunodeficiencies (ESID) immune status screening protocol includes various immunological tests, including measurement of immunoglobulin levels, IgG subclasses, response to pneumococcal polysaccharide vaccine and complement status.1 For the most part, interpretation of the results is clear. For example, low IgG levels (defined as an IgG serum concentration below 7.0 grams/l) are a well-known risk factor for recurrent infections. 2 However, other parts of the immune status screening protocol are less easily interpretable. MBL, as a part of the complement system, certainly plays a role in the immune response against encapsulated bacteria. Quantitative MBL deficiency is a relatively common trait affecting up to 20% of the population.3 Previ- ous studies reported conflicting results on the impact of an MBL-deficiency per se on infection risk.4,5,6 MBL deficiency does seem to increase infection risk in certain patient groups, who are already immunocompromised.7 It is unclear if MBL-deficiency is associated with recurrent respiratory tract infections. One study investigated the frequency of MBL-deficiency in patients with recurrent and/or severe infections, and found it to be significantly higher than in healthy controls.8 However, this population was very broadly defined, and is not en- tirely comparable with the patients with recurrent respiratory tract infections that are described in this thesis. As shown in Chapter 2, MBL-deficiency does not seem to be a contributing factor in patients presenting with recurrent respiratory tract infections. In this cohort of 81 patients the prevalence of MBL-deficiency was 22%, which is in the same order as in the general population. Clin- ical characteristics and outcomes of the immune status screening were similar between MBL-deficient and MBL-sufficient patients.

This study also showed that, in general, immune status abnormalities were a common finding. Twenty-eight percent of the patients had an impaired response to pneumococcal polysaccharide vaccination. These patients had a more 184 Chapter 11

severe clinical presentation compared to patients with a normal response to pneumococcal vaccination. IgG levels below cutoff (< 7.0 g/l) were seen in 29% of patients, IgM levels below cutoff (< 0.4 g/l) in 10% of patients and IgA levels below cutoff (< 0.7 g/l) in 11% of patients. IgG subclasses 1 and 2 were below cutoff (< 4.9 g/l and < 1.5 g/l, respectively) in 40 and 35% of patients. This illustrates that there is a rather high chance of an immunodeficiency in patients presenting with recurrent infections. The threshold for immunological screening should thus be low in these patients, who based on their clinical history qualify for this screening.

Another part of the immune status screening protocol is assessment of the response to pneumococcal polysaccharide vaccination. Usually only the antibody response of the IgG isotype is measured. An impaired response indicates failure to mount a functional antibody response, and this is associated with an increased risk for infections with encapsulated bacteria.9 When the response to vaccination is impaired, but the circulating levels of immunoglobulins are within the normal range, the patient is said to have a specific polysaccharide antibody deficiency.10 This entity has only been well described in the context of impaired IgG antibody response to vaccination. In Chapter 3 and Chapter 4, it is shown that the antibody response to vaccination can also be impaired in other isotypes, and also in IgG subclasses. Impaired IgA and IgG2 antibody responses seem to be of clinical relevance. In a cohort of 24 patients with recurrent respiratory tract infections and isolated low IgG1 levels (Chapter 3) hyporesponsiveness of the IgA isotype was associated with sinusitis, and hyporesponsiveness of the IgG2 subclass was associated with pneumonia. In another cohort of 26 patients with bronchiectasis of unknown etiology (Chapter 4), an impaired antibody response of the IgA and IgG2 subclass, even when the total IgG antibody response was normal, was associated with more severe bronchiectasis.

Thus, based on the findings of this thesis, the immune status screening protocol might be challenged on two points. First, the value of measurement of MBL-status in patients with recurrent respiratory tract infections is debatable. At this moment, an isolated MBL deficiency (i.e. when all other tested components of the immune system are functional) appears to have no therapeutic or prognostic consequences in these patients. Second, in case of otherwise normal results of the immune status investigation, measuring antibody response to vaccination for all immunoglobulin isotypes can be considered. However, the clinical consequences of impaired IgA and IgG subclass antibody responses have to be investigated in more detail, as well as the effect of antibody replacement therapy in these circumstances. Summary and general discussion 185

Immune status assessment in lung transplant candidates and recipients

Lung transplant candidates and recipients are a highly selected population, as all lung transplant candidates suffer from end-stage lung disease. After transplantation, all patients receive strong immunosuppressive therapy in order to prevent rejection. Therefore, these patients do not have normally functioning immune systems. However, the precise impact on the humoral immune system of immunosuppressive therapy after lung transplantation is not known. There are limited data on the long-term immunological follow up of lung transplant recipients.

To minimize post-transplantation infection risk, it is advised to update vaccinations prior to transplantation. These vaccinations include the 23-valent pneumococcal polysaccharide vaccination and the 13-valent pneumococcal conjugate vaccine. In healthy elderly (> 65 years) it is recommended to start with the conjugate vaccine, and to continue with the polysaccharide vaccine 4-6 weeks later. This is also recommended for patients that are immunocompromised, but there is little evidence supporting this schedule in these patients.11,12 There are no studies investigating the optimal vaccination schedule specifically in lung transplant recipients. The measurement of the response to pneumococcal vaccination is not a part of the standard screening protocol and so far, data on vaccination responses is scarce in this population.

As shown in Chapter 5 a majority of lung transplant candidates had one or more abnormalities in the immune status investigation, including response to pneumococcal polysaccharide vaccination. This cohort included 81 lung transplant candidates, and 62% had one or more abnormalities in the immune status investigation. The response to pneumococcal polysaccharide vaccination was impaired in 46% of patients, one fifth of patients had low IgG levels and two fifths of patients had low IgG1 and/or IgG2 levels. It is likely that these patients have an increased infection risk after lung transplantation.

Further, in Chapter 6 it is shown that humoral immune status significantly declines after lung transplantation. This was most outspoken in the first-year post-transplantation, when the immune suppressive regimen is the most in- tense. This study cohort included 55 lung transplant recipients. There was a significant decrease of all immunoglobulins, IgG subclasses and pneumococcal polysaccharide antibodies in the first year after transplantation. Thereafter, immunoglobulin and IgG subclass levels gradually increased, but remained significantly lower compared to pre-transplant values, even several years after transplantation. Furthermore, the response to pneumococcal polysaccharide vaccine was significantly lower post-transplantation compared to the pre-transplanta- 186 Chapter 11

tion response. Twenty-seven percent of patients had an impaired response to the first vaccination with 23vPPV before transplantation. The antibody response to the post-transplantation vaccination with 23vPPV was markedly lower: 87% of patients had an impaired response.

Based on the studies described in Chapter 5 and Chapter 6, the recommendations for immune status investigations and vaccinations in lung transplant candidates and recipients might be challenged on several points. First, despite the overall impairment in vaccination responses, most patients did show a partial response to vaccination. Therefore, pneumococcal vaccination is still likely to provide at least some degree of protection against pneumococcal infections in lung transplant recipients. However, based on the study results, it can be recommended that revaccination with pneumococcal vaccines should be considered one year post-transplantation. Secondly, because a significant proportion of patients showed a humoral immunodeficiency after lung transplantation, periodic monitoring of immunoglobulins, IgG subclasses and anti-pneumococcal antibodies should be included in routine clinical care. When patients are given pneumococcal vaccination, the vaccination response should also be routinely measured. And in case of recurrent infections in combination with hypogammaglobulinemia and/or an impaired antibody response, antibody replacement therapy should be considered. A flow chart of recommended immune status investigations and vaccinations based on the current findings is shown in Figure 1.

Antibody replacement therapy

Chapter 7 reviews the current literature on etiology, diagnosis, and treatment of primary antibody deficiency (PAD) and iatrogenic hypogammaglobulinemia (IHG).2 IHG can be caused by various immunosuppressive drugs, including the regimen given after lung transplantation.13 The clinical and laboratory manifestations of PAD and IHG comprise a broad spectrum and can vary between the type of PAD or IHG and even between patients with the same type of immune deficiency.14,15 The most common clinical features in both conditions are recurrent bacterial respiratory tract infections (mainly with encapsulated bacteria), but also viral and fungal infections. In PAD there is compelling evidence for the use of antibody replacement therapy, mainly for more severe forms of PAD.16-22 For milder forms of PAD the evidence is more limited. There are few studies on antibody replacement therapy performed in patients with IHG. Based on that limited evidence, antibody replacement therapy (ART) seems to be effective in reducing the infection frequency in IHG patients.13 Summary and general discussion 187

Figure 1. Immunological monitoring of lung transplantation patients.

Routine measurement of immunoglobulin levels is advised before and during immunosuppressive therapy. In this way, the development of IHG can be monitored and antibody replacement therapy can potentially be started before a patient presents with clinical manifestations of hypogammaglobulinemia. The best time of cessation of antibody replacement therapy in IHG is not always clear, and in some cases, prolonged substitution treatment may be warranted, even after immunosuppressive therapy has been stopped.

The study described in Chapter 8 provides new data on the efficacy of antibody replacement therapy in patients with mild or severe humoral immunodeficiency. The study included 87 patients for whom data on several measures of infection frequency were collected. Reported infection frequency, antibiotics use and hospital admissions were all significantly lower under antibody replacement therapy. When patients were subdivided into groups with severe (common variable immunodeficiency/idiopathic primary hypogammaglobulinemia) and mild (immunoglobulin G subclass deficiency/specific antibody deficiency) immunodeficiency, it was shown that antibody replacement therapy was effective in both groups. Median follow up time after the start of antibody replacement therapy was five years, and most patients had follow up of over two years. In total, the study included 385 patient-years on ART. The re- 188 Chapter 11

sults confirm the effectiveness of ART in more severe humoral immunodeficiency and adds to previously published literature that ART is also an effective therapy in patients with mild humoral immunodeficiency.23-25 Until now this category was considered at best “probably beneficial” (primary immune defects with normal IgG and impaired specific antibody production) or “may provide benefit” (isolated IgG subclass deficiency (IgG1, IgG2, IgG3) with recurrent infections).26 However, this study does not address the issue of stopping with ART. So far there are no strict criteria for stopping the treatment.

Remarkable cases of immune deficiency

Presentation with clinical history of recurrent, severe infections, mainly of the respiratory tract is a common manifestation of a (humoral) immunodeficiency during adulthood. Chapters 9 and 10 describe two patients with an interesting clinical presentation. Chapter 9 describes a previously healthy 35-year-old female who presented with invasive pneumonia caused by Streptococcus pneumoniae serotype 9V. This patient did not produce serotype specific antibodies against the infecting serotype during disease. After pneumococcal polysaccharide vaccination, she responded adequately to most other pneumococcal serotypes, but still had no response to the infecting serotype 9V. Upon reexamination nine years later, it was shown that she had a normal level of circulating antibodies against serotype 9V, which further increased after vaccination with a pneumococcal conjugate vaccine. A transient serotype-specific antibody hyporesponsiveness has been described in infants, and this has been linked to a high burden of circulating capsular polysaccharides.27 In this adult patient, it could not definitively be established if the temporary hyporesponsiveness was a contributing factor to the pneumonia or occurred due to a high load of capsular polysaccharides that was caused by the pneumonia.

Chapter 10 describes an unusual presentation of a patient with hypogammaglobulinemia. This patient was ultimately diagnosed through the clinical presentation of her newborn son. The child presented with two episodes of bacterial meningitis within the first two months of life. Serum analysis revealed a virtual absence of IgG. As infants largely depend on the prenatal transfer of maternal IgG in the first months of life, the mother was investigated. The mother was a healthy 37-year-old with an uncomplicated medical history. She reported no severe or recurrent infections, but was found to have a very low IgG level (0.57 g/l) with normal levels of IgM and IgA. Surprisingly, the patient initially mounted a normal antibody response to pneumococcal polysaccharide and diphtheria-tetanus-polio vaccines. However, follow up of anti-tetanus IgG antibodies showed a rapid decline. The patient has now received intravenous Summary and general discussion 189

immunoglobulins for sixteen years, and is still in excellent health. Over the years extensive and in depth immunological investigations were performed, but no underlying cause for the immunodeficiency has been found. This case illustrates that (severe) hypogammaglobulinemia does not necessarily lead to clinical manifestations of antibody deficiency, and that the molecular basis is still not known for a significant proportion of antibody deficiencies.

General considerations and future perspectives

As shown in this thesis an antibody deficiency (also a mild antibody deficiency) can be the cause of recurrent respiratory tract infections. Systematic investigation of the humoral immune status can lead to this diagnosis. Treatment with antibody replacement therapy can significantly decrease the burden of this disease and prevent future organ damage.22,28,29 A high index of suspicion is necessary for diagnosing an antibody deficiency in patients with recurrent respiratory tract infections.

However, there is still insufficient evidence of clinical relevance of all components of the humoral immune status protocol. As shown in this thesis, an isolated MBL deficiency does not seem to be of great relevance in this population. Maybe the genetic and functional tests are not sensitive enough to discriminate severe and complete deficiency from milder forms of MBL deficiency. Further- more, there are still other aspects of the immune status protocol that also have unknown clinical relevance. For example, in some patients an initially good response to pneumococcal vaccination can be seen, followed by a rapid decline in polysaccharide antibody titers in the first year after vaccination. These patients seem to have an unsustained response to vaccination, but whether this leads to an increased risk of infections has not yet been determined. It would therefore be advisable to measure an antibody response at its peak, i.e. 3-6 weeks after vaccination, but also at a later stage, 6-12 months later. Potentially, patients with rapidly declining antibodies (possibly due to absent long-lived plasmacells) could benefit from antibody replacement therapy.

An important step in the protocol for investigating the humoral immune status of patients with RTI is measuring the response to pneumococcal polysaccharide vaccination. An impaired response can be a reason to start antibody replacement therapy. The criteria for an impaired response (unfortunately) have varied over the years. The current criteria are stricter than before, leading to more patients being diagnosed with a humoral immunodeficiency. Which criteria are best remains to be determined. Potentially, the best method for interpreting pneumococcal polysaccharide vaccination responses is to use sero- 190 Chapter 11

type-specific cut-off and reference values. Thus, future studies are needed to evaluate the serotype specific pneumococcal polysaccharide response in a large group of healthy persons. This can help establishing reference values, which can then be verified in patients with recurrent respiratory tract infections.

Importantly, the diagnosis of an antibody deficiency can only be made by evaluating the response to polysaccharide vaccination. The pneumococcal polysaccharide vaccine is by far the most used polysaccharide vaccine for several reasons. First, the vaccine includes multiple serotypes, which allows a more weighed assessment of the humoral immune system. Second, there is much experience with using the pneumococcal polysaccharide vaccine, and, however imperfect, the response criteria are relatively well established.10,30,31 The introduction of the pneumococcal conjugate vaccine complicates matters, because this vaccine (also) induces a T-cell dependent antibody response. In order to keep using the pneumococcal polysaccharide vaccine for the diagnosis of antibody deficiencies two different approaches are possible: either patients suspected of having an antibody deficiency should receive a pneumococcal polysaccharide vaccine first, and only thereafter the conjugate vaccine, or interpretation of the response to pneumococcal vaccination should be based only on pneumococcal serotypes not included in the conjugate vaccine. The first option is rather impractical, because patients could have received the conjugate vaccine prior to being suspected of having an antibody deficiency.32 The second option seems better, but requires that the diagnostic laboratory is able to perform quantitative, serotype specific IgG antibody measurements against those serotypes. Alternatively, another type of polysaccharide vaccine could be used. However, at present no validated normal values are available for other polysaccharide vaccines.

As illustrated by the case described in Chapter 10 a (very) low IgG level does not always lead to recurrent infections. To some extent, this phenomenon was also observed in the cohort of lung transplant recipients (Chapter 6). A significant proportion of these patients had hypogammaglobulinemia or other immune status abnormalities, but this was not always accompanied by clinical manifestations of immunodeficiency (i.e. infections). These cases are indicative of a problem that is more commonly encountered in clinical practice, where patients are coincidentally found to have an abnormality in the immune status, such as low IgG and/or IgG subclass levels. In these cases, it is difficult to determine what is the best course of action. In some cases, such as the case from Chapter 10 it might indicated to start antibody replacement therapy, even in the absence of clinical manifestations of immunodeficiency. This is less clear if IgG levels are higher (but still decreased). In those patients, the best management is likely to withhold antibody replacement therapy and to regularly monitor immunoglobulin levels. In order to confirm this, future studies are needed Summary and general discussion 191

that monitor the natural course of asymptomatic hypogammaglobulinemia in relation to underlying disease, medication, age, other immune status abnormalities and clinical outcome.

Thus far, antibody replacement therapy is the only available therapy in patients with severe or mild humoral immunodeficiency. Immunoglobulin therapy provides the patients with a sufficient amount of antibody levels but does not cure the underlying defect in the immune system. The protective effect is mediated by antibodies which circulate in the general population, and therefore by definition is restricted to common prevalent pathogens. Emerging pathogens will therefore not be covered or at best with a certain delay. The same therapy is used for patients that have a wide variety of underlying immune defects. Diag- nosing the cause of the immunodeficiency may lead to recognizing the patients who would benefit most from ART. Ultimately, correction of the genetic defect would be a form of therapy with a lasting effect.

This thesis shows that in patients with recurrent respiratory tract infections assessment of the humoral immune status can lead to a diagnosis and subsequently a beneficial therapy. While a quantitative antibody deficiency will lead to a clinical immunodeficiency, our data show that also milder deficiencies can lead to severe clinical expression and a good response to antibody replacement therapy. Thus, future studies are needed to determine the clinical relevance of a more elaborate diagnostic protocol for humoral immune status investigation (including for instance antibody affinity determination, opsonophagocytosis capacity, ideally using autologous granulocytes and “autologous” pathogens). Subsequently the effectivity of antibody replacement therapy in these patients with “functional antibody deficiency” should be established.

Finally, the criteria for starting antibody replacement therapy may be relatively clear; the criteria for stopping are not. Lifelong prolongation of ART has, apart from financial implications also important consequences for the quality of life. Diagnostic vaccination with neoantigens, for which no antibody activity is present in IVIG preparations, could be investigated as a tool to guide with- drawal from ART. This might a safer method than stopping ART and monitoring infection frequency.

Replacement of antibodies in patients with a humoral immunodeficiency, as introduced by Bruton, has been the treatment of choice now for over 50 years. Data in this thesis indicate that this treatment is still clinically effective, also upon long-term use. This thesis confirms that awareness of the presence of a humoral immunodeficiency as cause of recurrent respiratory tract infections can lead to timely diagnosis and effective treatment. 192 Chapter 11

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1. de Vries E. European Society for Immunodeficiencies (ESID) members. Patient-centred screening for primary immunodeficiency, a multi-stage diagnostic protocol designed for non-immunologists: 2011 update. Clinical & Experimental Immunology. 2012;167(1):108-119. 2. Bonilla FA, Khan DA, Ballas ZK, et al. Practice parameter for the diagnosis and management of primary immunodeficiency. J Allergy Clin Immunol. 2015;136(5):1186-205.e1-78. 3. Heitzeneder S, Seidel M, Forster-Waldl E, Heitger A. Mannan-binding lectin deficiency – good news, bad news, doesn’t matter? Clin Immunol. 2012;143(1):22-38. 4. Endeman H, Herpers BL, de Jong BA, et al. Mannose-binding lectin genotypes in susceptibility to community-acquired pneumonia. Chest. 2008;134(6):1135-1140. 5. Mills TC, Chapman S, Hutton P, et al. Variants in the mannose-binding lectin gene MBL2 do not associate with sepsis susceptibility or survival in a large european cohort. Clin Infect Dis. 2015;61(5):695-703. 6. van Kempen G, Meijvis S, Endeman H, et al. Mannose-binding lectin and l-ficolin polymorphisms in patients with community-acquired pneumonia caused by intracellular pathogens. Immunology. 2017;151(1):81-88. 7. Keizer MP, Wouters D, Schlapbach LJ, Kuijpers TW. Restoration of MBL-deficiency: Redefining the safety, efficacy and viability of MBL-substitution therapy. Mol Immunol. 2014;61(2):174-184. 8. Hoeflich C, Unterwalder N, Schuett S,et al. Clinical manifestation of mannose-binding lectin deficiency in adults independent of concomitant immunodeficiency. Hum Immunol. 2009;70(10):809-812. 9. Ballow M. Primary immunodeficiency disorders: Antibody deficiency.J Allergy Clin Immunol. 2002;109(4):581-591. 10. Bonilla FA, Khan DA, Ballas ZK, et al. Practice parameter for the diagnosis and management of primary immunodeficiency. J Allergy Clin Immunol. 2015;136(5):1186-205.e1-78. 11. Elberse KE, Tcherniaeva I, Berbers GA, Schouls LM. Optimization and application of a multiplex bead-based assay to quantify serotype-specific IgG againststreptococcus pneumoniae polysaccharides: Response to the booster vaccine after immunization with the pneumococcal 7-valent conjugate vaccine. Clin Vaccine Immunol. 2010;17(4):674-682. 12. Centers for Disease Control and Prevention (CDC). Use of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine for adults with immunocompromising conditions: Recommendations of the advisory committee on immunization practices (ACIP). MMWR Morb Mortal Wkly Rep. 2012;61(40):816-819. 13. Compagno N, Malipiero G, Cinetto F, Agostini C. Immunoglobulin replacement therapy in secondary hypogammaglobulinemia. Front Immunol. 2014;5:626. 14. Bonilla FA, Bernstein IL, Khan DA, et al. Practice parameter for the diagnosis and management of primary immunodeficiency. Ann Allergy Asthma Immunol. 2005;94(5 Suppl 1):S1-63. 15. Durandy A, Kracker S, Fischer A. Primary antibody deficiencies. Nat Rev Immunol. 2013;13(7):519-533. 16. Bonilla FA, Khan DA, Ballas ZK, et al. Practice parameter for the diagnosis and management of primary immunodeficiency. J Allergy Clin Immunol. 2015;136(5):1186-205.e1-78. 17. Orange JS. Hossny EM. Weiler CR. Ballow M. Berger M. Bonilla FA. Buckley R. Chinen J. El-Gamal Y. Mazer BD. Nelson RP Jr. Patel DD. Secord E. Sorensen RU. Wasserman RL. Cun- ningham-Rundles C. Primary Immunodeficiency Committee of the American Academy of Allergy, Asthma and Immunology. Use of intravenous immunoglobulin in human disease: A review of evidence by members of the primary immunodeficiency committee of the american academy of allergy, asthma and immunology. J Allergy Clin Immunol. 2006;117(4 Suppl):S525-53. 18. Cunningham-Rundles C, Siegal FP, Smithwick EM, et al. Efficacy of intravenous immunoglobulin in primary humoral immunodeficiency disease. Ann Intern Med. 1984;101(4):435-439. 19. Nolte MT, Pirofsky B, Gerritz GA, Golding B. Intravenous immunoglobulin therapy for antibody deficiency. Clin Exp Immunol. 1979;36(2):237-243. 20. Ammann AJ, Ashman RF, Buckley RH, et al. Use of intravenous gamma-globulin in antibody immunodeficiency: Results of a multicenter controlled trial. Clin Immunol Immunopathol. 1982;22(1):60-67. Summary and general discussion 193

21. Roifman CM, Lederman HM, Lavi S, Stein LD, Levison H, Gelfand EW. Benefit of intravenous IgG replacement in hypogammaglobulinemic patients with chronic sinopulmonary disease. Am J Med. 1985;79(2):171-174. 22. Quinti I, Soresina A, Guerra A, et al. Effectiveness of immunoglobulin replacement therapy on clinical outcome in patients with primary antibody deficiencies: Results from a multicenter prospective cohort study. J Clin Immunol. 2011;31(3):315-322. 23. Olinder-Nielsen AM, Granert C, Forsberg P, Friman V, Vietorisz A, Bjorkander J. Immuno- globulin prophylaxis in 350 adults with IgG subclass deficiency and recurrent respiratory tract infections: A long-term follow-up. Scand J Infect Dis. 2007;39(1):44-50. 24. Abdou NI, Greenwell CA, Mehta R, Narra M, Hester JD, Halsey JF. Efficacy of intravenous gammaglobulin for immunoglobulin G subclass and/or antibody deficiency in adults. Int Arch Allergy Immunol. 2009;149(3):267-274. 25. Abrahamian F, Agrawal S, Gupta S. Immunological and clinical profile of adult patients with selective immunoglobulin subclass deficiency: Response to intravenous immunoglobulin therapy. Clin Exp Immunol. 2010;159(3):344-350. 26. Perez EE, Orange JS, Bonilla F, et al. Update on the use of immunoglobulin in human disease: A review of evidence. J Allergy Clin Immunol. 2017;139(3S):S1-S46. 27. Borrow R, Stanford E, Waight P, et al. Serotype-specific immune unresponsiveness to pneumococcal conjugate vaccine following invasive pneumococcal disease. Infect Immun. 2008;76(11):5305-5309. 28. Resnick ES, Moshier EL, Godbold JH, Cunningham-Rundles C. Morbidity and mortality in common variable immune deficiency over 4 decades.Blood . 2012;119(7):1650-1657. 29. Brent J, Guzman D, Bangs C, et al. Clinical and laboratory correlates of lung disease and cancer in adults with idiopathic hypogammaglobulinaemia. Clin Exp Immunol. 2016;184(1):73-82. 30. Bonilla FA, Bernstein IL, Khan DA, et al. Practice parameter for the diagnosis and management of primary immunodeficiency. Ann Allergy Asthma Immunol. 2005;94(5 Suppl 1):S1-63. 31. Orange JS, Ballow M, Stiehm ER, et al. Use and interpretation of diagnostic vaccination in primary immunodeficiency: A working group report of the basic and clinical immunology interest section of the american academy of allergy, asthma & immunology. J Allergy Clin Im- munol. 2012;130(3 Suppl):S1-24. 32. Schaballie H, Wuyts G, Dillaerts D, et al. Effect of previous vaccination with pneumococcal conjugate vaccine on pneumococcal polysaccharide vaccine antibody responses. Clin Exp Im- munol. 2016;185(2):180-189.

CHAPTER 12

Nederlandse samenvatting

Nederlandse samenvatting 197

Nederlandse samenvatting

De longen zijn een essentieel orgaan voor de ademhaling. Ze staan hiervoor in constante verbinding met de buitenwereld. Naast opname van zuurstof en af- gifte van koolzuur aan de buitenlucht betekent dit ook een levenslange exposi- tie aan mogelijk schadelijke (pathogene) micro-organismen zoals virussen en bacteriën. In de dagelijkse praktijk van de longarts worden vaak patiënten gezien met steeds terugkerende (recidiverende) luchtweginfecties. Deze recidiverende luchtweginfecties kunnen velerlei oorzaken hebben en zijn ook het meest voorkomende symptoom van een (primaire) afweerstoornis (immuun- deficiëntie).

Bij recidiverende luchtweginfecties kunnen op langere termijn complicaties optreden zoals het ontstaan van bronchiëctasieën: een onomkeerbare beschadiging en verwijding van de luchtwegen. Andere mogelijke complicaties op langere termijn zijn immuungemedieerde aandoeningen zoals een lymfoom. Het is van groot belang dat de behandelend arts zich bewust is van de mogelijk- heid van een (primaire) afweerstoornis als oorzaak voor steeds terugkerende luchtweginfecties, zodat tijdig gestart kan worden met behandeling, waardoor complicaties op langere termijn kunnen worden voorkomen.

Normaliter heeft de mens verschillende afweermechanismen om zichzelf be- schermen tegen micro-organismen: • mechanische afweer, met name een intacte huid en slijmvliezen • aangeboren afweersysteem (immuunsysteem), zoals de fagocyterende witte bloedcellen • verworven afweersysteem (immuunsysteem), waartoe de T- en B-lymfocyten behoren

De mechanische afweer vormt de eerste verdedigingslinie van de luchtwegen. De binnenkant van de luchtwegen is bekleed met slijmvlies (epitheel) bestaande uit steuncellen met trilharen (cilia) en cellen die slijm produceren. Deze bekleding van de luchtwegen vormt een mechanische barrière voor stof- deeltjes en micro-organismen. De trilharen zorgen voor transport van de stof- deeltjes en/of slijm (mucus) naar de keel en kunnen daar worden opgehoest of doorgeslikt, de zogenaamde mucociliaire klaring. Het epitheel produceert daarnaast ook eiwitten, de zogenaamde host-defence proteins zoals onder andere lysozym, lactoferrine en surfactant protein A en D (SP-A and SP-D) die belangrijk zijn voor eliminatie van micro-organismen uit de luchtwegen.

198 Chapter 12

Figuur 1

Het aangeboren immuunsysteem (Figuur 1) kan snel reageren op micro-organismen die het lichaam (de luchtwegen) binnen dringen en bestaat uit een cellulair deel (met name de fagocyten) en een humoraal deel (met name de eiwitten van het complementsysteem). De fagocyten herkennen de pathogene micro-organismen met behulp van een receptor op hun celmembraan. De humorale component van het aangeboren immuunsysteem bestaat uit verschillende in het bloed aanwezige eiwitten zoals lactoferrine, collectines (waaronder MBL= mannose-bindend lectine) en het complementsysteem. Het aangeboren systeem reageert snel, is weinig specifiek en bouwt maar een beperkt immunologisch geheugen op. Dit wil zeggen dat bij een tweede infectie met hetzelfde micro-organisme geen snellere of betere immuunreactie optreedt dan bij het eerste contact.

Het complementsysteem bestaat uit een groep eiwitten die als taak hebben om pathogene micro-organismen op te ruimen. Dit kan op een directe manier door het dodelijk beschadigen van het micro-organisme of op een indirecte manier door een bacterie te bekleden met complementeiwitten zodat deze bacterie makkelijker kan worden worden gefagocyteerd en gedood door fagocyterende witte bloedcellen. Het complementsysteem kan op drie manieren worden geactiveerd: • klassieke weg activatie via antilichamen • lectine weg activatie via MBL (mannose-bindend lectine) of ficolinen • alternatieve weg activatie (spontane activatie)

MBL is een eiwit dat kan binden aan suikers (mannose) in het kapsel (omhulsel) van bepaalde typen bacteriën en door activatie van het complementsysteem Nederlandse samenvatting 199

bijdraagt aan het onschadelijk maken van deze bacteriën. Bijzonder aan MBL is dat in 15-20% van de menselijke populatie (door een kleine genetische variatie) de concentratie van dit eiwit in het bloed veel lager dan normaal is. Het is nog niet duidelijk of deze lage MBL-concentraties bijdragen aan het ontstaan van steeds terugkerende luchtweginfecties.

Het verworven afweersysteem (Figuur 1) bestaat ook uit een cellulaire en een humorale component: witte bloedcellen (lymfocyten) en eiwitten. Er zijn twee hoofdgroepen lymfocyten: T- en B-lymfocyten. Elke lymfocyt kan specifiek een bepaalde lichaamsvreemde stof (antigeen, zoals een pathogeen micro-organisme) herkennen en kan een immuunreactie daarop in gang zetten. Sommige T-lymfocyten kunnen, net als de fagocyten van het aangeboren afweersysteem, pathogene micro-organismen onschadelijk maken. Andere T-lymfocyten kunnen helpen om B-lymfocyten te activeren tot het maken van antilichamen. Wan- neer een bepaalde B-lymfocyt geactiveerd wordt, gaat deze zich vermenigvuldi- gen en ontwikkelt deze zich tot plasmacel (klonale expansie). De plasmacellen produceren dan antilichamen gericht tegen de specifieke lichaamsvreemde stof. Deze antilichamen vormen de humorale component van het verworven immuunsysteem. Het verworven afweersysteem reageert langzamer dan de aangeboren afweer, maar werkt specifieker en kan een geheugen opbouwen. Dit houdt in dat na een eerste kennismaking met een antigeen sommige T- en B-lymfocyten zich ontwikkelen tot geheugencellen. Deze geheugencellen zorgen bij een volgend contact met hetzelfde antigeen voor een snellere en betere reactie van het verworven afweersysteem. Dit is het principe dat ten grondslag ligt aan de effectiviteit van vaccinatie. Als vuistregel geldt dat de respons van de B-lymfocyten (de humorale respons) vooral gericht is tegen bacteriën en de respons van de T- lymfocyten (de cellulaire respons) tegen virussen.

De antilichamen of immuunglobulinen (Ig), geproduceerd door plasmacellen, kunnen worden onderverdeeld in 5 hoofdklassen: IgM, IgG, IgD, IgE en IgA. IgG wordt onderverdeeld in vier subklassen. De immuunglobulinen en subklassen hebben allen een eigen effectormechanisme tegen de verschillende specifieke antigenen. IgM en IgG zijn belangrijk voor het elimineren van bacteriën. IgG komt het meest voor in het bloed: meer dan 75% van de immuunglobulinen in het bloed is IgG. IgG kan onderverdeeld worden in de subklassen: IgG1, IgG2, IgG3 en IgG4. Er worden bijvoorbeeld met name IgG2 antistoffen gemaakt tegen het suikerkapsel van de pneumococ. Dit is een bacterie die ernstige luchtweginfecties kan veroorzaken. De functie van IgD als antilichaam is nog niet geheel duidelijk. IgA komt met name voor in lichaamsvloeistoffen zoals speeksel, tra- nen en moedermelk. IgA speelt een belangrijke rol in de afweerreactie in de slijmvliezen, zoals de luchtwegen en de darmen. IgE speelt een rol bij allergische reacties en ook in de afweer tegen parasitaire infecties (zoals worminfecties). 200 Chapter 12

Een tekort aan antilichamen door een stoornis in het humorale deel van het afweersysteem kan leiden tot het frequent voorkomen van infecties. De meest voorkomende infecties zijn die van de luchtwegen. Dit kan dus een uiting zijn van een (primaire) afweerstoornis (immuundeficiëntie). Bij een patiënt die zich presenteert op de polikliniek longziekten met steeds terugkerende (recidiverende) luchtweginfecties kan het geïndiceerd zijn om aanvullend onderzoek naar het afweersysteem te laten verrichten. Voordat hiertoe wordt overgegaan, dienen andere oorzaken te zijn uitgesloten, zoals aangeboren afwijkingen van de trilharen, niet goed behandeld astma of aandoeningen van het keel- neus- oor gebied of maag.

Een immuundeficiëntie kan ook ontstaan ten gevolge van geneesmiddelen, gif- tige stoffen, of blootstelling aan radioactiviteit. Dit noemen we een secundaire immuundeficiëntie. Een voorbeeld hiervan zijn patiënten die een longtransplantatie hebben ondergaan. Deze patiënten worden levenslang met medicatie behandeld die het eigen afweersysteem onderdrukt. Het doel hiervan is afstoting van de long te voorkomen. Echter, door het gebruiken van deze medicatie heeft het immuunsysteem ook een minder sterke reactie tegen ziekteverwek- kers. Patiënten die een longtransplantatie hebben ondergaan, hebben daarom meestal een secundaire immuundeficiëntie. Door de verminderde functie van het afweersysteem is echter de kans op infecties verhoogd. Een te hoge infectie frequentie kan afstoting van de nieuwe long tot gevolg hebben.

Het immunologisch onderzoek naar een (primaire) afweerstoornis (immuunstatusonderzoek) wordt uitgevoerd volgens het protocol van de European So- ciety for Immunodeficiencies (ESID). Dit protocol adviseert een aantal aanvul- lende onderzoeken, waaronder het meten van de immuunglobulinen (inclusief IgG subklassen), en complementfunctie. Daarnaast wordt geadviseerd om specifieke antilichamen te meten tegen pneumococcen, voor en na de toediening van een pneumococcenvaccin. Bij deze vaccinatie wordt een klein onderdeel van de bacterie Streptococcus pneumoniae (pneumococ) toegediend. De pneumococ heeft buiten de celwand nog een kapsel bestaande uit suikers: de po- lysacchariden (Figuur 2). Op grond van een verschillende samenstelling van deze suikers in het kapsel van de pneumococ kunnen wij tot op heden 93 verschillende soorten pneumococcen (serotypes) onderscheiden.

Voor meting van de respons voor en na vaccinatie maken we gebruik van het 23-valent polysaccharide pneumococcenvaccin. Hierin zijn de kapselpolysac- chariden van 23 serotypes van de pneumococ opgenomen die meer dan 85-90% van de infecties veroorzaken. Niet ieder type pneumococ veroorzaakt dus bij de mens even vaak infecties. Door de antilichaamconcentratie tegen verschillende pneumococcenserotypes (titers) te meten voor en na vaccinatie kan een indruk Nederlandse samenvatting 201

Figuur 2. Elektronenmicroscopische opname van twee pneumococcen, omgeven door polysaccharide kapsel.

worden verkregen van de functionele staat van de humorale afweer. Voor de beoordeling van de respons worden op dit moment de volgende criteria gebruikt: voldoende hoge concentraties antistoffen (≥1.3 microgram/ml) en een tenminste tweevoudige stijging van de concentratie na vaccinatie, elk voor ten minste 70% van de gemeten serotypes. Bij een gestoorde antistofrespons is met name de kans verhoogd op infecties met bacteriën met een suikerkapsel (po- lysaccharidekapsel) zoals pneumococcen.

Wanneer er een primaire of een secundaire immuundeficiëntie is vastgesteld, kan dit een indicatie zijn om te starten met gammaglobuline substitutietherapie. Gammaglobuline wordt gemaakt uit het bloedplasma van een grote groep gezonde donoren. Zo kunnen dus de antilichamen die een patiënt zelf niet kan maken, worden aangevuld (gammaglobuline substitutie). Dit kan zorgen voor een betere bescherming tegen infecties met onder andere gekapselde bacteriën.

Dit proefschrift evalueert het humorale afweersysteem bij patiënten met aandoeningen van de luchtwegen en bij patiënten voor en na longtransplantatie. Daarnaast wordt de behandeling van een primaire afweerstoornis met gammaglobuline substitutietherapie onderzocht.

De these is onderverdeeld in 4 delen:

1. Screening van het humorale afweersysteem in patiënten met recidiverende luchtweginfecties en: a. MBL-deficiëntie (Hoofdstuk 2) b. milde IgG1-deficiëntie (Hoofdstuk 3) c. een gestoorde polysaccharide antistofrespons (Hoofdstuk 4) 202 Chapter 12

2. Screening van het humorale afweersysteem in patiënten in het traject rond longtransplantatie: a. op de wachtlijst voor transplantatie (Hoofdstuk 5) b. na longtransplantatie (Hoofdstuk 6)

3. Gammaglobuline substitutietherapie: a. literatuuroverzicht van gammaglobuline substitutietherapie bij een primaire afweerstoornis en veroorzaakt door medicatie (iatrogeen) (Hoofd- stuk 7) b. lange termijn resultaten van behandeling met gammaglobuline substitutietherapie (Hoofdstuk 8)

Casusbespreking van 2 patiënten met een bijzondere presentatie (Hoofdstuk 9 en Hoofdstuk 10)

In Hoofdstuk 2 wordt een groep beschreven van 81 patiënten met recidiverende luchtweginfecties waarbij immuunstatusonderzoek is gedaan. Er is in deze patiënten ook gekeken naar het MBL-eiwit, dat een onderdeel is van het complementsysteem. Het belang van MBL bij de afweer is niet altijd duidelijk. In de normale populatie heeft ongeveer 15-20% een verlaagd MBL. Eerdere studies laten tegengestelde resultaten zien wat het infectierisico betreft in patiën- ten met een verlaagd MBL: in groepen patiënten die om andere redenen al een verminderde afweer hebben lijkt dit verhoogd, maar in andere patiënten niet. In ons cohort van 81 patiënten had 22% een verlaagd MBL-gehalte. De resultaten van het immuunstatusonderzoek verschilden niet tussen patiënten met een verlaagd en een normaal MBL-gehalte. Wel was opvallend dat er veel afwijkingen werden gevonden in de overige onderdelen van het immuunstatusonderzoek. Negenentwintig procent van de totale groep had een verlaagd IgG in het bloed en 28% had een gestoorde antistofrespons na vaccinatie met een 23-valent pneumococcenvaccin. De symptomatologie in deze laatste groep pa- tiënten was ernstiger, maar dit was niet significant geassocieerd met een verlaagd MBL-gehalte.

Een gestoorde antistofrespons bij normale waarden voor immuunglobulinen in het serum wordt een specifieke polysaccharide antistofdeficiëntie genoemd (SPAD) genoemd. Hierbij gaat het om de respons na vaccinatie met een 23-valent pneumococcenvaccin. Normaal gesproken wordt alleen de totale hoeveel- heid pneumococcenantistoffen van de IgG klasse gemeten. In Hoofdstuk 3 en Hoofdstuk 4 wordt aangetoond dat de respons ook gestoord kan zijn in een andere immuunglobulineklasse of subklasse. In Hoofdstuk 3 wordt een groep patiënten beschreven met een verlaagde IgG1 subklasse met recidiverende luchtweginfecties. Binnen deze groep hadden patiënten met recidiverende Nederlandse samenvatting 203

klachten van de neusbijholten een verminderde respons van het immuunglobuline IgA na pneumococcenvaccinatie, en hadden patiënten met longontste- kingen een verminderde respons van de IgG2 subklasse. Het is nog niet duidelijk of deze afwijkingen voldoende reden zouden zijn om te starten met gammaglobuline therapie en wat het effect daarvan zou zijn. In Hoofdstuk 4 wordt een groep van 26 patiënten met bronchiëctasieën van onbekende oorzaak beschreven. Deze patiënten werden eveneens gevaccineerd met het 23-valente pneumococcenvaccin. Meerdere patiënten hadden een verminderde respons in het IgA en/of IgG2, terwijl de totale antistofrespons van het IgG binnen de normale grenzen was. Een verminderde IgA en/of IgG2 respons was geassocieerd met een ernstiger beeld van de bronchiëctasieën.

Patiënten op de wachtlijst voor longtransplantatie en patiënten die longtransplantatie hebben ondergaan vormen een bijzondere categorie patiënten. Alle patiënten op de wachtlijst voor longtransplantatie lijden aan een eindstadium longziekte. Na transplantatie is er geen sprake meer van een ernstige longziekte maar wordt de normale afweer tegen infecties onderdrukt door de medicijnen die afstoting van de donorlong moeten voorkomen. Om bescherming te bieden tegen infecties na longtransplantatie wordt geadviseerd om patiënten voor longtransplantatie te vaccineren met onder andere het 23-valente pneumococcenvaccin. Tot op heden wordt niet geadviseerd om te beoordelen hoe de respons op deze vaccinatie is en daarom is er nog weinig bekend over de vaccina- tierespons in deze patiëntengroepen. Ook de overige onderdelen van het immuunstatusonderzoek zijn in deze patiënten nog niet uitgebreid onderzocht.

In Hoofdstuk 5 worden de resultaten van immuunstatusonderzoek in een groep van 81 patiënten beschreven die op de wachtlijst staan voor longtransplantatie. Een vijfde van deze groep had een verlaagd IgG in het bloed en twee vijfde had verlaagde IgG1 en/of IgG2 subklassen. Het MBL was verlaagd bij 7%. De respons op de pneumococcenvaccinatie was gestoord bij 46%. Het is dus waarschijnlijk dat deze patiënten een verhoogd infectierisico hebben na longtransplantatie.

In Hoofdstuk 6 wordt een groep van 55 patiënten na longtransplantatie beschreven. In deze patiënten is immuunstatusonderzoek gedaan voor transplantatie. De immunologische parameters zijn gevolgd na transplantatie. Deze data laten zien dat het humorale immuunsysteem sterk onderdrukt wordt na longtransplantatie en dat dit effect het grootst is in het eerste jaar na transplantatie, wanneer de patiënten de hoogste dosering immuunsuppressieve medicijnen krijgen. Dit effect was te zien in de spiegels van immuunglobulinen en subklassen en in de spiegels van specifieke pneumococcenantistoffen. De respons op 204 Chapter 12

vaccinatie met het 23-valente pneumococcenvaccin was duidelijk verminderd na transplantatie: voor transplantatie had 27% van de patiënten een gestoorde respons; na transplantatie was dit 87%. Het lijkt daarom gewenst om deze immunologische parameters te vervolgen en na 1 jaar patiënten te beoordelen voor een nieuwe pneumococcenvaccinatie. De respons op deze vaccinatie is welis- waar verminderd maar kan nog wel een positief effect hebben op de infecties met pneumococcen.

Hoofdstuk 7 geeft een overzicht van de literatuur met betrekking tot de etio- logie, diagnose en behandeling van een primaire afweerstoornis en bij een iatrogene afweerstoornis. Iatrogeen betekent dat de aandoening is ontstaan ten gevolge van een medische behandeling. Bijvoorbeeld patiënten die een longtransplantatie hebben ondergaan gebruiken dagelijks en levenslang immuunsuppressiva om afstoting van de donorlong tegen te gaan. Zij worden hierdoor vatbaar voor infecties. Bij zowel een primaire afweerstoornis als bij een iatrogene afweerstoornis komen recidiverende luchtweginfecties voor met vooral gekapselde bacteriën. Voor de ernstige vormen van een primaire afweerstoornis is er overtuigend wetenschappelijk bewijs dat behandeling met gammaglobuline het aantal infecties vermindert. Echter bij mildere vormen van een primaire afweerstoornis is er minder bewijs voor de effectiviteit van behandeling met gammaglobuline.

Er zijn een aantal studies gedaan naar de behandeling van een iatrogene afweerstoornis met gammaglobuline, die laten zien dat dit zorgt voor een afname van het aantal infecties. Voor het starten van een behandeling met immuunsuppressiva is het van belang de immuunglobulinen in het bloed te meten. Ook moeten deze tijdens de behandeling worden gemonitord. Wanneer een iatrogene afweerstoornis ontstaat, kan tijdig gestart worden met gammaglobuline, voordat er ernstige infecties optreden. Het is minder duidelijk wanneer de behandeling moet worden gestaakt. Soms blijft de afweerstoornis aanwezig terwijl de immuunsuppressiva al zijn gestaakt, bijvoorbeeld bij rituximab, een middel dat gebruikt wordt bij sommige vormen van longfibrose. Het kan dan nodig zijn om door te behandelen met gammaglobuline.

In Hoofdstuk 8 wordt een groep van 87 patiënten beschreven die zijn behandeld met gammaglobuline. Deze patiënten werden verwezen voor analyse van recidiverende luchtweginfecties in de periode van 1992 tot 2014. Bij al deze pa- tiënten werd een humorale afweerstoornis gediagnosticeerd. In 43 patiënten was er sprake van een ernstige afweerstoornis en in 37 patiënten van een milde afweerstoornis (7 patiënten vielen buiten deze categorieën). Bij de patiënten met een ernstige afweerstoornis werd gammaglobuline via de bloedbaan toegediend (intraveneus). Meestal vond dit éénmaal per maand plaats in het ziekenhuis. Nederlandse samenvatting 205

Bij de mildere vorm was er sprake van wekelijkse toediening onder de huid (subcutaan) via een pompje. Hiervoor was geen opname nodig.

De werkzaamheid van gammaglobuline substitutietherapie is beoordeeld door het scoren van de gerapporteerde infectiefrequentie, het antibioticagebruik en het aantal opnames in het ziekenhuis. Deze parameters zijn voor en tijdens (en eventueel na) de behandeling met gammaglobuline substitutietherapie beke- ken. De gemiddelde follow-up was 5 jaar.

In deze studie bleek gammaglobuline een effectieve therapie, zowel voor ernstige als voor milde vormen van immuundeficiëntie. In beide groepen patiën- ten was er een significante daling van het aantal gerapporteerde infecties, het antibioticagebruik, en het aantal ziekenhuisopnames tijdens de behandeling met gammaglobuline. De resultaten bevestigen de effectiviteit bij de ernstige vormen van een humorale immuundeficiëntie, zoals bekend uit de literatuur, maar laten zien dat de therapie ook werkzaam is bij mildere vormen van humorale immuundeficiëntie.

In Hoofdstuk 9 en Hoofdstuk 10 worden twee patiënten beschreven met een bijzondere presentatie. De patiënte in Hoofdstuk 9 is een 35-jarige vrouw die zich presenteerde met een ernstige pneumonie, welke werd veroorzaakt door pneumococcen (serotype 9V). Tot het moment van opname verkeerde zij in goede gezondheid. Tijdens de ziekte bleek zij tegen dit specifieke pneumococcen serotype geen antistoffen te maken. Een maand na ontslag uit het ziekenhuis werd zij op de polikliniek longziekten gezien voor immuunstatusonderzoek. Na vaccinatie het 23-valent pneumococcenvaccin maakte zij nog steeds geen antistoffen tegen serotype 9V, terwijl zij wel voldoende antistoffen maakte tegen de andere pneumococcen serotypes in het vaccin. Bij nieuw immuunstatusonderzoek 9 jaar later had zij normale antistoffen in het bloed tegen het pneumococcen serotype 9V. Zij werd opnieuw gevaccineerd en in het bloed was er een stijging te zien van de antistoffen tegen serotype 9V. Geconcludeerd kan worden dat zij een tijdelijke stoornis had in haar afweer tegen pneumococcen serotype 9V, wat mogelijk heeft bijgedragen aan het krijgen van een ernstige pneumonie.

In Hoofdstuk 10 wordt een 37-jarige patiënte beschreven die net was bevallen van haar derde kind. Deze zoon ontwikkelde 2 episodes met een hersenvlies- ontsteking in de eerste twee maanden van zijn leven. Het immuunglobuline G bleek in zijn bloed niet aantoonbaar. Kinderen hebben pas na 6 maanden normale immuunglobulinespiegels in het bloed en worden tot dan beschermd door immuunglobulinen (IgG) van de moeder, welke tijdens de zwangerschap de placenta kunnen passeren. Naar aanleiding van de afwezigheid van IgG in het bloed van het kind werd daarom de moeder onderzocht. 206 Chapter 12

De moeder was gezond en had geen klachten op het moment dat zij werd onderzocht. Ze had geen recidiverende of ernstige infecties behoudens ieder jaar een tonsillitis. Ze bleek echter een zeer ernstig verlaagd IgG te hebben, met normale hoeveelheden IgM en IgA. Bij verder onderzoek bleek zij normale IgG antistoffen te kunnen maken na vaccinaties met het 23-valente pneumococcenvaccin en het difterie-tetanus-polio vaccin. Het vervolgen van de concentraties van deze antistoffen toonde een snelle afname van de anti-tetanus IgG antistoffen.

Deze patiënte wordt inmiddels al meer dan 16 jaar behandeld met gammaglobuline therapie. Daarbij heeft ze geen infecties en een prima kwaliteit van leven. In de jaren na de diagnose heeft diepgaand immunologisch onderzoek plaatsgevonden naar de oorzaak van haar ziekte. Tot op heden is deze echter niet gevonden. Deze ziektegeschiedenis laat zien dat een zeer ernstige afweerstoornis niet altijd leidt tot ernstige infecties. Mogelijk zou deze patiënte later wel ernstige infecties hebben gekregen, als haar afweerstoornis niet (per toeval) vroeg was ontdekt.

Conclusie

In dit proefschrift worden de resultaten van immuunstatusonderzoek in verschillende groepen patiënten met recidiverende luchtweginfecties beschreven. In een belangrijk deel van deze patiënten kon een milde tot ernstige, afweerstoornis worden aangetoond. Van sommige bevindingen bij het immuunstatusonderzoek was de klinische betekenis echter niet geheel duidelijk. Door uit- breiding van het huidige protocol voor immuunstatusonderzoek met meer specifieke testen, zoals functionele antistoftesten (opsonofagocytose) of vaccinatie met een antigeen waar de patiënt nog niet eerder aan blootgesteld is (neo-antigeen), zouden in de toekomst mogelijk meer patiënten met een klinisch relevante immuundeficiëntie kunnen worden gediagnosticeerd.

Het proefschrift toonde verder aan dat behandeling met gammaglobuline in pa- tiënten met een afweerstoornis zorgt voor een afname van de infectiefrequentie. Het is aannemelijk dat daarmee onherstelbare beschadiging van de longen wordt voorkomen.

Een bijzondere groep waarbij immuunstatusonderzoek werd verricht waren patiënten die in aanmerking komen voor longtransplantatie. Bij immuunstatusonderzoek vóór transplantatie werden veel afwijkingen gevonden. Mogelijk kan hiermee een aanwijzing worden verkregen voor een verhoogd infectierisico na transplantatie. Bij immuunstatusonderzoek nà transplantatie werd vooral in het eerste jaar een aanzienlijke afname van de functie van het humo- Nederlandse samenvatting 207

rale immuunsysteem vastgesteld. Mogelijk hebben sommige van deze patiën- ten derhalve baat bij behandeling met gammaglobuline therapie. Verder klinisch wetenschappelijk onderzoek bij deze kwetsbare groep patiënten is nodig om dit te bevestigen.

Recidiverende luchtweginfecties zijn het meest voorkomende symptoom van een humorale afweerstoornis. Dit proefschrift bevestigt het belang van immuunstatusonderzoek voor het stellen van deze diagnose in de klinische praktijk. Voor het verlenen van optimale zorg is het belangrijk dat de behandelend arts zich daarvan bewust is en blijft.

Dankwoord

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Dankwoord

Allereerst gaat mijn bijzondere dank uit naar de patiënten in het St. Antonius Ziekenhuis die hun gegevens ter beschikking stelden. Zonder hun medewer- king was deze promotie niet mogelijk geweest.

In chronologische volgorde wil ik graag mijn dank overbrengen aan de volgende personen.

Allereerst aan wijlen prof. dr. J.M.M. van den Bosch. In 1992 gaf hij mij de gele- genheid de polikliniek klinische immunologie longziekten over te nemen. Die sloot naadloos aan op mijn belangstelling voor de zorg voor patiënten met recidiverende luchtweginfecties. Hij was een man met visie, die de basis legde voor het longtransplantatieprogramma in Nederland, en ook voor het Interstitial Lung Diseases Centre of Excellence in het St. Antonius Ziekenhuis. De woorden “gewoon volhouden” en vooral de reden van deze woorden zijn voor velen nog dagelijks relevant.

Drs. H. van Velzen-Blad, beste Heleen, jouw vasthoudendheid om laboratorium onderzoek en kliniek te verbinden hebben de basis gelegd voor deze promotie. Het begrip dat je ten toon spreidde voor de dokter die altijd druk was, was bijzonder. Dank voor de vele jaren dat we hebben samengewerkt.

Prof. dr. G.T Rijkers, geachte promotor, beste Ger, dank voor de beoordeling van echt alle manuscripten. Je was er sinds het begin. Jouw inzicht in het immuunsysteem, maar ook je vaak verrassende kijk op resultaten, leidden altijd tot verbetering van het onderzoek. Deze goede samenwerking tussen een klinisch immunoloog uit de academie en de perifere longarts is een mooi voorbeeld van hoe het beste van twee werelden bij elkaar kan komen.

Prof. dr. J.C. Grutters, geachte promotor, beste Jan, dank dat ook jij mijn promotor bent. Dankzij jouw inspanning kreeg ik gedurende een jaar een dag per week tijd om aan deze promotie te werken. Dat heeft ervoor gezorgd dat ik hier sta. Jouw wetenschappelijk inzicht en je altijd opbouwende kritiek zijn van grote waarde geweest. De oprechtheid waarmee jij patiënten en collega’s bena- dert, is zoals het hoort.

Dr. P. Zanen, beste Pieter, dank dat ik gebruik mocht maken van jouw kennis van de statistiek. Jouw analyses waren onmisbaar bij de totstandkoming van meerdere artikelen van deze promotie. Je was altijd bereid tot overleg. Ik hoop dat we ook bij toekomstig onderzoek gebruik mogen maken van jouw expertise. 212 Dankwoord

T.W. Hoffman, beste Thijs, dank voor je inzet bij deze promotie. Het aantal uren dat wij samen hebben gewerkt, is niet te tellen. Toen je Nederland verliet voor een stage in het University Teaching Hospital in Lusaka, Zambia, werden de bijeenkomsten zonder problemen van lijfelijk naar digitaal omgezet. Ideeën ontstaan door discussie en dat hebben we veel gedaan. Ik kijk met plezier uit naar de onderzoeken die we nu samen voorbereiden.

Veel dank ook aan de overige medeauteurs van de artikelen in dit proefschrift: drs. P.E. Horikx†, dr. A.J. van Houte, drs. C.S. de Graaff, dr. E.A. van de Graaf, dr. J.M. Kwakkel-van Erp, dr. J.D. Oudijk, dr. B. Meek, dr. S. van Mens, dr. M.J.D. van Tol, dr. G. Vidarsson en dr. C.M. Jol-van der Zijde.

De leden van de leescommissie onder leiding van prof. dr. M.J.M. Bonten wil ik danken voor hun beoordeling van het manuscript. De leescommissie werd verder gevormd door prof. dr. X. Bossuyt, prof. dr. E.A.M. Sanders, prof. dr. F.W.J.M. Smeenk en prof. dr. J-W.J. Lammers. Ook dank aan prof. dr. D.H. Biesma, dr. E.H.J. van Haren en dr. R.E. Jonkers voor hun bereidheid zitting te nemen in de corona.

De afdeling Medische Microbiologie en Immunologie in ons ziekenhuis ben ik veel dank verschuldigd voor de ruimte die zij gaven aan dit onderzoek. Zonder alle laboratoriumbepalingen die noodzakelijk waren voor de analyse van pati- ënten met recidiverende luchtweginfecties was dit boekje er niet geweest.

Dank aan mijn collega longartsen en arts-assistenten longziekten van het St. Antonius Ziekenhuis en het Universitair Medisch Centrum Utrecht voor jullie belangstelling en geduld. Hopelijk kunnen we onze samenwerking nog lang continueren. Dank voor de toegewijde ondersteuning van het secretariaat longziekten en na- tuurlijk van ons eigen team longtransplantatie.

Lieve paranimfen: Joost Jacobs en Thijs Hoffman, ik ben zeer vereerd dat jullie mij bijstaan op deze bijzondere dag.

Dank aan mijn familie en vrienden voor jullie warme belangstelling. De tijd die ik straks over heb hoop ik met jullie te mogen doorbrengen.

Bij een promotie op deze leeftijd is het bijna onvermijdelijk dat sommigen hier niet meer bij aanwezig kunnen zijn. Ik gedenk mijn ouders en dank hen voor hun liefdevolle opvoeding. Een grote afwezige is mijn echtgenoot Frans Jozef Jacobs. Zijn betekenis in mijn leven laat zich niet vastleggen in een dankwoord. Dit boekje is daarom opgedragen aan hem en aan onze kinderen Isabelle en Joost. Curriculum vitae

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Curriculum vitae

Diana van Kessel was born on December 13th 1955 in Culemborg, the Nether- lands. In 1974 she completed her secondary education at the Rijksscholenge- meenschap Tiel. After one year of extra courses in physics, chemistry and biology at the Stichting Aanvullend Onderwijs Utrecht in 1975, she studied medicine at Utrecht University from 1975 until June 1983. From July 1983 until January 1985 she performed research in the field of sarcoidosis at the department of Pulmonology in the St. Antonius Hospital in Utrecht/Nieuwegein (head prof. dr. J.M.M. van den Bosch). From January 1985 until October 1990 she specialized in pulmonary medicine in the St. Antonius hospital in Nieuwegein (head dr. R.G.J.R.A. Vanderschueren), which started with 2 years of internal medicine (head dr. O.J. Meeuwisen). From October 1990 until September 1992 she worked as a pulmonologist at the Diakonessenhuis in Utrecht and Tergooi Hospital in Blaricum, the Nether- lands. From September 1992 until present she works as a pulmonologist in the St. Antonius Hospital in Nieuwegein and from 2002 also at the Division Heart & Lungs, University Medical Center in Utrecht, the Netherlands. Her main fields of interest are recurrent respiratory tract infections, lung volume reduction surgery and lung transplantation.