Background
More than half the world’s middle-aged population is infected with the gram-negative bacterium
Helicobacter pylori. Overall estimate of prevalence of infection is 78% in developing and 58% in developed countries [
1]. Prevalence of infection steadily declines in the industrialized Western World and emerging economies [
2]. The bacterium is ingested orally and is transmitted within families mostly by the mother [
3,
4].
H. pylori infection is acquired in childhood and if untreated persists lifelong as a chronically active infection [
5]. Although the majority of infections are asymptomatic, the chronic inflammatory changes of the gastric mucosa hold the risk for serious diseases of the gastrointestinal tract. Clinical manifestations begin with acute gastritis, which in a fraction of cases evolves to chronic atrophic gastritis. Gastric ulcer develops in 10% of infected individuals, and gastric adenocarcinoma in 2% and rarely mucosa-associated lymphoid tissue (MALT) lymphoma is induced [
6]. It is still under debate if and when to screen and whom to treat for
H. pylori to reach maximum benefit [
7,
8]. For development of disease a permanent gastric inflammatory response to infection appears to be essential [
9] and inflammation is enforced by a complex interplay of bacterial virulence factors, host cofactors (such as mediators of inflammation), genetic predispositions (such as IL-1ß polymorphisms), and dietary factors [
10].
The
H. pylori genome is of high plasticity and genomic changes such as recombination, mutation and uptake even of exogenous DNA modulate the interaction with the host and adapt the bacterium to environmental changes that occur with duration of infection and stage of disease [
10,
11]. These interactions with the host might change the complex immune response with age and might be reflected in specific antibody patterns which have so far rarely been investigated in the context of age and gender.
We have recently developed
H. pylori multiplex serology [
12]. In contrast to conventional serological diagnosis of infection, multiplex serology simultaneously quantifies antibodies directed against arrays of protein antigens [
13]. Bacterially expressed, affinity-purified glutathione-
S transferase (GST) fusion proteins presenting conformational epitopes [
14] are used as antigens. They are bound to individual sets of fluorescent polystyrene beads and antigen-loaded bead mixtures are exposed to human serum in a single reaction. For each bead set, antibodies bound to the respective antigen are quantified by streptavidin-R-phycoerythrin labelled monoclonal antibodies to human immunoglobulin. Multiplex serology allows analysis of 2000 sera per day for antibodies to up to 100 different antigens and thus provides a high-throughput platform for detection of antibody patterns in large epidemiological studies.
Using
H. pylori multiplex serology [
12], we have previously identified antibodies to HcpC and GroEL as new independent virulence factors that, in combination with the established markers anti-CagA and anti-VacA, were highly predictive of chronic atrophic gastritis risk [
15]. We also found anti-CagA and anti-GroEL to be independent predictors of gastric cancer in a German case–control study [
16]. Antibodies to all fifteen
H. pylori proteins were associated with gastric cancer in a Swedish population-based cancer case–control study [
17] and seropositivity to six proteins (Omp, HP305, HyuA, HpaA, CagA and VacA) may be a risk marker for distal gastric cancer in the high-incidence population of China [
18].
To characterize the dynamics of the immune response as reflected in age and gender specific antibody patterns to fifteen different
H. pylori proteins in a healthy population, we analysed 1,797 German individuals of a cross-sectional study representative for the general population covering the range from 1–82 years of age [
19] with
H. pylori multiplex serology.
Discussion
Previous serological studies describing
H. pylori prevalence in a general population relied on crude bacterial lysates as antigens in ELISA. In contrast, our
H. pylori multiplex serology is based on the detection of antibodies to fifteen affinity-purified
H. pylori proteins. Definition of
H. pylori seropositivity is based on antibody reactivity with at least four
H. pylori proteins. Compared to a conventional combination of screening ELISA and Western blot confirmation multiplex serology has higher specificity without loss of sensitivity [
12]. A further strength of multiplex serology is its ability to allow qualitative and quantitative antibody pattern analysis [
16].
The 1573 adult subjects (aged 18–82 years) analysed here are representative for the non-institutionalized adult German population [
19] concerning age but not gender distribution since sera were an unbiased subset of a population-based study conducted between 1985 and 1989 with a female predominance. Children sera were collected 6 to 17 years later from hospitalised individuals without gastrointestinal disease and their ability to represent the general population is therefore limited. We cannot exclude that the longer storage times and the higher number of freeze-thaw cycles in the sera from adults might have reduced antibody reactivities, however the observed increase from children to adults in number of antigens recognized and in antibody reactivities against the individual antigens is opposite to an reducing effect of longer storage times and more freeze-thaw cycles. The increases with age among adults were observed in samples all originating from the same study with same storage and freeze-thaw cycle history.
We observed an overall
H. pylori seroprevalence of 48%, 12% in children <15 years and 52% among adults >25 years. Several population-based studies were conducted in industrialized countries between 1982 and 1991. In 4,742 subjects from Northern Ireland (12-64 years) [
20], and 2237 subjects from San Marino (23- > 70 years) [
21] similar
H. pylori prevalences of 50.5% and 51%, respectively, were reported, in contrast to lower prevalences of 35% in 3589 Danish adults (30-60 years) [
22] and 38% in 273 Australian adults (20-80 years) [
23].
In comparison to other studies from Germany, we observed 72% of
H. pylori prevalence for the group 51-61 years which is higher than the 60% reported for 260 healthy adult German blood donors that were younger and might be healthier than the population analyzed here [
24]. However, the 12% seroprevalence estimated here in children younger than 14 years is comparable to the 13% described for 216 children from Germany before [
25].
H. pylori seroprevalence increased strongly with age, differing by 39% between the groups of 25-34 and 55-64 years of age. Similar age-specific prevalence estimates have been described for other developed countries [
20,
22,
24,
26] with the highest prevalence of infection in the older age groups.
The age-dependent differences in seroprevalence are either caused by a birth cohort effect [
27,
28] due to stronger
H. pylori exposure in the past decades or by cumulating risk of infection with age. The prevalence of infection depends on the rate of acquisition and loss of infection. The rate of seroconversion is higher in children throughout the world [
29‐
31] although
H. pylori infection can be acquired at all ages [
22,
32]. The rate of seroconversion slows down with age in all cohorts [
33,
34] to small values of 0.2-1.0% per year estimated as cross sectional prevalence increase per year in adults of developed countries [
30] which is slightly lower than a mean prevalence increase of 1.2% per year estimated here.
The age-dependent increases of seroprevalence were observed also for all
H. pylori proteins reaching maximum values in males older 65 years. In this oldest age group an extraordinary steep increase in prevalence occurred for the majority of proteins in males that we did not observe in females and that lead to significant gender differences for eleven of the fifteen proteins. Previous meta-analyses also highlight a male predominance of
H. pylori infection in 18 publications describing adult populations [
35] as a global phenomenon, but not for children in ten paediatric populations [
36]. In this study none of the proteins seemed to be associated with young age as a marker of childhood infection.
The conserved, surface localized lipoprotein H. pylori adhesion A (HpaA) does not follow the general finding of a seroprevalence increase by age in H. pylori seropositives. HpaA seroprevalences significantly decrease with age in males and at least do not increase in females for unknown reasons. We performed analyses stratified by HpaA status (data not shown), and in overall H. pylori seropositives. Antibodies to HpaA seem to be a marker for increased immune response, i.e. they are associated with higher seroprevalence to other H. pylori proteins and recognition of higher numbers of antigens for both genders. In contrast, HpaA serostatus does not impact seroprevalence for other H. pylori proteins in H. pylori overall seronegatives.
The major finding of our study is the observation of a qualitative increase in the immune response to
H. pylori with age. The degree of multiple seropositivity and strength of antibody reactivity increased with age among seropositives for GroEL, HyuA
, CagM, Catalase, NapA and UreA. These proteins are necessary for bacterial colonization and initiation of the host immune response. GroEL belongs to the chaperone family and promotes refolding of misfolded proteins under stress conditions. It seems to be associated with the adhesion of
H. pylori to human gastric epithelial cells [
37] and the induction of inflammatory responses [
38]. NapA also mediates the binding of
H. pylori to the host cell [
39], activates neutrophils and monocytes and antagonizes oxidative stress [
40]. Catalase is necessary for long term colonization as part of the antioxidant defence mechanisms of
H. pylori[
41]. Survival of
H. pylori in the acidic gastric habitat is secured by its enzyme urease, composed of the two heterogeneous subunits UreA and UreB, which metabolizes urea to carbon dioxide and ammonia [
42]. CagM is necessary for the translocation of the CagA protein into the host cell via the type IV secretion system [
43] and HyuA belongs to the oxoprolinase family and is important for aminoacid biosynthesis [
44].
This cross-sectional study does not allow distinguishing whether the increase of antibody reactivities is related to age or birth cohort effect. However our observation that this phenomenon also occurs in seropositives provides evidence that it is related to age, and immunosuppression or lower load of
H. pylori infection in younger people as examples of birth cohort related effects are unlikely. The increase of antibody reactivities with age is opposed to the need of
H. pylori to escape from the permanent defence of the innate and adaptive immune response. The niche of the gastric mucous layer protects
H. pylori and makes it inaccessible to specific antibodies and thereby leaves the humoral immune response ineffective [
9]. The qualitative increase of antibody reactivities with age might also reflect higher immunogenicity of bacterial antigens in individuals infected with multiple
H. pylori strains which might be more prevalent in older age groups of the German population. Colonization with multiple
H. pylori strains is possible [
45] and change of strains may occur during chronic infection [
46].
H. pylori adapts its genome to its host continuously by point mutation, and intragenomic and intergenomic recombination [
8,
11]. These mechanisms are discussed to be responsible for lifelong bacterial immune evasion and development of one’s “individual strain”. So far,
H. pylori multiplex serology is based on antigens conserved among strains and the analysis of strain multiplicity needs to be addressed in a more comprehensive evaluation including antigens that allow for detection of strain specific antibodies.
Competing interest
The authors declare that they have no competing interests.
Authors’ contributions
Conceived and designed the experiments: MP LG. Performed the experiments: AM TW. Analyzed the data: AM TW MP. Contributed reagents/materials/analysis tools: TW HB LG MP. Wrote the paper: AM, TW, MP. Drafted the article: AM TW MP. Critically revised the article: TW HB LG MP. All authors read and approved the final manuscript.