Helicobacter pylori Vaccines: Lessons from Small Animal Models

The discovery of H. pylori nearly 20 years ago has revolutionized our understanding of peptic ulcer disease and gastric cancer. Whereas the acid theory of ulcers held sway from the 1940s onward, the contribution of an infectious disease became apparent in the 1980s. Direct evidence associating H. pylori infections with gastritis and ulcers first came from the studies of Marshall and Morris [1, 2] who satisfied Koch's postulates by creating histologically confirmed gastritis in themselves following ingestion of a suspension of viable organisms. Studies worldwide have now documented an association of H. pylori infection with chronic gastritis and peptic ulcers [reviewed in 3]. In addition, epidemiologic studies have also shown an association between infection with H. pylori and gastric cancers [reviewed in 4]. H. pylori is a highly motile, gram negative microaerophilic spiral bacterium which colonizes the antral region near the pylori valve of the human stomach. It is estimated that half or more of the world's population, particularly in developing countries, are infected. H. pylori lives within the layer of the mucus overlying gastric epithelial cells to which it may adhere but does not invade. In order to survive in the acidic environment of the stomach, H. pylori produces a large amount of the enzyme urease, which cleaves the urea into CO2 and ammonia, thus buffering its environment and perhaps also providing a source of nitrogen. H. pylori also expresses a toxin which can produce vacuoles in epithelial cells and expresses other virulence factors, some associated with the cag pathogenicity island which codes for elements of a type IV secretion system [5, 6]. Perhaps because of it's unique ecological niche within the mucus layer where it is out of reach of many innate and adaptive host responses, H. pylori establishes chronic infections which can last for decades. It is well adapted to survival in this environment but is quite fastidious and grows slowly in vitro; which is probably the main reason it was not ‘discovered’ until 1982. In the majority of infected hosts there are no overt symptoms, although histologic gastritis is always present. Approximately 10% of the infected individuals eventually develop peptic ulcers secondary to chronic inflammatory responses and other host and bacterial factors, and < 1% develop gastric adenocarcinomas or MALT lymphomas. Shortly after its discovery, a search for therapies to eradicate of H. pylori ensued. After nearly two decades, no simple, single-agent regimen has emerged. Rather, combinations of two or three drugs including antibiotics and bismuth salts initially, but more recently antisecretory agents which suppress acid secretion have been used. While these therapies can achieve eradication rates as high as 90%, they are quite complex and expensive [7]. In addition, patient compliance is often poor owing to severe diarrhoea and nausea, leading to treatment failures. Furthermore, emergence of antibiotic resistant H. pylori is relatively common. Finally, studies in animal models indicate that although antibiotics can cure Helicobacter infections, immunity to re-infection does not develop [8–10]. Thus, in the early 1990s we and others began to investigate vaccination as an alternate therapy for control of H. pylori infections. Since gastric Helicobacters live in close association with the epithelial mucous membrane of the stomach, the goal of the initial Helicobacter vaccine studies was induction of secretory immunoglobulin (Ig)A antibodies in gastric secretions. These antibodies would presumably be able to block infection. It is becoming clear that this premise is at best only partially correct (see Mechanisms below). Because they are relatively inexpensive, with a wealth of immunological reagents available, the mouse has become the animal model of choice for early stage preclinical vaccine studies. It has only been possible to reliably infect mice with H. pylori since 1995, so that many vaccine studies have employed a related organism, H. felis, which establishes a vigorous infection in many strains of mice. The first murine vaccine studies employed H. felis bacterial lysates delivered to mice orogastrically along with cholera toxin (CT) as a mucosal adjuvant [11, 12]. These studies demonstrated 80–100% protection from infection. Perhaps foreshadowing recent results, these early studies also showed that multiple i.p. immunizations with live H. felis also protected approximately half of immunized mice [12]. In another study using germ-free pigs, a modest 10-fold reduction in the bacterial load was observed in H. pylori challenged pigs which had been immunized subcutaneously with formalin killed H. pylori in incomplete Freund's adjuvant. Many others investigators have replicated the more successful orogastric immunization results using either CT or the related heat labile toxin of E. coli as adjuvants [reviewed in 13, 14]. Other routes of mucosal delivery of vaccine antigens were also successful, including intranasal, rectal, intra-Peyer's patch and a true oral immunization in which the vaccine deposited in a mouse's mouth was then swallowed [15–17]. All of the successful immunization strategies reported to date which have used killed organisms/bacterial proteins have employed some type of adjuvant. Immunization in the absence of adjuvant has not been protective even in those cases where it could be shown that a vigorous anti-Helicobacter antibody response was induced [18]. For mucosal immunization, CT in concert with orogastric immunization was the first adjuvant to be used. This combination had previously been shown to be effective at inducing systemic and mucosal antibody responses in several experimental systems [reviewed in 19, 20]. The toxicity of CT and a related molecule from E. coli, heat labile toxin (LT), almost certainly precludes their use in humans, as recently demonstrated in a human clinical trial using LT where 2/3 of the volunteers experienced significant diarrhoea [21, see accompanying Frontlines review by Cynthia Lee [22]]. This fact has led to the investigation of many alternative adjuvants and delivery systems. One strategy has been to try to eliminate the toxicity of CT and LT. Thus, when the nontoxic cell-binding B subunit (CTB) purified from holotoxin was used as an oral adjuvant for bacterial lysate, a reduction in H. felis infection was observed [23]. This oral adjuvant activity of CTB was probably owing to contaminating holotoxin, however, as when recombinant CTB or recombinant LTB were used as oral adjuvants they were ineffective [24, 25]. A second approach to eliminate the toxicity of CT and LT has been to prepare site-directed toxin mutants with reduced or absent enzymatic activity and thus low or no toxicity. One nontoxic mutant LT-K63, where lysine has replaced serine at amino acid residue 63, has been used successfully to orogastrically immunize mice versus H. pylori both prophylactically and therapeutically [26, 27]. Many additional mutant toxins have been prepared and in general they have more adjuvant activity when used intranasally than orally [reviewed in 20]. Two related approaches are to prepare CT or LT adjuvant molecules which rely on fusion proteins incorporating either the A subunit or the B subunits of these toxin molecules [28, 29]. It is probable that one or more of these modified toxin approaches will lead to an adjuvant which is safe in humans and could be employed in H. pylori vaccines. Published reports on other adjuvants which have been evaluated mucosally include poly (D,L-lactide-coglycolide) microspheres in mice [30] and muramyl dipeptide (MDP) in infant ferrets [31]. Mice orally immunized with bacterial sonicate incorporated into microspheres developed enhanced anti-H. pylori antibody responses but animals were not challenged [30]. MDP had no measurable effect on anti-H. mustelae antibody responses or numbers of gastric bacteria, and immunized challenged ferrets exhibited more severe disease than nonimmunized challenged animals [31]. As an alternative to mucosal adjuvants for killed/subunit vaccines, attenuated Salmonella vectors expressing recombinant H. pylori urease have been shown to be effective at inducing protective Helicobacter immunity in mice after either oral or intranasal immunization [32, 33]. Based on these encouraging results, two small scale human trials with Salmonella strains expressing urease have been conducted; in one case no antiurease response was detected and in the other, modest responses were induced [34, 35]. See accompanying Frontlines Topic Review by Cynthia Lee [22] for additional details. The initial focus of Helicobacter vaccination studies was on gastric IgA antibodies. However, as evidence began to emerge that gastric IgG antibodies [36] or even nonantibody mediated mechanisms might be involved in protective immunity (see Mechanisms below), several groups of investigators have begun to revisit parenteral immunization. In one study, rodents immunized subcutaneously with urease and the saponin QS21 or a glyco-lipid-peptide showed 1.5–2.5 log reductions in H. pylori numbers [37] and nearly total cure when QS21 was used therapeutically [38]. In another study subcutaneous urease adsorbed to alum yielded a modest, 10-fold reduction in bacterial colonization [39], whereas in a third study subcutaneous bacterial sonicate with either alum or complete Freund's adjuvant yielded moderate to good protection [40]. In other experiments mice were immunized subcutaneously with urease and either LT, LTB or a combination of LT and LTB [25]. Equivalent protection (1.5 log reduction in gastric cfu's of H. pylori) was provided by urease administered with 5 µg of subcutaneous LT, 50 µg of subcutaneous LTB, or 1 µg of oral LT. Ten ng of LT mixed with 50 µg of LTB appeared to be even more effective [25]. In primates, urease administered 4 times intramuscularly with alum was ineffective at protecting rhesus monkeys from H. pylori infection although when animals were immunized once orally with urease/LT followed by three intramuscular urease/alum booster immunizations, a 20-fold reduction in bacterial numbers was observed [41]. A novel delivery system which might be used either mucosally or parenterally consists of recombinant poliovirus in which UreB (the B subunit of H. pylori urease) was substituted for the viral capsid protein [42]. In initial studies, two intramuscular injections of recombinant urease/polio virus ‘replicons’ followed by a single intramuscular injection of recombinant urease without adjuvant induced detectable antiurease serum antibody responses and spleen cell interferon(IFN)-γ responses; no H. pylori challenge experiments were performed [42]. Parenteral immunization with an H. pylori hsp60 peptide plus Freund's complete adjuvant was also shown to reduce bacterial load up to 20-fold [43]. More recently, parenteral immunization with plasmid DNA encoding either the H. pylori hspA or hspB genes also resulted in a 4–20-fold reduction in gastric H. pylori colony counts in challenged mice [44]. Parenteral DNA vaccination has also been reported using gene sequences for H. pylori urease [45]. In summary, it appears that both mucosal and parenteral immunization or perhaps combined mucosal/parenteral immunization protocols may be effective vaccination strategies for H. pylori. Virtually all vaccines in use today are prophylactic; they are designed to prevent acquisition of infection. There is growing interest, however, in therapeutic vaccines which might be used to treat pre-existing infections. Owing to the large numbers of people who are already infected with H. pylori, the absence of simple, single-drug treatment protocols, and the documented high probability for development of drug-resistant H. pylori, H. pylori infection is a good candidate for therapeutic vaccination. Indeed, using oral immunization protocols which were relatively unchanged from those developed for prophylactic immunization several groups of investigators have shown that pre-existing H. felis, H. mustelae, and H. pylori infections and/or gastritis can be substantially reduced or eliminated by therapeutic immunization [26, 46–52]. These studies have evaluated oral immunization using bacterial lysates or purified antigens combined with CT, LT or a mutated toxin. In spite of these multiple successful therapeutic immunizations in mice and ferrets, therapeutic oral immunization of rhesus monkeys with urease/LT was ineffective [10]. A phase I therapeutic oral immunization trial in humans resulted in a modest reduction in bacterial density in some subjects [21]. The accompanying Frontlines Topic Review by Cynthia Lee [22] summarizes primate and human H. pylori vaccine trials in more detail. Given the generally positive results in small animal models, the more modest results of therapeutic vaccine trials in monkeys and humans are disappointing, but illustrate the difficulties in extrapolating from mice to men. On a positive note, no postimmunization gastritis was observed in any of the therapeutic immunization studies, which is in marked contrast to mouse prophylactic Helicobacter immunization where it is a relatively common finding (see Post-Immunization Gastritis below). Initial Helicobacter vaccine studies employed bacterial lysates delivered orally with CT adjuvant. The relatively undefined nature of whole-cell vaccines and their potential for reactogenicity makes it more likely that defined purified antigen(s) will be preferred in H. pylori vaccines. There are several approaches which can be taken for identification of candidate vaccine antigens including evaluation of putative virulence factors, screening for antigens using sera/monoclonal antibodies (MoAbs), and genomics (see accompanying Frontlines Topic Review on genomics by Richard Ferrero [53]). Several putative H. pylori virulence factors have been identified [reviewed in 5]. These include urease, motility (flagella), various adhesins, the vacuolating toxin VacA, and gene products of the pathogenicity island possibly including CagA. Bacterial enzymes such as catalase and superoxide dismutase which may aid H. pylori in resisting toxic reactive oxygen molecules produced by host inflammatory responses have also been identified as potential virulence factors, as has a recently identified outer membrane protein, oipA, which appears to regulate the severity of inflammation [54]. From this list, urease and its subunits [15, 55, 56], CagA and VacA [57], and catalase [58] have all emerged as candidate vaccine antigens. No published reports yet exist on the potential for H. pylori adhesins or flagellar proteins to severe as vaccine candidates. HspB, which is associated and may copurify with H. pylori UreB has also been shown to be a vaccine candidate [56]. Another putative virulence factor which has been shown to be a vaccine candidate is the H. pylori neutrophil activating protein, NAP [59]. An alternate approach for identifying candidate vaccine antigens is to screen the sera of infection-convalescent individuals or immunized, protected animals for dominant antigens. Spontaneous clearance of H. pylori is thought to be a rare event but nonetheless reactivity to urease was identified in the sera of one such patient and a MoAb specific for urease could passively protect mice [60, 61]. Additional antigens recognized by antibody secreting cells from the gastric mucosa or serum of H. pylori infected patients could also serve as vaccine candidates [62, 63]. In a variation of this approach, sera from mice immunized with H. felis lysate plus CT were used to screen an H. pylori expression library [64]. Positive colonies were picked and the antigens were expressed and tested for vaccine efficacy in the mouse H. pylori model. In addition to the known vaccine candidates UreB and HspB, a lipoprotein named Lpp20, a ribosomal protein, two putative membrane proteins and one secreted protein of unknown were to be vaccine candidates by this [64]. was also identified as a of H. pylori outer membrane and a vaccine candidate date screening approaches have on The growing that the response an in Helicobacter immunity (see Mechanisms that screening anti-H. pylori responses might also vaccine candidates. A approach for identifying H. pylori candidate vaccine antigens on genomics and The complete sequences of two H. pylori have been published In an accompanying Frontlines Topic Review Richard Ferrero this of can be to vaccine and summarizes the results to As animal models of H. pylori infection have provided evidence that either mucosal or parenteral immunization protocols using several adjuvants can either prevent or reduce the of infection and may also be able to eliminate an infection. The initial oral Helicobacter immunization was that mucosal immunization would IgA antibodies which when secreted into the gastric mucus and/or might block the infection. The fact that IgA also administered into the gastric could prevent H. felis infection of mice this [11, A 10-fold reduction in gastric H. pylori in animals a secreting an IgG antibody specific for also that an antibody can a in clearance of H. In addition there is evidence in both animals and humans that after immunization or clearance the of anti-Helicobacter antibodies is to the infection Several other of evidence a for antibodies in only low of antibodies can be detected in gastric of immunized mice to challenge and After antibody although the of both and nonimmunized challenged mice eventually reach Furthermore, a intranasal immunization high of mucosal antibodies yet not protect mice from infection [18]. Thus, there is a poor between antibodies and the fact that therapeutic immunization can reduce or eliminate a pre-existing Helicobacter pre-existing antibodies infection [26, 46–52]. In addition, recent evidence that after there is no in bacterial load between prophylactically immunized and nonimmunized only after to 4 the of H. pylori in immunized mice to and in had previously been in the mouse H. felis model where is Thus, antibodies are not infection. Finally, animals which are in IgA or which antibodies of any can be protected from H. felis and H. pylori by either prophylactic or therapeutic immunization These results not a for antibodies in Helicobacter but clearance of infection by mechanisms than of infection is likely in H. antibodies are not is for Helicobacter Studies using complex I and mice which are in and demonstrated a for cells but not cells Furthermore, reduced bacterial were observed in or which were challenged with H. felis or H. pylori after of cells from immunized These studies a for immunity but not cells are antibodies are not for protection versus gastric Helicobacter infections, immunization does the of the response Perhaps immunization also the of the in experimental animals and humans, infection with H. pylori or H. felis and other inflammatory This response has been shown to to disease severity in animals After either prophylactic or therapeutic oral immunization versus H. felis using CT as an reduced bacterial load with a than a response However, when mice were parenterally immunized, responses of either a or a response could be associated with the adjuvant used Several from have possible for either or in protection from Helicobacter in published reports that or may be In other reports the of the response was by infections with other agents These reports not investigate vaccination but bacterial load and gastritis after experimental mice with H. felis and the the response a response and reduced the severity of gastric inflammation The of the H. felis however, in the mice infected with H. In infection with H. felis and a the bacterial and responses, but not reduce the gastritis in these mice Thus, the response appears to to both Helicobacter disease and protecting animals presumably from Helicobacter infections but the mechanisms as the experimental results are often and One by nearly all investigators who successfully immunized mice or other small animals versus H. felis or H. pylori was the of gastritis and and in While the and severity of the gastritis these studies, its nearly that it may be involved in the In many the severity of the gastritis in and mice was than that observed in animals with no immunization to This enhanced gastritis has been although it more be and challenge because no gastritis was observed after immunization in the absence of from several that this gastritis appears to be cells but not B cells postimmunization and challenge gastritis is a for because the chronic nature of H. pylori gastritis is thought to be in the development of both peptic ulcers and gastric cancer. An to in these studies is the of the gastritis to the bacterial investigators have evaluated both the protection and the gastritis at a single 1 to 4 after the of Helicobacter along with enhanced gastritis have been One early study that the bacterial load in immunized and challenged mice was at challenge than the first Another study that the enhanced postimmunization and challenge gastritis observed early after bacterial challenge with H. felis, several that in an immunized challenge with a gastric Helicobacter will lead to an enhanced gastric inflammatory response which may be for of the challenge to this Helicobacter may a infection in immunized which is by elements of the enhanced gastric inflammatory eradication is successful, the bacterial antigens which are the gastritis will and the inflammation will to the are only reduced in but not the gastric inflammation may with this when H. felis immunized and mice with gastritis were with antibiotics to eliminate any bacteria, the gastritis In an immunization study where the animals were not for protection and gastritis until no gastritis was observed [44]. to our the animals had been evaluated at an gastritis may have been present. In a related study, and recently observed that mice could be of a pre-existing H. pylori infection after of spleen cells into animals The cure of the infection was associated with an which then to a of several Finally, we recently a of bacterial load and gastritis in H. pylori immunized mice and in In these studies we observed a infection in immunized mice which with an enhanced we were to either H. pylori or significant inflammation in the of immunized mice. these studies our that the inflammatory response may be for the of a Helicobacter but that once the is eliminated the gastritis may which elements of the inflammatory response are for the of the are not yet known but the appears to be and is the of investigation by many