Helicobacter pylori overcomes natural immunity in repeated infections

Key Clinical Message Repeated experimental reinfection of two subjects indicates that Helicobacter pylori infection does not promote an immune response protective against future reinfection. Our results highlight the importance of preventing reinfection after eradication, through public health initiatives, and possibly treatment of family members. They indicate difficulties for vaccine development, especially therapeutic vaccines.


Introduction
Billions of humans in the world are infected with Helicobacter pylori [1]. While H. pylori infection typically leads to lifelong asymptomatic colonization of the stomach, 10% of infections result in peptic ulcers, 1-5% of infections result in gastric cancer, and more rarely, infection leads to mucosaassociated lymphoid tissue (MALT) lymphoma [1,2].
Treatment of H. pylori-induced gastritis with antibiotics is effective [3]. However, recurrence after treatment does occur [4]. This could be due to failed eradication or reinfection. Our aim was to assess whether H. pylori infection in humans produces an immune response protective against future reinfection. Anti-H. pylori antibodies are present in chronically infected persons, but fail to clear the infection [5]. However, the protective role of natural immune responses to H. pylori has not previously been tested experimentally in humans.
To assess whether H. pylori could overcome natural immunity, we eradicated the asymptomatic H. pylori infection in otherwise healthy subjects and then rechallenged them with their own strain of H. pylori. We assumed immunity would be maximal against an individual's own strain and would lead to the failure of reinfection attempts.
Our findings have clinical significance in relation to strategies to combat H. pylori. They illuminate the significance of reinfection and inform vaccine development attempts, as an effective immune response is necessary for vaccination to work.

Methods
Healthy volunteers (n = 312) were recruited through advertising. Three hundred and ten applicants were excluded (Table 1), leaving two largely asymptomatic adult subjects with H. pylori infection proven by C 14 urea breath test (PYtest â Kimberly Clark, USA) (UBT) [6]. Figure 1 provides a timeline. In brief, subjects underwent a baseline endoscopy with mucosal biopsies of the antrum and corpus taken for rapid urease test (CLOtest, Kimberly Clark, USA), H. pylori culture, histology, and immunohistochemistry. Culture and antibiotic susceptibility testing was performed as previously described [1]. Isolates were screened for toxin genes (cagA and vacA) by PCR [5,7]. For histology, mucosal biopsies were fixed in 10% formalin and processed routinely. They were stained with hematoxylin and eosin (H&E) to assess morphology and toluidine blue to demonstrate bacteria.
Biopsies for immunohistochemistry were frozen at À80°C and later processed according to standard methods, with regulatory T cells enumerated [8]. The specific antibodies used for immunohistochemistry were CD3polyclonal, rabbit, Dako; CD4+ -SP35, monoclonal, rabbit, Cell Marque; FoxP3-SP97, Spring Bioscience, rabbit. FoxP3+ and CD4+ T cells from mucosal biopsies were counted and compared to overall T-cell (CD3+) populations. T cells were enumerated manually across five independent microscopic fields at 40x magnification, and averages reported.
Blood samples were also collected at this and subsequent endoscopies and used to monitor liver function, kidney function, electrolytes, and full blood count. Processing of samples was performed in accordance with the standard practice at PathWest (a certified laboratory).
Serum samples were evaluated by ELISA (IgG) as previously described [9].
At the initial (and final endoscopy), endoscopic gastric secretion tests (EGT) [10] were conducted: Gastric acid secretion over a 10-min period was measured after subcutaneous pentagastrin injection, followed by a 20-min delay. The EGT value was multiplied by six to provide a value equivalent to maximal acid output (MAO).
After the initial endoscopy, there was a delay to ensure successful bacterial culture. Subjects then took a sevenday eradication therapy consisting of esomeprazole (20 mg), amoxicillin (1000 mg), and clarithromycin (500 mg) twice daily. Tinidazole (500 mg) twice daily was added for days five to seven. Eradication was confirmed by two UBTs at least 4 weeks apart. Subsequently, the subjects drank a dose of 10 9 colony-forming units of H. pylori culture suspended in 10 mL of commercially available beef broth. This followed ingestion of 40 mg of famotidine the night before. Two weeks later, infection status was determined by UBT. Subjects then underwent another endoscopy with biopsy collection followed by eradication therapy. At least 124 days post-therapy, subjects again drank their H. pylori and repeated the cycle.
Diary cards were used to record symptoms for 14 days at baseline, for 7 days during each antibiotic treatment, and for 14 days after each reinfection.
Written informed consent was given by the subjects. The study was approved by the Sir Charles Gairdner Hospital Human Research Ethics Committee (study number 2007-045). The trial is registered with the Australia New Zealand Clinical Trials Registry [11] (trial number ACTRN12607000467437).

Baseline results
Baseline investigation confirmed that subjects had H. pylori infections, but did not have other significant pathology or antibiotic-resistant H. pylori. Subject 1, a 58-year-old female, experienced occasional heartburn prior to the study. Subject 2, a 30-year-old male, was completely asymptomatic. Initial endoscopic examination of subject 1 showed a small hiatus hernia, two tiny erosions at the gastroesophageal junction, erythema of the gastric antral mucosa, and petechiae in the duodenum. Erythema plus mild nodularity of the proximal antrum was reported in subject 2.
Gastric mucosal biopsies of both subjects showed active chronic gastritis with H. pylori (Fig. 2, Table 2), and all samples were also positive for H. pylori by histology, RUT, and culture ( Table 2).
PCR studies showed that subject 1's strain was cagA+ with the vacA s1 m2 subtype, while subject 2's strain was cagAand had the vacA s2 m2 subtype.

Eradication and reinfection cycles
Subjects were proven free of H. pylori after the first antibiotic treatment by UBT (Table 2). Subjects were then reinfected with their own H. pylori strain. Reinfection was confirmed by UBTs after two weeks and at endoscopy ( Table 2, Fig. 2). A second antibiotic treatment was given, and subjects were proven free of H. pylori by UBT (  Subjects underwent a second cycle of reinfection proven by UBT and biopsies (Table 2, Fig. 2). Final eradication of H. pylori was confirmed by UBT.

Clinical symptoms
Subject 1 reported increased heartburn after the second reinfection. She noted softer stools after both reinfections, and diarrhea, for 2 days, and vomited once after the second reinfection. Subject 2 reported halitosis after both reinfections.

Blood chemistries
Blood chemistry remained within the normal range for both subjects.

Immunological studies
Regulatory T cells (13-25%) were present in both subjects at baseline (Fig. 3) and typically increased upon reinfection.
Total T-cell numbers remained stable. Subjects showed persistent antibody titers throughout the study (Fig. 4).

Gastric acid secretion
Gastric acid secretion was within normal ranges (normal range for MAO is 5-30 mmol/h in women and 7-47 mmol/h in men [12]. At initial endoscopy, MAO was calculated as 37.1 mEq/h for subject 1 and 27.4 mEq/h for subject 2. At visit 13, it was 28.1 mEq/h for subject 1 and 33.9 mEq/h for subject 2.

Discussion
We showed that, in two subjects with prior H. pylori infection and high levels of H. pylori-specific antibodies, reinfection was possible on two occasions. These cases demonstrate, for the first time experimentally, that natural immunity in humans is not protective.
The results are consistent with epidemiological studies [4]. Recurrence of infection has been observed at low-to- moderate levels in various populations. However, differentiation between recrudescence due to treatment failure and reinfection has been problematic. Even where molecular fingerprinting techniques confirm the bacteria are the same over time, one cannot differentiate between failed therapy and reinfection from a family member with the same strain [6]. Study of the rate of recurrence over multiple years suggests that while recrudescence may explain the majority of cases in the developed nations, and reinfection is more common in developing populations [6].
Our study supports the view, as it provides direct evidence to prove that reinfection is possible. The results are also congruent with the successful experimental reinfection in monkeys [13,14]. However, our study is unique, not only because it was in humans, but also because we tested immunity to the original strain. Deliberate infection in humans had previously only been performed with a strain from an external source (BCS100) [15,16]. During our study, a sustained immune regulatory environment was maintained with high levels of regulatory T cells. This offers a potential mechanism for the lack of protection. Similarly, regulatory T cells are elevated during chronic infection and aid the bacteria to survive in the host [8,17]. However, determining the exact mechanism requires further study, as the bacterium is known to exhibit a complex array of evasion strategies [18,19].
When might natural immunity be effective? In natural infection, exposure to numerous H. pylori strains probably occurs in childhood until the subject is colonized by a strain suited to that individual's gastric mucosa. It may be able to evade the host's immune system because of a particular combination of bacteria, host, or environmental factors. If so, it could be that our subjects always remained susceptible to their own strains, but would be able to eliminate others.
Other reports indicate that innate immune responses may offer protection and that acute infections are more likely to be cleared than chronic infections. Marshall spontaneously cleared his H. pylori infection, despite failing to seroconvert [20]. Similarly, Okuda et al. found a high (75%) rate of transient infections in infants (but could not rule out false positives) [21].
Our findings have implications for public health strategies to control H. pylori. As eradication therapy does not prevent future reinfection, antibiotic use needs to be  combined with measures to stop reinfection. New epidemiological research is needed to determine the key transmission routes. There is evidence for and against a range of routes (see Mitchell 2001 [22] for a review). Development of screening techniques that allow isolation and culture of H. pylori from contaminated specimens from the environment and subjects is vital to further this research [22]. In the meantime, we suggest that eradication should be combined with education to improve hygiene, provision of clean water supplies and sanitation, and possibly synchronized eradication across families. This accords with Statement 7 of the Second Asia-Pacific Guidelines for H. pylori infection, which states that in areas of high prevalence, eliminating H. pylori infection through improvements in public health and education will have the greatest impact in reducing the burden of gastric cancer [23]. The statement focuses on the benefits of preventing first infections in patients who may never be treated, but our results also highlight the problem of reinfection.
The obvious alternative to antibiotics is vaccination. Unfortunately, the ability of H. pylori strains to repeatedly infect humans despite the presence of antibodies is reason for pessimism concerning the development of a therapeutic H. pylori vaccine. Vaccination against a high burden of H. pylori, as is the case in therapeutic vaccination, will be difficult to achieve, probably because of the down-regulated immune environment (35, 36). Our results are in accordance with reviews lamenting failure to develop an effective H. pylori vaccine [2,24,25]. However, recently, Zeng et al. reported the success of a prophylactic H. pylori vaccine in a field trial in schoolaged children [26]. This demonstrates that under conditions of low bacterial burden and acute infection, H. pylori infection may be prevented. Certainly, the trial of Zeng et al. represents an extremely promising advance. However, as Sutton has outlined there is still some way to go before reaching a marketable vaccine [27]. We agree with the Maastrict IV/Florence Consensus Report (Statement 20) that the profound benefits of an H. pylori vaccine merit continued effort in this area [28]. Our results and others [29] indicate that protection independent of B-cell responses should be considered, with a focus on prophylactic vaccination. While host immune regulation by H. pylori may be challenge for vaccine development, it may have potential benefits [29]. The immune modulatory properties of H. pylori may be exploited in the fight against inflammatory diseases such as allergies. Similarly, exploiting H. pylori's ability to evade the immune system may permit the use of safe genetically modified strains as a live delivery agent [30] that can be used repeatedly.
The study had several limitations. It was limited to two subjects as asymptomatic adults with H. pylori were hard to find. The scarcity of H. pylori -infected volunteers in Western Australia has previously been reported [31].
A larger study would increase generalizability of the results and could include subjects with greater symptoms indicative of immune response. These patients may not be so susceptible to reinfection. In addition, infection with novel strains would test whether subjects are able to eliminate other strains, even while susceptible to their own. However, this question may be better addressed in animal models, given the greater risk of harm. Safety was a priority in our study. Hence, we started with largely asymptomatically infected subjects and only reinfected them with their own strains. If we could not eradicate H. pylori after reinfection, they would not be in a worse state than before the study.
We hypothesize that the presence of a strong immune regulatory environment contributed to successful reinfection. However, our immunological studies were only preliminary, and more comprehensive assays at more time points are necessary.
As we assumed reinfection might be difficult, we used high doses of bacteria. The minimum bacterial dose required to reinfect a person was not determined.

Conclusion
We have shown in two subjects that the natural immune response to H. pylori does not protect against reinfection with the same strain. Our findings highlight the importance of avoiding reinfection from the environment and from family members following eradication. The immune regulatory properties of H. pylori and the lack of natural immunity suggest that therapeutic vaccination against H. pylori will be difficult. However, prophylactic vaccines may be possible.