**Differential Hallmarks of Celiac Versus Non-Celiac Gluten Sensitivity**

Mahesh Mohan and Karol Sestak

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67545

#### **Abstract**

Non-celiac gluten sensitivity (NCGS) is an intestinal tissue transglutaminase (TG2) and IgE-independent form of GS. NCGS is approximately 6× more prevalent than the classical celiac disease (CD), and its incidence is on the rise. Because of its high relative prevalence and striking resemblance to other forms of GS, there is a greater need to develop new and accurate diagnostic assays to facilitate its definitive diagnosis. As the presence of serum anti-gliadin antibodies (AGA) in the absence of TG2 antibodies is suggestive of NCGS, several reports have recommended AGA immunoassays for differential diagnosis. Although AGA immunoassays are in general suitable for diagnostic purpose, to corroborate NCGS and to distinguish it from CD, a simultaneous use of CD-specific diagnostics, i.e., TG2 antibody-based assay, is also required. Due to lower accuracy of AGA assays than those of TG2-based ones, there will always be a chance (estimated to 5–10%) of misdiagnosing NCGS. Moreover, AGA-based diagnostics would not take into consideration the fact that NCGS is potentially triggered by not only gluten but also other molecules such as fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs). Therefore, a second generation of assays needs to be developed to differentiate NCGS from CD with high accuracy.

**Keywords:** celiac, gluten, NCGS, tissue transglutaminase, differential diagnosis, gut microbiome, gluten-free, diet, IBS, chronic inflammation, small intestine, GI tract

## **1. Introduction: NCGS, CD, and irritable bowel syndrome**

Similarities between non-celiac gluten sensitivity (NCGS) and irritable bowel syndrome (IBS) were first noted in 1978 when it was reported that an adult female patient with IBS but not

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

celiac disease (CD) showed dramatic relief of chronic diarrhea and abdominal pain symptoms after administration of gluten-free diet (GFD) [1–6]. More recent studies corroborated that some but not all IBS patients show significant onset of clinical diarrhea upon mucosal challenge with gluten [7, 8]. There is an emerging consensus that tissue transglutaminase (TG2) antibody-negative and anti-gliadin antibodies (AGA)-positive (TG2−AGA+) IBS patients with DQ2/8-negative haplotype qualify as NCGS candidates [3]. Such an assumption can be confirmed by placing suspect NCGS patients on GFD with subsequent relief of clinical/immunological symptoms. Conversely, if AGA test is used alone, without other corroborative/exclusionary assays, its predictive value for NCGS is poor [4]. Taken together, it appears that NCGS and IBS patients share several clinical and histopathological symptoms. NCGS should therefore be differentiated from IBS based on complete CD/NCGS serology, and diagnosis can be confirmed by performing a mucosal gluten challenge. To simplify and to expedite diagnostic steps, new molecular assays need to be developed to differentiate NCGS from IBS and CD.

## **2. Composition of host gut microbiome and NCGS/CD**

Given the unprecedented rise of food allergies and autoimmune disorders in urban populations during recent decades, several studies have indicated that a potential causative association exists between some of these disorders and composition of the host's gut microbiome [9, 10]. Since both CD and NCGS are inflammatory disorders of not only gastrointestinal (GI) tract but also other organs, including dysfunction of the gut-brain axis [11, 12], studies aimed at identification of specific hallmarks of gut dysbiosis of these disorders are the focus of current investigations.

It has been reported that bacteria involved in gluten metabolism predominantly belong to phylum *Firmicutes*, in particular, those from the genus *Lactobacillus*, followed by *Streptococcus*, *Staphylococcus,* and *Clostridia* [13, 14]. Recently, it was shown that GFD treatment significantly altered proportions of these bacterial groups and that restoration of normal bacterial flora took many months and possibly years [14, 15]. It was also shown that increased presence of some of the bacterial species involved in gluten metabolism leads to enteritis [13]. Our group recently demonstrated that *Streptococcaceae* and *Lactobacillaceae* families were enriched in GS rhesus macaque model of CD, while *Coriobacteriaceae* predominated in healthy animals [14]. In the future, studies to elucidate specific dysbiotic pathways that distinguish NCGS from CD need to be done.

## **3. Host luminal shedding of fecal microRNAs**

Recently, a novel concept concerning the capability of intestinal epithelial cells to release luminal regulatory microRNAs (miRNAs) was described [16]. It was demonstrated that these fecal miRNAs could potentially enter bacterial cells and regulate their replication and growth. In this context, it is possible that inflammation-induced miRNAs could enter commensal bacteria and posttranscriptionally suppress or promote their growth by binding to specific sequences on bacterial genes [16]. This in turn, depending on the outcome, may give pathogenic bacteria an opportunity to expand leading to dysbiosis. [16]. These findings have therapeutic implications as oral supplementation of stable miRNA mimics capable of targeting specific dysbiotic or probiotic members of the gut microflora relevant to disease relapse and/or remission may be implemented. In our recently published studies, we hypothesized that GS disorders including CD and NCGS have their own unique signatures of dysbiosis. In addition, it is also likely that regulatory miRNAs secreted by host epithelial cells in response to dysbiotic events are also disease specific. Recently, we identified and reported several miRNAs (miR-203, miR-204, miR-23b, and miR-29b) with perfect complementarity between miRNA seed nucleotides (5′ prime nt position 2–7) and 16S rRNA sequence of dysbiotic bacterial species in the rhesus macaque model of CD (**Figure 1**) [14].

celiac disease (CD) showed dramatic relief of chronic diarrhea and abdominal pain symptoms after administration of gluten-free diet (GFD) [1–6]. More recent studies corroborated that some but not all IBS patients show significant onset of clinical diarrhea upon mucosal challenge with gluten [7, 8]. There is an emerging consensus that tissue transglutaminase (TG2) antibody-negative and anti-gliadin antibodies (AGA)-positive (TG2−AGA+) IBS patients with DQ2/8-negative haplotype qualify as NCGS candidates [3]. Such an assumption can be confirmed by placing suspect NCGS patients on GFD with subsequent relief of clinical/immunological symptoms. Conversely, if AGA test is used alone, without other corroborative/exclusionary assays, its predictive value for NCGS is poor [4]. Taken together, it appears that NCGS and IBS patients share several clinical and histopathological symptoms. NCGS should therefore be differentiated from IBS based on complete CD/NCGS serology, and diagnosis can be confirmed by performing a mucosal gluten challenge. To simplify and to expedite diagnostic steps, new molecular assays need to be developed to differentiate

Given the unprecedented rise of food allergies and autoimmune disorders in urban populations during recent decades, several studies have indicated that a potential causative association exists between some of these disorders and composition of the host's gut microbiome [9, 10]. Since both CD and NCGS are inflammatory disorders of not only gastrointestinal (GI) tract but also other organs, including dysfunction of the gut-brain axis [11, 12], studies aimed at identification of specific hallmarks of gut dysbiosis of these

It has been reported that bacteria involved in gluten metabolism predominantly belong to phylum *Firmicutes*, in particular, those from the genus *Lactobacillus*, followed by *Streptococcus*, *Staphylococcus,* and *Clostridia* [13, 14]. Recently, it was shown that GFD treatment significantly altered proportions of these bacterial groups and that restoration of normal bacterial flora took many months and possibly years [14, 15]. It was also shown that increased presence of some of the bacterial species involved in gluten metabolism leads to enteritis [13]. Our group recently demonstrated that *Streptococcaceae* and *Lactobacillaceae* families were enriched in GS rhesus macaque model of CD, while *Coriobacteriaceae* predominated in healthy animals [14]. In the future, studies to elucidate specific dysbiotic pathways that distinguish NCGS from CD

Recently, a novel concept concerning the capability of intestinal epithelial cells to release luminal regulatory microRNAs (miRNAs) was described [16]. It was demonstrated that

**2. Composition of host gut microbiome and NCGS/CD**

disorders are the focus of current investigations.

**3. Host luminal shedding of fecal microRNAs**

NCGS from IBS and CD.

4 Celiac Disease and Non-Celiac Gluten Sensitivity

need to be done.

Dysbiotic bacterial species that could be potentially regulated in this fashion by inflammatory miRNAs included members of the *Streptococcaceae* and *Lactobacillaceae* families that are known to play roles in metabolism of gluten [13]. As biological and regulatory functions of miRNAs include host cell effects such as expression of epithelial tight junction proteins, more work remains to be performed to characterize regulatory relationships and pathways pertinent to miRNA molecules that influence dysbiotic gut microbiota in NCGS and CD individuals.

**Figure 1.** Small intestinal epithelial cells of gluten-sensitive rhesus macaque (A) were visualized by immunofluorescent triple labeling of cytokeratin-1 (red), tight junction protein claudin-1 (green), and nuclear DNA (blue) antigens. Epithelial cells of gluten-sensitive but not healthy, normal primates produced regulatory fecal microRNAs (miRNA) species complementary with dysbiotic bacterial species such as *Streptococcus leuticeae* (B) and others. It was proposed that intensity of such interactions can shape the gut microbiome dysbiosis either toward remission or relapse [14, 16].

## **4. Dietary gluten and neurodevelopmental disease markers**

The first report suggesting an association between increased occurrence of neurodevelopmental disorders and consumption of gluten-containing cereal grains dates back to 1966 [17]. In the same year, it was reported that some but not all GS patients develop neurological dysfunctions referred to as gluten ataxia, gluten neuropathy, or gluten encephalopathy [18, 19]. Since then, several studies have suggested that symptoms of the autism spectrum disorders (ASD) could be improved upon changes in diet. One of these diets is GFD [20]. Despite its widespread use, the efficacy of GFD for the treatment and prevention of ASD has not been conclusively proven. More recently, a case report involving NCGS patients with gluten psychosis was reported [21]. The molecular mechanisms underlying ASD/psychosis vs. dietary gluten relationship are highly complex and understudied [22, 23]. Therefore, a transition from the "clinical phenomena" to "basic research" type of studies is needed. We propose that perturbation levels (measured by the extent of mRNA expression) of ASD predisposition genes need to be elucidated in preclinical, humanlike models first in the context of experimental introduction/withdrawal of dietary gluten.

For this and other purposes, we developed the rhesus macaque (*Macaca mulatta*) model of GS [14, 24–30]. The presence of AGAs, gluten-sensitive enteropathy (GSE), increased intestinal permeability, and genetic predisposition were all documented. Consistent with human disease, GSE in macaques is characterized by a wide range of severity, ranging from the subclinical to severe form that includes decreased absorption of nutrients, decreased xenobiotic metabolism, cancer predisposition, diarrhea, dermatitis, decreased diversity of gut microbiome, as well as the perturbations in expression of several neurodevelopmental disorder-associated genes including those of ASD and down syndrome. One of these genes that showed significant upregulation in GS rhesus macaques was the Ca2+-dependent activator protein for secretion 2 (*CADPS2*). In humans, the *CADPS2* gene is located within the autism susceptibility locus 1 on chromosome 7q. It was shown that *Cadps*2-knockout mice exhibit cellular and behavioral traits consistent with ASD [31]. The CADPS2 protein regulates exocytosis of synaptic vesicles in neurons and neuroendocrine cells. In accordance with these findings, analysis of the ASD-associated genetic predisposition factors by a group at Harvard School of Medicine revealed that ASD is not restricted to not only humans but also apes, monkeys, and dolphins [32]. Remission and relapse stages of GSE can be accomplished in GS macaques by feeding gluten-free and gluten-containing diets, respectively. Similar to human gluten-sensitive patients, AGA and GSE are reversibly dependent in GS macaques by exposure to dietary gluten [24, 33, 34]. Thus, an extensive use of GS rhesus macaque model in experimental and translational studies involving neurodevelopmental disorder-associated genes and their corresponding pathways is desired—as a new preclinical tool for not only ASD research but also for the development of NCGS vs. CD differential diagnostics.

## **5. NCGS vs. CD microbial signatures**

Based on the assumption that CD is caused by an autoimmune reaction to TG2, while NCGS is caused by chronic bacterial intestinal infections, a recent study by Columbia University researchers focused on the identification of differential, bacterial byproduct-specific diagnostic markers to distinguish the two conditions [35]. Their findings suggested that enteropathy could occur in individuals who report GS in the absence of CD, while it is associated with increased serum antibodies recognizing bacterial lipopolysaccharide (LPS) and/or its CD14 ligand [35]. Although several antibodies were evaluated for their potential to be used as differential diagnostic tools including anti-LPS, anti-flagellin, and anti-soluble CD14 (sCD14), the best predictive values were attributed to antibodies targeting LPS and sCD14. These results corroborated that NCGS and CD have common and differential features that can be further exploited for the development of more sensitive and accurate differential diagnostic assays.

## **Author details**

**4. Dietary gluten and neurodevelopmental disease markers**

introduction/withdrawal of dietary gluten.

6 Celiac Disease and Non-Celiac Gluten Sensitivity

**5. NCGS vs. CD microbial signatures**

The first report suggesting an association between increased occurrence of neurodevelopmental disorders and consumption of gluten-containing cereal grains dates back to 1966 [17]. In the same year, it was reported that some but not all GS patients develop neurological dysfunctions referred to as gluten ataxia, gluten neuropathy, or gluten encephalopathy [18, 19]. Since then, several studies have suggested that symptoms of the autism spectrum disorders (ASD) could be improved upon changes in diet. One of these diets is GFD [20]. Despite its widespread use, the efficacy of GFD for the treatment and prevention of ASD has not been conclusively proven. More recently, a case report involving NCGS patients with gluten psychosis was reported [21]. The molecular mechanisms underlying ASD/psychosis vs. dietary gluten relationship are highly complex and understudied [22, 23]. Therefore, a transition from the "clinical phenomena" to "basic research" type of studies is needed. We propose that perturbation levels (measured by the extent of mRNA expression) of ASD predisposition genes need to be elucidated in preclinical, humanlike models first in the context of experimental

For this and other purposes, we developed the rhesus macaque (*Macaca mulatta*) model of GS [14, 24–30]. The presence of AGAs, gluten-sensitive enteropathy (GSE), increased intestinal permeability, and genetic predisposition were all documented. Consistent with human disease, GSE in macaques is characterized by a wide range of severity, ranging from the subclinical to severe form that includes decreased absorption of nutrients, decreased xenobiotic metabolism, cancer predisposition, diarrhea, dermatitis, decreased diversity of gut microbiome, as well as the perturbations in expression of several neurodevelopmental disorder-associated genes including those of ASD and down syndrome. One of these genes that showed significant upregulation in GS rhesus macaques was the Ca2+-dependent activator protein for secretion 2 (*CADPS2*). In humans, the *CADPS2* gene is located within the autism susceptibility locus 1 on chromosome 7q. It was shown that *Cadps*2-knockout mice exhibit cellular and behavioral traits consistent with ASD [31]. The CADPS2 protein regulates exocytosis of synaptic vesicles in neurons and neuroendocrine cells. In accordance with these findings, analysis of the ASD-associated genetic predisposition factors by a group at Harvard School of Medicine revealed that ASD is not restricted to not only humans but also apes, monkeys, and dolphins [32]. Remission and relapse stages of GSE can be accomplished in GS macaques by feeding gluten-free and gluten-containing diets, respectively. Similar to human gluten-sensitive patients, AGA and GSE are reversibly dependent in GS macaques by exposure to dietary gluten [24, 33, 34]. Thus, an extensive use of GS rhesus macaque model in experimental and translational studies involving neurodevelopmental disorder-associated genes and their corresponding pathways is desired—as a new preclinical tool for not only ASD research but also for the development of NCGS vs. CD differential diagnostics.

Based on the assumption that CD is caused by an autoimmune reaction to TG2, while NCGS is caused by chronic bacterial intestinal infections, a recent study by Columbia University Mahesh Mohan1 and Karol Sestak2 \*

\*Address all correspondence to: ksestak.tulane@gmail.edu

1 Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA

2 Division of Microbiology, Tulane National Primate Research Center, Covington, Louisiana, USA

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[8] Biesiekierski JR, Newnham ED, Irving PM, Barrett JS, Haines M, et al.: FRACP1 gluten causes gastrointestinal symptoms in subjects without celiac disease: A double-blind ran-

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[11] Daulatzai MA: Non-celiac gluten sensitivity triggers gut dysbiosis, neuroinflammation, gut-brain axis dysfunction, and vulnerability for dementia. CNS Neur Disord.

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[13] Caminero A, Herran AR, Nistal E, Perez-Andres J, Vaquero L, et al.: Diversity of the cultivable human gut microbiome involved in gluten metabolism: Isolation of microorganisms with potential interest for coeliac disease. FEMS Microbiol Ecol 2014;**88**:309–319.

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[17] Dohan FC: Wheat "consumption" and hospital admissions for schizophrenia during

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[20] Catassi C, Bai JC, Bonaz B, Bouma G, Calabro A, et al.: Non-celiac gluten sensitivity: The

[21] Lionetti E, Leonardi S, Franzonello C, Mancardi M, Ruggieri M, Catassi C: Gluten psy-

[22] Genuis SJ, Lobo RA: Gluten sensitivity presenting as a neuropsychiatric disorder.

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## **Chapter 2**

## **Celiac Disease and HBV Vaccination**

Caterina Anania, Francesca Olivero, Eugenia Olivero and Lucia Pacifico

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67348

#### **Abstract**

Celiac disease (CD) is an immune-mediated systemic disorder elicited by gluten and related prolamins in genetically susceptible individuals, characterized by the presence of a variable combination of gluten-dependent clinical manifestations, CD-specific antibodies, HLA-DQ2 and HLA-DQ8 haplotypes, and enteropathy. Hepatitis B virus (HBV) infection is an important global public health problem that can cause chronic liver disease, and it is associated to a high risk of death from cirrhosis and hepatocellular carcinoma. Since 1982, a safe and effective HBV vaccine has been available, and recommendation for HBV vaccination has been extended to all infants to achieve protection against HBV infection. HBV vaccination is highly effective in eliciting a sustained immune response in immune-competent individuals. However, research papers have suggested that celiac patients may have low rate of protective antibodies after HBV vaccination. The failure of CD subjects to respond to HBV vaccination has great importance for public health policies as the nonresponders could be regarded as a reservoir for HBV. The aim of our work is to revise and to discuss the scarce literature on this field in order to provide clinical practice guidelines to establish the best surveillance program of response to HBV vaccine in CD pediatric patient.

**Keywords:** celiac disease, children, hepatitis B vaccine, HLA, gluten-free diet

#### **1. Introduction**

Celiac disease (CD) is an immune-mediated systemic disorder elicited by gluten and related prolamins in genetically susceptible individuals, characterized by the presence of a variable combination of gluten-dependent clinical manifestations, CD-specific antibodies, HLA-DQ2 and HLA-DQ8 haplotypes, and enteropathy. Genetic, immunological, and environmental factors therefore appear to be responsible for the disease. HLA-DQ2 is present in 90%–95% of patients with CD, whereas 5% carry the HLA-DQ8 haplotype and the remaining 5% at least one of the two DQ2 alleles [1, 2]. The prevalence of CD is high in the European and

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

North American population (1%), reaching 10%–15% in patients who have first-degree relatives with this disease [1, 2].

HLA system has a fundamental role in identifying the antigens inoculated with the vaccines and in the production of specific antibodies [3, 4], and some HLA phenotypes seem to be predictive of a less effective immunological response [5].

In particular, the immunogenic peptides in the hepatitis B (HBV) vaccine determine the protective immune response to the virus through the HLA-DR and DQ molecules [6, 7], with the DR3-DQ2 and DR7-DQ2 haplotypes generally having a lower response rate [7–10].

HBV infection is one of the major causes of chronic liver disease, associated with a high risk of death from cirrhosis and hepatocellular carcinoma, and therefore represents an important global public health problem [11, 12]. To prevent it, since 1982, a safe and effective hepatitis B vaccine has been available. The one currently in use is a recombinant vaccine that contains HBV surface antigen (HBsAg) and causes the production of specific antibodies (anti-HBs) that protect against the infection [13]. Many epidemiologic studies have been conducted to determine the efficacy of the vaccine. A positive immune response to the vaccine is defined as the development of HBV anti-HBs at a titer of >10 mIU/mL, after a complete and appropriate immunization schedule, measured preferably 1–3 months after the last vaccine administration [14, 15]. The optimum response, conferring seroprotection against HBV infection, is defined as an anti-HBs titer ≥100 IU/l [14, 15]. Subjects that develop an anti-HBs titer between 10 and 100 IU/ml are referred to as "poor responders." Vaccinated subjects with an anti-HBs titer <10 mIU/ ml after completion of primary vaccine series are called "nonresponders" [16]. HBV vaccination is very effective, showing a sustained immune response in immune-competent individuals: the antibody response has been found to occur in more than 90% of the healthy subjects vaccinated with the standard dose regimen of 20 μg HBV vaccine given at 0, 1, and 6 months of intervals [17, 18]. However, among healthy immunocompetent subjects, approximately 4–10% do not produce protective levels of anti-HBs after immunization [19] depending on age, male gender, obesity, inappropriate vaccine storage conditions, route of administration, smoking, drug abuse, state of immunosuppression, and presence of specific HLA haplotypes.

## **2. Responses to vaccinations in celiac children**

Data concerning antibody response of patients with CD to vaccine are scanty. Most studies in this field are addressed to HBV vaccination response, while fewer works are available about the immunological response to other vaccinations.

Several research papers have suggested that celiac patients may have low rate of protective antibodies after vaccinations such as HBV. The failure of CD subjects to respond to HBV vaccination has great importance for public health policies as the nonresponders could be regarded as a reservoir for HBV [20]. The studies that have addressed the relation between CD and HBV vaccination in children are summarized in **Table 1** [21–29]. In the earliest report involving 26 celiac patients aged 9.2 ± 4.6 years and 18 age-matched controls, receiving the full complement of childhood vaccination (HBV, tetanus, rubella, *Haemophilus influenzae* type b), Park et al. [21] demonstrated that a significantly higher proportion of subjects in the CD group failed to respond to HBV vaccine compared with controls (53.9% versus 11.1%; *p* < 0.05). However, all subjects in both groups tested positive for other vaccinations. These results led the authors to support the role of HLA haplotypes in response to HBV vaccine. Nemes et al. [22] evaluated HBV vaccine response in CD patients in relation to disease activity and examined the possible role of dietary gluten in the failure to achieve protective antibody titers. The authors studied 128 biopsy-proven CD children and adolescents and 113 age-matched control subjects; 22 patients with CD were prospectively vaccinated with a recombinant HBV vaccine after the diagnosis of CD during dietary treatment, while 106 CD patients received a recombinant HBV vaccine unrelated to CD diagnosis and dietary compliance. They found that a seroconversion rate for anti-HBs was 95.5% (95% CI: 78.25–99.2%) after vaccination in the patients prospectively immunized, while the response rate was 50.9% and correlates with gluten intake (untreated patients 25.9%, non-strict diet 44.4%, strict diet 61.4%) when HBV immunization was performed unrelated to diagnosis and diet status suggesting that disease activity may play a primary role in vaccination failure rather than specific HLA alleles [22]. Subsequently, Ertem et al., to assess the response to HBV vaccine prospectively in a group of CD children and to explore the potential link between CD and HBV vaccine nonresponse, evaluated serologically for anti-HBs status 63 previously biopsy-proven CD patients on a strict gluten-free diet (GFD) and 54 healthy children. CD children who were anti-HBs negative at baseline were fully vaccinated prospectively and reevaluated for the response to HBV vaccine. The authors found that the response rate to HBV vaccine in CD patients prospectively vaccinated was 96.9%, which was as high as the response rate obtained in healthy population, and they concluded that treatment with GFD and compliance to the treatment rather than the specific HLA alleles may improve the immune response to HBV vaccine in CD patients [23]. Balamtekin et al. conducted a study to compare the response rates to HBV vaccination in the first year of life, using two different immunization protocols. The total study group included 64 CD children (group 1 who received HBV vaccination at birth, 2 and 9–12 months of life, and group 2 at birth, 1 and 6 months of life) and 49 healthy controls. The authors found that the response rate to HBV vaccine and anti-HBs titers in CD patients who completed the HBV vaccination before 1 year of age were significantly lower compared to healthy controls, whereas no statistically significant difference was observed with the two different HBV vaccination schedules [24]. Ertekin et al. compared the response to HBV vaccine between children with CD and healthy children and investigated the relationship between the patients' responses to HBV vaccine, the clinical presentation of CD, and the dietary compliance in the patients. They evaluated the production of specific anti-HB surface antigen (HBsAg) in 52 CD patients and 20 age- and sex-matched healthy children who received HBV vaccination according to the standard immunization schedule. The authors found that anti-HBs titers of CD patients were positive in 32 (61%) and negative in 20 (38.5%) patients, while 18 (90%) of control subjects had positive anti-HBs titers. They found also statistically significant differences between negative anti-HBs titers, clinical presentation of CD, and dietary compliance in patients with CD (*P* < 0.05). Therefore, they concluded that, in children with CD, the immune response to HBV vaccination may be improved by compliance to the GFD [25]. Leonardi et al. [26] in a retrospective report confirmed that CD patients have a lower percentage of response to HBV vaccination than healthy subjects. In fact, they found that 30 (50%) of 60 CD patients were nonresponders to HBV vaccination, compared to 7 (11.6%) of 60 controls. The same authors also found that a significantly higher number of nonresponders in adolescent patients older than 14 years and concluded that a very early

North American population (1%), reaching 10%–15% in patients who have first-degree rela-

HLA system has a fundamental role in identifying the antigens inoculated with the vaccines and in the production of specific antibodies [3, 4], and some HLA phenotypes seem to be pre-

In particular, the immunogenic peptides in the hepatitis B (HBV) vaccine determine the protective immune response to the virus through the HLA-DR and DQ molecules [6, 7], with the

HBV infection is one of the major causes of chronic liver disease, associated with a high risk of death from cirrhosis and hepatocellular carcinoma, and therefore represents an important global public health problem [11, 12]. To prevent it, since 1982, a safe and effective hepatitis B vaccine has been available. The one currently in use is a recombinant vaccine that contains HBV surface antigen (HBsAg) and causes the production of specific antibodies (anti-HBs) that protect against the infection [13]. Many epidemiologic studies have been conducted to determine the efficacy of the vaccine. A positive immune response to the vaccine is defined as the development of HBV anti-HBs at a titer of >10 mIU/mL, after a complete and appropriate immunization schedule, measured preferably 1–3 months after the last vaccine administration [14, 15]. The optimum response, conferring seroprotection against HBV infection, is defined as an anti-HBs titer ≥100 IU/l [14, 15]. Subjects that develop an anti-HBs titer between 10 and 100 IU/ml are referred to as "poor responders." Vaccinated subjects with an anti-HBs titer <10 mIU/ ml after completion of primary vaccine series are called "nonresponders" [16]. HBV vaccination is very effective, showing a sustained immune response in immune-competent individuals: the antibody response has been found to occur in more than 90% of the healthy subjects vaccinated with the standard dose regimen of 20 μg HBV vaccine given at 0, 1, and 6 months of intervals [17, 18]. However, among healthy immunocompetent subjects, approximately 4–10% do not produce protective levels of anti-HBs after immunization [19] depending on age, male gender, obesity, inappropriate vaccine storage conditions, route of administration, smoking,

DR3-DQ2 and DR7-DQ2 haplotypes generally having a lower response rate [7–10].

drug abuse, state of immunosuppression, and presence of specific HLA haplotypes.

Data concerning antibody response of patients with CD to vaccine are scanty. Most studies in this field are addressed to HBV vaccination response, while fewer works are available about

Several research papers have suggested that celiac patients may have low rate of protective antibodies after vaccinations such as HBV. The failure of CD subjects to respond to HBV vaccination has great importance for public health policies as the nonresponders could be regarded as a reservoir for HBV [20]. The studies that have addressed the relation between CD and HBV vaccination in children are summarized in **Table 1** [21–29]. In the earliest report involving 26 celiac patients aged 9.2 ± 4.6 years and 18 age-matched controls, receiving the full complement of childhood vaccination (HBV, tetanus, rubella, *Haemophilus influenzae* type b), Park et al. [21] demonstrated that a significantly higher proportion of subjects in the CD group failed to

**2. Responses to vaccinations in celiac children**

the immunological response to other vaccinations.

tives with this disease [1, 2].

12 Celiac Disease and Non-Celiac Gluten Sensitivity

dictive of a less effective immunological response [5].

diagnosis of CD seems to increase significantly the percentage of responders suggesting that a short time of gluten introduction seems to play a favorable effect on the antibody memory [26]. Leonardi et al. [27] in a subsequent retrospective study, including 66 CD patients and 50 healthy children, analyzed and compared the immunologic response against obligatory vaccination (HBV, diphtheria and tetanus component, and *Bordetella pertussis*) and against recommended vaccination (*Measles virus*, *Paramyxoviridae*, and *Rubella virus*) in the two groups. The authors found similar response to obligatory and recommended vaccines into the two groups, except for HBV vaccine. Moreover, they compared patients whose diagnosis was made before or after 18 months of age and found that an early or a delayed diagnosis does not significantly modify the immunological response, except for that one involved in the HBV vaccination. Thus, the immunologic response did not seem to be influenced by the natural history of CD [27]. Urganci and Kalyoncu determined the rate of response to hepatitis A (HBA) and HBV vaccine, the duration of protection against HAV and HBV, and the incidence of acute HAV or HBV infections during follow-up in 30 pediatric patients with CD and compared them with 50 healthy age-, sex-, and body mass index-matched controls [28]. They found that 14 (46%) of 30 CD patients and 15 (30%) of the controls had natural immunity for HAV, whereas all patients and controls did not show evidence of earlier exposure to HBV. Sixteen patients and 35 controls received HAV vaccine, and HBV vaccine was administered to all CD patients and controls; protective anti-HAV antibodies were developed in 12 (75%) of the patients and all the controls (75% versus 100%, respectively). Thirty patients and 50 controls received HBV vaccine, and 70% of the patients versus 90% of the controls achieved seroprotection. The authors concluded that the rate of seroconversion to the HBV and HAV vaccine is lower in CD patients than in healthy controls. Finally, in a very recent paper, Leonardi et al. comparing a group of patient affected by diabetes mellitus type 1 (DMT1) and CD and a group affected by DMT1 without CD (both groups had similar HLA haplotype) found a higher nonsignificant percentage of nonresponders in DMT1/CD group than in DMT1 (53.3% versus 38.2%); comparing the DMT1/CD group with CD group, the authors found a similar percentage (53.3% versus 50%) of nonresponders, and this result indirectly confirmed that gluten can favor a further decrease of efficacy to HBV vaccine, beyond the HLA system [29].



diagnosis of CD seems to increase significantly the percentage of responders suggesting that a short time of gluten introduction seems to play a favorable effect on the antibody memory [26]. Leonardi et al. [27] in a subsequent retrospective study, including 66 CD patients and 50 healthy children, analyzed and compared the immunologic response against obligatory vaccination (HBV, diphtheria and tetanus component, and *Bordetella pertussis*) and against recommended vaccination (*Measles virus*, *Paramyxoviridae*, and *Rubella virus*) in the two groups. The authors found similar response to obligatory and recommended vaccines into the two groups, except for HBV vaccine. Moreover, they compared patients whose diagnosis was made before or after 18 months of age and found that an early or a delayed diagnosis does not significantly modify the immunological response, except for that one involved in the HBV vaccination. Thus, the immunologic response did not seem to be influenced by the natural history of CD [27]. Urganci and Kalyoncu determined the rate of response to hepatitis A (HBA) and HBV vaccine, the duration of protection against HAV and HBV, and the incidence of acute HAV or HBV infections during follow-up in 30 pediatric patients with CD and compared them with 50 healthy age-, sex-, and body mass index-matched controls [28]. They found that 14 (46%) of 30 CD patients and 15 (30%) of the controls had natural immunity for HAV, whereas all patients and controls did not show evidence of earlier exposure to HBV. Sixteen patients and 35 controls received HAV vaccine, and HBV vaccine was administered to all CD patients and controls; protective anti-HAV antibodies were developed in 12 (75%) of the patients and all the controls (75% versus 100%, respectively). Thirty patients and 50 controls received HBV vaccine, and 70% of the patients versus 90% of the controls achieved seroprotection. The authors concluded that the rate of seroconversion to the HBV and HAV vaccine is lower in CD patients than in healthy controls. Finally, in a very recent paper, Leonardi et al. comparing a group of patient affected by diabetes mellitus type 1 (DMT1) and CD and a group affected by DMT1 without CD (both groups had similar HLA haplotype) found a higher nonsignificant percentage of nonresponders in DMT1/CD group than in DMT1 (53.3% versus 38.2%); comparing the DMT1/CD group with CD group, the authors found a similar percentage (53.3% versus 50%) of nonresponders, and this result indirectly confirmed that gluten can favor a further decrease

of efficacy to HBV vaccine, beyond the HLA system [29].

14 Celiac Disease and Non-Celiac Gluten Sensitivity

**Year Country Study design Patient population** 

2007 Japan Prospective 26 (mean age 9.2 ± 4.6

2008 Finland Prospective 22 (mean age 8.8

**and sample size**

years) treated CD prospectively immunized; 27 (mean age 16.7 years) untreated CD; 79 (mean age 16.7 years)

treated CD vs

113 (mean age 16.1 years) controls

years) untreated CD vs 18 (mean age 10.4 ± 3.8) controls

**Vaccine (%) of** 

HBV 0.5%

**nonresponders**

24.8%; *P* < 0.001, P < 0.001, *P* = 0.102

HBV 53.9% vs 11.1%; *P* < 0.05

> 74.0% 38.6% vs

**HLA**

NA

Group 1 (22 treated CD): HLA DQ2 Group 2 (53/106 treated and untreated CD): 51: HLA DQ2 2: HLA DQ8

**Author/ references**

Park et al. [21]

Nemes et al. [22] *HBV* hepatitis B virus; *CD* celiac disease; *HLA* human leukocyte antigen; *NA* nonavailable; *DMT1* diabetes mellitus type 1.

**Table 1.** Response to HBV vaccination in CD children and adolescents compared to healthy subjects.

## **3. Pathogenetic role of HLA system in vaccination unresponsiveness in celiac disease**

The mechanism for hepatitis B vaccination failure in patients with CD is not clear. A few hypotheses have been proposed. Multiple candidate genes influence the ability to respond to the recombinant HBV vaccine [9, 30–32]. HLA is believed to contribute significantly to the genetic susceptibility immune response variations to the vaccine [33]. Poor or nonresponsiveness to HBV vaccine has been associated with HLA-DQ2, DR3, and DR7 alleles, which are also associated with CD [9, 10, 34]. In particular, HLA genotype DQ2, found in 90–95% of celiac patients, may have a fundamental role in the predisposition to a weaker immunization to recombinant hepatitis B vaccine in these patients. The HLA is coded by the major histocompatibility complex (MHC) group of genes located on chromosome 6 in the human genome, and they are essential for determining the specificity of an individual's immune response [35]. There are three classes of HLA: HLA class I, HLA class II, and HLA class III. Among them, HLA class II molecules have the task of presenting antigens to the T lymphocytes from outside the cell. Antibody-producing B cells are then stimulated to produce specific antibodies by these antigens [36]. HLA-DQ2 haplotype would be responsible for the failure of induction of the Th2 response needed to promote the differentiation of B cells and the formation of memory B cells necessary for immunization.

Defective or insufficient HBsAg-specific T-helper cells, inadequate T-helper 1, and T-helper 2 cytokine production [37–39], or diminished expression of cell contact signal between activated T and B cells, like CD40L [40] may also be responsible for the lack of response to HBsAg [41, 42]. On this regard, interleukin genotypes (IL10, IL12, IL18) were associated with the anti-HBs antibody development in response to HBsAg in hemodialysis patients [43, 44]. Chen et al. in 2011 found that serum anti-HBsAg response to HBV vaccine in healthy population was closely related to four specific single-nucleotide polymorphism (SNPs) in the IL4, IL4RA, IL13, and Toll-like receptor (TLR2) genes and suggested that variation in these structures may influence the duration and intensity of HBV vaccine-induced immune response [45].

Other studies suggested that compliance with a GFD is responsible for the response to the hepatitis B vaccine in patients with CD. Several studies have hypothesized gluten intake as a cause of failed immunity upon vaccination. Gluten may be implicated because both HBsAg protein fragments and gliadin peptides bind to HLA-DQ2 molecules and induce proliferation of T lymphocytes. Defective antibody production may result from competition between the proteins [22, 23].

## **4. New approaches in hepatitis B vaccination in celiac children**

Inadequate response to HBV immunization in CD patients represent a public health concern because the group of nonresponder patients could act as an HBV infection reservoir. For this reason, response to HBV vaccine should be investigated in children with CD. To protect this population and to achieve the goal of universal protection, new immunization strategies were proposed for CD: the first one is the use of booster and/or higher doses of HBV vaccine by intramuscular (IM) route, and the second one addresses on the use of intradermal route (ID). The studies that have addressed new immunization strategies in CD are summarized in **Table 2** [22, 23, 46, 47].


to the recombinant HBV vaccine [

16 Celiac Disease and Non-Celiac Gluten Sensitivity

memory B cells necessary for immunization.

also associated with CD [

2 cytokine production [37

response [45].

proteins [22, 23].

**Table**

**2** [22, 23, 46, 47].

9, 30

genetic susceptibility immune response variations to the vaccine [33]. Poor or nonresponsive

ness to HBV vaccine has been associated with HLA-DQ2, DR3, and DR7 alleles, which are

celiac patients, may have a fundamental role in the predisposition to a weaker immunization to recombinant hepatitis B vaccine in these patients. The HLA is coded by the major histocom

patibility complex (MHC) group of genes located on chromosome 6 in the human genome, and they are essential for determining the specificity of an individual's immune response [35]. There are three classes of HLA: HLA class I, HLA class II, and HLA class III. Among them, HLA class II molecules have the task of presenting antigens to the T lymphocytes from out

side the cell. Antibody-producing B cells are then stimulated to produce specific antibodies by these antigens [36]. HLA-DQ2 haplotype would be responsible for the failure of induction of the Th2 response needed to promote the differentiation of B cells and the formation of

Defective or insufficient HBsAg-specific T-helper cells, inadequate T-helper 1, and T-helper

vated T and B cells, like CD40L [40] may also be responsible for the lack of response to HBsAg [41, 42]. On this regard, interleukin genotypes (IL10, IL12, IL18) were associated with the anti-HBs antibody development in response to HBsAg in hemodialysis patients [43, 44]. Chen et al. in 2011 found that serum anti-HBsAg response to HBV vaccine in healthy population was closely related to four specific single-nucleotide polymorphism (SNPs) in the IL4, IL4RA, IL13, and Toll-like receptor (TLR2) genes and suggested that variation in these structures may influence the duration and intensity of HBV vaccine-induced immune

Other studies suggested that compliance with a GFD is responsible for the response to the hepatitis B vaccine in patients with CD. Several studies have hypothesized gluten intake as a cause of failed immunity upon vaccination. Gluten may be implicated because both HBsAg protein fragments and gliadin peptides bind to HLA-DQ2 molecules and induce proliferation of T lymphocytes. Defective antibody production may result from competition between the

Inadequate response to HBV immunization in CD patients represent a public health con

cern because the group of nonresponder patients could act as an HBV infection reservoir. For this reason, response to HBV vaccine should be investigated in children with CD. To protect this population and to achieve the goal of universal protection, new immunization strategies were proposed for CD: the first one is the use of booster and/or higher doses of HBV vaccine by intramuscular (IM) route, and the second one addresses on the use of intradermal route (ID). The studies that have addressed new immunization strategies in CD are summarized in

**4. New approaches in hepatitis B vaccination in celiac children**

–32]. HLA is believed to contribute significantly to the

9, 10, 34]. In particular, HLA genotype DQ2, found in 90–95% of

–39], or diminished expression of cell contact signal between acti






**Table2.** Seroconversion rate in CD children and adolescents after IM or ID HBV vaccination.

Nemes et al. administered intramuscularly to 37 nonresponder CD children on GFD, the booster dose of 20 μg of recombinant HBV vaccine, and found that 36 out 37 (97.3%) showed seroconversion 4 weeks after vaccination. However, success with the booster vaccination after controlled GFD suggests that disease activity may play a primary role in vaccination failure [22]. Few studies that exist about HBV vaccine administered by ID route in CD patients unresponsive to IM recombinant vaccine. Leonardi et al. revaccinated 20 CD children and adolescents with a 2 μg dose of recombinant intradermal HBV vaccine. After 4 weeks they found that 15 out 20 patients (75%) showed a protective titer of anti-HBs [22, 23].

Subsequently, Leonardi et al. conducted a prospective, randomized study on 58 CD patients, vaccinated in the first year of life, without protective HBV antibodies as demonstrated by blood analysis. They performed in all patients randomly an HBV vaccination booster dose by ID or IM route. In 30 CD children, a 2 μg dose of recombinant HBV vaccine was administered by the ID route, while 28 CD patients received by IM route 10 μg dose of the same vaccine. Four weeks after every booster dose, 90% of ID patients and 96.4% of IM subjects showed a protective anti-HBs titer after a third booster dose. The authors concluded that both routes are effective in revaccinating CD patients; however, the ID route seems to produce a significantly higher percentage of higher responders [47].

Data suggest that the ID route offers greater immunogenicity due to direct delivery of antigen to the skin immune system, using even lower doses of antigen than IM route [47]. Moreover, the presence of a skin reaction on the site of the intradermal injection could represent a less expensive strategy to test serum anti-HBs response after the booster dose [48]. Economic studies suggest that the substantial cost-saving benefits could be achieved using a fraction of the IM dose via an ID route [48, 49].

## **5. Conclusions**

The available literature shows that HBV vaccine response is lower in celiac subjects compared with healthy ones. Some authors hypothesize that the failure to respond to HBV vaccination is related to specific HLA association, whereas others argue that exposure to gluten at the time of vaccination may play an important role in unresponsiveness to the HBV vaccine. Therefore, nonresponsiveness to the HBV vaccination in CD patients represents a serious public health problem because of the large diffusion of CD that affects about 1% of the European population. Consequently, new vaccination strategies have been proposed to achieve full protection in this context, including the administration of booster doses of HBV vaccine by the intramuscular or the intradermal route. An evaluation of the response to HBV vaccine should be considered as a routine assessment in children newly diagnosed with CD who were previously vaccinated for HBV. Whenever unresponsiveness occurs, certain measures must be taken into account, such as revaccination utilizing ID route, which offers a potentially greater immunogenicity than the IM one, even using lower doses, due to the direct delivery of antigen to the skin immune system. Moreover, the revaccination should be done after the decrease of specific antibodies, which usually occurs after about 1 year of GFD, seen as some studies support GFD as crucial to vaccine responsiveness. More randomized controlled studies with a prospective design are needed for CD patients in order to clarify this topic.

## **Author details**

Nemes et al. administered intramuscularly to 37 nonresponder CD children on GFD, the booster dose of 20 μg of recombinant HBV vaccine, and found that 36 out 37 (97.3%) showed seroconversion 4 weeks after vaccination. However, success with the booster vaccination after controlled GFD suggests that disease activity may play a primary role in vaccination failure [22]. Few studies that exist about HBV vaccine administered by ID route in CD patients unresponsive to IM recombinant vaccine. Leonardi et al. revaccinated 20 CD children and adolescents with a 2 μg dose of recombinant intradermal HBV vaccine. After 4 weeks they found

Subsequently, Leonardi et al. conducted a prospective, randomized study on 58 CD patients, vaccinated in the first year of life, without protective HBV antibodies as demonstrated by blood analysis. They performed in all patients randomly an HBV vaccination booster dose by ID or IM route. In 30 CD children, a 2 μg dose of recombinant HBV vaccine was administered by the ID route, while 28 CD patients received by IM route 10 μg dose of the same vaccine. Four weeks after every booster dose, 90% of ID patients and 96.4% of IM subjects showed a protective anti-HBs titer after a third booster dose. The authors concluded that both routes are effective in revaccinating CD patients; however, the ID route seems to produce a significantly

Data suggest that the ID route offers greater immunogenicity due to direct delivery of antigen to the skin immune system, using even lower doses of antigen than IM route [47]. Moreover, the presence of a skin reaction on the site of the intradermal injection could represent a less expensive strategy to test serum anti-HBs response after the booster dose [48]. Economic studies suggest that the substantial cost-saving benefits could be achieved using a fraction of the

The available literature shows that HBV vaccine response is lower in celiac subjects compared with healthy ones. Some authors hypothesize that the failure to respond to HBV vaccination is related to specific HLA association, whereas others argue that exposure to gluten at the time of vaccination may play an important role in unresponsiveness to the HBV vaccine. Therefore, nonresponsiveness to the HBV vaccination in CD patients represents a serious public health problem because of the large diffusion of CD that affects about 1% of the European population. Consequently, new vaccination strategies have been proposed to achieve full protection in this context, including the administration of booster doses of HBV vaccine by the intramuscular or the intradermal route. An evaluation of the response to HBV vaccine should be considered as a routine assessment in children newly diagnosed with CD who were previously vaccinated for HBV. Whenever unresponsiveness occurs, certain measures must be taken into account, such as revaccination utilizing ID route, which offers a potentially greater immunogenicity than the IM one, even using lower doses, due to the direct delivery of antigen to the skin immune system. Moreover, the revaccination should be done after the decrease of specific antibodies, which usually occurs after about 1 year of GFD, seen as some studies support GFD

that 15 out 20 patients (75%) showed a protective titer of anti-HBs [22, 23].

higher percentage of higher responders [47].

IM dose via an ID route [48, 49].

18 Celiac Disease and Non-Celiac Gluten Sensitivity

**5. Conclusions**

Caterina Anania1 , Francesca Olivero1 , Eugenia Olivero2 and Lucia Pacifico<sup>1</sup> \*


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**Gluten Determination Techniques**
