Identification of anti-Helicobacter pylori antibody signatures in gastric intestinal metaplasia

Our two-stage study represents the most comprehensive proteome-level analysis to identify anti-H. pylori antibodies to distinguish IM from NAG. We found moderate-to-strong associations with IM for 11 IgG and 2 IgA antibodies against several outer membrane proteins (OMPs) and proteins essential for bacterial survival. A combined model of the 11 IgG antibodies moderately discriminated IM from NAG. Another unique feature of our study was the comparison between current and past H. pylori infections. We identified 15 IgG and 1 IgA antibodies associated with current infection, including responses to several proteins related to colonization and persistence.

H. pylori coevolved with humans and developed efficient mechanisms to avoid the surveillance of the host immune system. As a consequence, H. pylori infection typically does not elicit a strong humoral response [8], which is consistent with the limited number of immunogenic proteins observed in our study (4%; 62/1528). Also, expanding our previous findings in gastric cancer using the same NAPPA array [5], we confirmed that IgG antibodies were more informative in determining gastric pathology compared with IgA antibodies.

Anti-CagA antibodies are sustained for a long time even after successful H. pylori eradication [9, 10]. A potential mechanism for the persistent antigenic stimuli may be due to the remnants of CagA-positive strains in the deepest gastric glands surviving long years of infection. Our finding that a higher CagA seropositivity was moderately discriminative between IM cases and NAG controls aligns with two previous cross-sectional analyses [11, 12] within the Linxian Nutrition Intervention Trial cohort. Pan et al. compared antibodies against HP0547/CagA, HP0887/VacA, GroEL, UreA, HcpC, and HP1118/Ggt in patients with precancerous lesions and those with superficial gastritis, and reported a positive IM association with anti-HP0547/CagA and an inverse IM association with anti-HP1118/gGT [11]. Using a different platform, a 15-plex Luminex fluorescent bead-based immunoassay, Epplein et al. reported positive associations of IM with antibodies against HP1564/Omp, HP0547/CagA, HP0887/VacA, HcpC, HP0305, GroEL, NapA, HyuA, Cad, and HpaA [12]. These studies confirmed the important role of CagA in gastric carcinogenesis.

H. pylori OMPs serve as key factors for nutrient scavenging and evasion of host defense mechanisms [13]. In our study, two antibodies to OMPs were positively associated with IM. HP1177/Omp27/HopQ binds to carcinoembryonic antigen-related cell-adhesion molecules with high specificity [14]. Type I HopQ may be associated with carcinogenesis by transferring CagA or triggering type IV secretion system to initiate and maintain chronic inflammation mainly by activating toll-like receptor 9 via the canonical NF-KB pathway [15, 16]. Taxauer et al. showed that the HopQ-CEACAM interaction is also important for the activation of the non-canonical NF-KB pathway [17]. HP1125/PalA/Omp18 is expressed by all known H. pylori strains and is involved in persistent colonization [18]. In particular, Omp18 may alter interferon-γ levels and optimize virulence phenotype (i.e., CagA), survive oxidative stress, and anti-phagocytosis. pH taxis is another important mechanism for the long-term persistence of H. pylori [19]. HP0103/TlpB is required in chemorepulsive responses to acid, as well as the quorum-sensing molecule autoinducer-2 [20]. Interestingly, binding of polymeric G-repeats regulator to the upstream of tlpB is sufficient to regulate TlpB both at the transcript and protein level [21]. HP0596/Tipα is another tumor promoter secreted as dimers and enters the gastric cells, a process mediated through NF-KB activation [22], that also has DNA-binding activity [23]. Similar to CagA, antibodies to HP0596/Tipα may persist after bacterial eradication [24]. Notably, our analyses showed anti-HP0596/Tipα to be moderately positively correlated with other antibodies (HP1125/PalA/Omp18 and HP0103/TlpB). Although our best discriminatory model suggests independent effects for anti-HP1177/Omp27/HopQ, HP0547/CagA, HP0103/TlpB, and HP1125/PalA/Omp18, given the complex networks of these antigens, their orchestrated synergistic contribution cannot be dismissed.

In the current study, we report several inverse antibody associations with IM. These findings may indicate loss of antigenic stimuli potentially related to atrophy progression, use of antibiotics, or immune protection against carcinogenesis. In our previous NAPPA gastric cancer study, we found several inverse associations including antibodies against HP0371/FabE/AccB, HP0243/NapA, and HP0153/RecA that were also identified in this IM study. HP0371/FabE/AccB is a biotin carboxyl carrier protein of acetyl-CoA carboxylase, which is involved in fatty acid biosynthesis [25]. NapA plays dual roles, recruiting host neutrophils/monocytes and stimulating the production of reactive oxygen intermediates, but on the other hand, sequestering iron and stress-resistant to oxidative stress [26]. NapA may also promote the formation of H2O2-induced biofilm and contribute to multidrug resistance [27]. RecA is a protein that is necessary for repairing DNA damage or facilitating recombination. In addition, mutants of recA are hypersensitive to DNA-damaging agents such as metronidazole, ultraviolet, or ionizing radiation [28].

We also found some novel antibodies that were inversely associated with IM. HypB is a GTPase with a key role in nickel homeostasis [29]. H. pylori hypB-deficient mutant exhibits significantly decreased urease activity as there is a physical interaction between urease and HP0900/HypB [30]. HpaA is a lipoprotein in the flagellar sheath and outer membrane and plays an essential role in the adhesion and colonization of the gastric mucosa [31]. Although poorly characterized, HP0709 encodes an enzyme that is involved in either DNA methylation or synthesis of some branched amino acids. HP0385 is a hypothetical protein with unknown functions. The findings for HpaA or RecA are inconsistent with a previous study showing a positive association of these two proteins with IM [12]. Considering the dual-faceted roles of these antigens, along with their various parts in coordination with other proteins and the external environment, further studies should investigate host-antigen interactions of these antigens. It is also possible that these proteins are important for colonization and establishment of successful infection but less relevant when the microenvironment changes due to hypochlorhydria and other tissue transformations related to atrophy.

Extensive IM confers a higher risk of gastric cancer. Our exploratory case-case analysis identified three antibody associations (anti-HP0516/HslU, anti-HP0385, and anti-HP1453/homD) with antral restricted IM (vs. corpus extension). The corresponding bacterial antigens have not been well studied. In particular, homD is a conserved gene and little is known about its encoding OMP (HP1453/homD) [32]. Further studies are warranted to address the potential differences in the humoral responses to H. pylori by IM anatomical subsite.

Although H. pylori infection can be detected by many methods, their sensitivities and specificities are variable. Taken together, the results of four different H. pylori tests assessing current and past infections, the high-risk study population seems to have had an almost universal bacterial exposure. There are no bacterial proteins that are recognized in all H. pylori-infected individuals. Differential humoral responses to specific H. pylori antigens could be related to variable protein expression influenced by the stomach microenvironment. Loh et al. [33], documented increased expression and translocation of CagA in response to high salt conditions in rodent models. Noto et al. [34], reported a similar increased expression of CagA in strains growing under iron-limiting conditions. Our NAPPA anti-GroEL fairly agreed with a commercial wcELISA. Non-invasive identification of H. pylori-specific antibodies accurately classifying individuals with current infection could assist with clinical management (e.g., H. pylori eradication). We identified 16 antibodies as markers of current H. pylori infection, including some novel candidates. Our results align with previous studies assessing a limited number of antibodies. Comparing results of a 13-plex Luminex fluorescent bead-based immunoassay and urea breath test, Butt et al. found that seropositivity to VacA, HP0010/GroEL, HcpC, and HP1564/ PlpA indicate current infection [35]. Using combined results of wcELISA, urease, and culture to define current infection, Shafaie et al. found that seropositivity to HP0547/CagA, HP0596/Tip-α, and HP0175/ PpiC was associated with current infection [36]. In currently infected individuals, flagellar antigens exhibit seropositivity as high as 90% [37]. Our NAPPA array included a total of 28 flagellar antigens and found positive associations with HP0870/FlgE, HP0601/FlaA, HP0295/Fla that showed overall prevalences ranging from 40 to 58% in all individuals, and from 50 to 71% in currently infected individuals. Based on in vitro studies, HP1118/gGT seems to be a pathogenic factor associated with H. pylori-induced peptic ulcer disease [38]. As mentioned above, Pan et al. reported an inverse IM association with anti-HP1118-gGT [11]. Similarly, we found a suggestive association in this study and a significant inverse association in our previous NAPPA gastric cancer study [5]. In our current study, HP1341/TonB/TonB2 has shown higher seroresponse in individuals with current infection compared to those with past infection. In our previous report on gastric cancer, we only had H. pylori serology results, thus we could not determine whether the subjects had a current or past infection [5]. We speculate the slightly lower seroprevalence of HP1341/TonB/TonB2 in gastric cancer is likely due to the loss of H. pylori colonization. To our knowledge, no data are available on the functions of anti-HP1110. Future research may explore the role of these antigens in vaccine development.

Up to date, there are two bacterial Genome-Wide Association Studies (GWAS) of gastric cancer. Berthenet et al. identified 32 significant loci (HP0068, HP0102, HP0269, HP0290, HP0468, HP0524, HP0527, HP0528, HP0531, HP0532, HP0540, HP0541, HP0544, HP0555, HP0569, HP0615, HP0709, HP0747, HP0797, HP0906, HP0936, HP1004, HP1046, HP1055, HP1149, HP1177, HP1184, HP1243, HP1331, HP1421, HP1460, HP1572) by comparing hpEurope genomes from patients with gastric cancer (n = 49) with genomes from patients with gastritis (non-atrophic and atrophic with and without IM; n = 124) [39]. On the other hand, Tuan and Yahara et al. [40] identified 12 significant loci (HP0082, HP0130, HP0231, HP0463, HP0490, HP0776, HP0807, HP0915, HP1250, HP1440, HP1467, HP1523; 11 single nucleotide polymorphisms and three DNA motifs) by comparing hspEAsia genomes from patients with gastric cancer (n = 125) and genomes from patients with duodenal ulcer (n = 115). Likely due to differences in the control group (gastritis vs. duodenal ulcer) and the underlying genetic structures of hpEurope and hspEAsia populations, there are no common hits between the GWAS. Notably, two of our candidate antibodies, anti-HP1177/Omp27/HopQ (positive association with IM) and anti-HP0709 (inverse association with IM and positive association with current H. pylori infection) represent hits in the hpEurope GWAS. HP1177/Omp27/HopQ was identified under the comparison including IM in the case group, while HP0709 was identified under the comparison including IM in the control group. One of our additional candidates, anti-HP0231/dsbK/dsbG (positive association with current infection), represents a gastric cancer hit in the hspEAsia GWAS.

The strengths of our IM study include the use of a well-validated and highly reproducible state-of-the-art microarray technology. Secondly, we employed a two-stage approach with discovery and independent validation using blinded testing to ensure the rigor of our findings. Importantly, we tested well-characterized samples with a high prevalence of H. pylori infection and data from several complementary tests documenting current and past H. pylori infections. Despite these strengths, our study has some limitations. First, although comprehensive, our H. pylori-NAPPA did not include a universal set of proteins, as the panproteome of H. pylori is undetermined. Second, our findings may not be generalizable to all populations as there is a difference in the immunogenic profile among races/ethnicities [41,42,43]. Third, although we combined four tests, a potential misclassification in our definitions of current and past H. pylori infections cannot be averted. The sensitivity and specificity of just urease and histology combined (both > 95%) are comparable to the urea breath test [44]. Moreover, individuals with H. pylori-negative gastritis could have been included in the group with no evidence of H. pylori exposure. In addition, information on past treatment of H. pylori infection was not available. Antibiotics may alter humoral responses, thus altering the association between the antibodies and IM. We addressed antibody associations with advanced IM based on the anatomical subsite. Additional heterogeneity should be evaluated for different histological types of IM (complete vs. incomplete).

Antibodies to several specific H. pylori proteins are associated with modest gastric cancer risk in prospective cohort studies [41, 45]. To date, none of the proposed antibodies have enough discriminatory power to distinguish gastric cancer patients from cancer-free individuals for screening. Our data suggest only moderate gain in discriminating IM and NAG with a combined IgG antibody model compared to an anti-CagA-only model. Additional candidate anti-OMP antibodies could be tested in prediagnostic samples.

Besides the etiological importance of our identified H. pylori antibodies in carcinogenesis, a non-invasive blood test for IM based on the antibodies may have a potential translation to noninvasive detection of patients with this premalignant lesion. After additional validation, the candidate antibodies in combination with other biomarkers (e.g., pepsinogens), could have a direct clinical application for triaging high-risk individuals for further diagnostic procedures, particularly in places where mass gastric cancer screening resources are limited.

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