Recent Advances that Favor Development of a Vaccine for Helicobacter Pylori Infection

Helicobacter pylori is a motile Gram-negative spiral bacterium that colonizes the human gastric epithelium. It was first reported to be successfully cultured from patient biopsies under microaerophilic conditions in 1984 [1]. Infection is believed to occur predominantly in early childhood, and it persists for life unless successfully treated with antimicrobial therapy. H. pylori infection is one of the most common infections in human beings with over half the Earth‘s population being infected. The prevalence of H. pylori infection is less than 50% in the USA and many other industrialized nations, but in developing countries more than 80% of the adult population can be infected [2]. H. pylori is recognized as the etiologic agent of peptic ulcer disease independent of nonsteroidal anti-inflammatory drugs (NSAIDs) [3]. Although no generalized causal connection has been established with dyspepsia, all infected individuals develop histologic gastritis that can progress to atrophic gastritis and ultimately to gastric cancer [4]. In fact, H. pylori is classified by the WHO as a Group I carcinogen [5].

Helicobacter pylori colonization and pathogenesis is enhanced by several unique virulence factors. The large, multisubunit urease enzyme converts host urea to ammonia and carbon dioxide, which helps create a microenvironment that is less acidic than the gastric lumen. Urease expression is essential for colonization and the importance of this protein is illustrated by the fact that 6% of the bacterial mass is due to the urease enzyme [6]. Vacuolating cytotoxin A (Vac A) has been shown to have many effects on host cells, including arresting T-cell activation, vacuolating epithelial cells, membrane channel formation, interference with cell signaling, and induction of apoptosis [7]. Not all strains express Vac A and its role in vivo remains ill defined, but the numerous interactions with host cells described in vitro indicate that it plays an important role in H. pylori virulence. One of the most interesting virulence factors, however, is the type four secretion system (TFSS) and the cytotoxin-associated gene type A (CagA) protein encoded by the pathogenicity island [7]. Elements of the TFSS induce inflammatory signals from the epithelial cells, such as IL-8 production. The TFSS also serves as a conduit to introduce the Cag A protein into the cytoplasm of the epithelial cells where it becomes phosphorylated. Once phosphorylated, it will participate in the phosphorylation of host-cell proteins and, ultimately, influence cell morphology and cell cycle regulation. The presence of the CagA protein, and the type of phosphorylation sites it contains, have been positively associated with increased gastric cancer risk.

Gastric cancer is the fourth most common cancer, and the second most frequent cause of cancer deaths worldwide [8]. In the USA, gastric cancer ranks as the 14^th most frequent cause of cancer mortality but it is much more common in Japan, central Europe, Scandinavia, and in South and Central America [9]. H. pylori infection and noncardia cancer have a significant positive association with an overall odds ratio (OR) of 2.97 (95% CI: 2.34–3.77) [10]. No such association has been demonstrated for H. pylori and cardia cancer. A more recent meta-analysis showed that chronic infection with more virulent CagA-positive strains increases the risk of noncardia gastric cancer an additional 1.64-fold (95% CI: 1.21–2.24) [11]. Recently, data from clinical trials have shown that eradication of H. pylori significantly reduces the risk of gastric cancer development (OR: 0.35; 95% CI: 0.16–0.75). There are some data, however, that show there is no significant difference of the incidence of gastric cancer between H. pylori eradication treatment and the control group [12]. Experimental animal models have allowed further investigation to support an association between H. pylori infection and gastric cancer. The multistep model of gastric carcinogenesis described above, in which chronic gastritis progresses to atrophic gastritis, intestinal metaplasia, dysplasia and finally gastric cancer, has been clearly reproduced in Mongolian gerbils infected with H. pylori[13]. In the murine model, transgenic expression of the H. pylori oncoprotein CagA by gastric epithelial cells results in the development of precancerous and cancerous lesions [14].

The significant morbidity and mortality associated with gastric cancer in many parts of the world provide a compelling argument in favor of developing a vaccine against H. pylori, even though its prevalence is declining markedly in the West. However, there are at least two issues that give the scientific community pause about moving forward with vaccine development. The first is the concept that while H. pylori and its associated diseases are rapidly declining in the developed world, there has been a coincident increase in esophageal disease. The decline of H. pylori infections is clearly related to the cohort effect and the evidence that socioeconomic status is the only well-defined risk factor for the acquisition of H. pylori infection. As the socioeconomic status improves in a given population, the rate of H. pylori acquisition will drop accordingly. But there is recent evidence, albeit controversial, that there is also an inverse relationship between H. pylori infection and the development of gastroesophageal reflux disease (GERD), as well as esophageal and gastric cardia cancers. Currently, the rate of esophageal and gastric cardia cancers is considerably lower than antral gastric cancer. And while disease such as GERD can have profound impacts on one‘s quality of life, there should be no question that diseases such as GERD would be preferable to gastric cancer.

The second issue that raises concerns about a vaccine for H. pylori is feasibility. When investigators first began considering a vaccine for H. pylori they were confronted with two onerous realities. First, the host responds to H. pylori infection with robust gastric inflammation and both a local and systemic adaptive immune response. In spite of the response, H. pylori persists for the life of the host, thus many believed that if the active host immune response could not eradicate the bacteria, then a response generated by vaccination probably would not provide any greater advantage. Second, H. pylori is a mucosal pathogen in the strictest sense in that it remains at the apical surface of the gastric epithelium and generally does not invade the host tissue. While vaccines have been one of the greatest public health success stories in history, most successful vaccines work for pathogens that are either systemic, such as the Smallpox Variola virus, Clostridium tetani, and Corynebacterium diphtheriae, or become systemic after gaining entry via the mucosa, such as the Poliovirus. Few vaccines have been developed for mucosal pathogens such as venereal diseases and gastrointestinal infections. Experimental vaccines for these types of pathogens need to be delivered to the mucosal tissue, such as by oral immunization, so the intestinal lymphoid tissue can be targeted. These types of vaccines are notoriously weak, generally require multiple boosts, can be compromised by the low pH of the stomach and duodenum, and often involve controversial mucosal adjuvants such as cholera toxin in order to improve efficacy.

However, against this backdrop, we and others demonstrated that rodents could achieve significant immunity by administering bacterial lysate with cholera toxin adjuvant by the oral route [15,16]. This prototype vaccine has since been modified many times and has been used extensively to show that purified proteins can also be used along with many types of novel delivery vehicles for oral immunization. The vaccine works equally well when administered therapeutically as it does prophylactively. It has even achieved some success in ferrets and nonhuman primates. Unfortunately, protective immunity in these studies is generally measured as a significant reduction in bacterial load, and although greater than 99% of bacteria can be eradicated, the animals often remain infected. Animal studies in which complete eradication is achieved are becoming more common, but this type of success has not been extended to higher order mammals. In fact, in the most notable human clinical trial to date, administration of a therapeutic vaccine to infected subjects worked about as well as the murine vaccine [17]. Subjects were immunized with a subunit vaccine consisting of H. pylori urease in combination with low doses of Escherichia coli labile toxin (LT), a bacterial toxin similar to cholera toxin often used as a mucosal adjuvant. A significant reduction in bacterial load was achieved but no sterilizing immunity. Additionally, many subjects experienced diarrheal symptoms from the LT, leaving many to believe that further progress would require better mucosal adjuvant technology.

Recent studies, however, indicate that it may in fact be possible to develop an effective vaccine. It is worth noting that although many H. pylori vaccine trials in animals and humans have not generated complete protection, they have significantly reduced the bacterial load and, therefore, immunization is having a positive impact on the host immune response. We now know from studies in both mice and humans that the host immune response to H. pylori, while clearly evident histologically and as demonstrated by anti-H. pylori antibodies, is actually downregulated by the presence of CD25⁺ Treg cells. These naturally occurring regulatory T cells play an important role in limiting the host response to commensal bacteria in the gut and preventing certain autoimmune reactions [18,19,20]. In vitro analysis of T cells from mice and humans has demonstrated that when the Treg cells are depleted, the remaining H. pylori-specific T cells are able to respond to H. pylori antigens more strongly [21–23]. In vivoexperiments in mice have shown that when CD25⁺ T cells are depleted, or are prevented from becoming active, the mice become capable of reducing or eradicating the bacteria in the absence of immunization [21,22,24,25]. It is encouraging that immunization is able to overcome this type of immune regulation to the extent that it does. Gaining additional knowledge about this regulation and how to over-ride it could be an important step in developing a truly protective vaccine against H. pylori for human use.

As noted above, successful vaccination for mucosal pathogens is difficult for many reasons and these challenges have prevented the development of vaccines for many enteric and urogenital pathogens. Lymphocytes tend to be committed to either peripheral or mucosal tissues and, therefore, systemic immunizations generally do not result in the migration of activated lymphocytes to the mucosal sites where immunity is needed. Activation of mucosal lymphocytes requires local stimulation and this has typically been achieved by oral immunization targeting the intestines, or through intranasal immunization targeting the upper respiratory tract. Over the past 15 years however, we and others have tested numerous vaccine formulations and routes of delivery. Protective immunity against H. pylori can be provided not only by oral immunization, but by intranasal, rectal and, most surprisingly, by systemic delivery. We have tested subcutaneous and intraperitoneal immunization in adult and neonatal mice and achieved immunity comparable to mice immunized by mucosal routes [26,27]. It may be that challenge of immunized mice induces a generalized response in which memory H. pylori-specific T cells are attracted to the gastric mucosa not because they are mucosal lymphocytes but because they are responding to inflammation signals.

The true significance of demonstrating protection by systemic immunization is the possibility of using established, safe, human-use adjuvants and being able to avoid the many hurdles involved in developing an oral vaccine. Towards this end, along with experimental oil-based adjuvants, we tested H. pylori antigens emulsified in ‘alum’. The adjuvant alum is a hydrophilic, particulate composed of aluminum hydroxide or other aluminum components and is the only adjuvant currently approved for human use in the USA. It has been used for decades and has an excellent record for safety when used for systemic vaccination. We found alum to be an effective adjuvant for systemic delivery of an H. pylori vaccine in mice [26,27]. Recently, several different recombinant H. pylori subunit vaccines adsorbed to alum were each tested for safety and immunogenicity after intramuscular immunization in a human clinical trial with uninfected volunteers [28]. No adverse reactions were described and most subjects responded to vaccination with increased antibody responses to each vaccine as well as memory lymphocyte responses. Ultimately, an efficacious vaccine against H. pylori will require a continuing evolution of design and delivery technology.

However, previous work is encouraging. Initial clinical trials reveal that the immune system can be manipulated towards the detriment of the bacteria as the bacterial load was shown be significantly decreased. Although the mucosal adjuvant LT induced untoward effects, there is promise that H. pylori immunity may be achieved by systemic immunization, thus negating the requirement for a mucosal adjuvant.

These new developments make vaccine development much more practical than previously believed. As our understanding of how the host immune response to H. pylori is regulated, vaccine technology can be adjusted to circumvent such downregulation. While a vaccine against H. pylori will undoubtedly require continued development and repeated trials, it is likely that a successful vaccine can be produced. The potential to eliminate or dramatically reduce gastric cancer in large populations of the world continues to make this a worthwhile endeavor. There is the likelihood that a reduced incidence of H. pylori within a population will also result in an increased incidence of esophageal malignancies. These diseases, while serious, are unlikely to occur in frequencies that would offset the overwhelming benefits of preventing gastric cancer.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

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The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.