Rectal and Intranasal Immunizations with Recombinant Urease Induce Distinct Local and Serum Immune Responses in Mice and Protect against Helicobacter pylori Infection

To determine the optimal inductive sites for immunization against Helicobacter pylori infection, the protective efficacy of recombinant urease (rUre) was assessed for mice given the vaccine by either the oral (p.o.), intranasal (i.n.), or rectal route. When mice were immunized with rUre (25 μg p.o. or rectally or 10 μg i.n.) plus heat-labile toxin from Escherichia coli as the mucosal adjuvant, all routes afforded protection against challenge with H. pylori, as indicated by a significant reduction in gastric urease activity (P < 0.0005) compared to that of sham-immunized controls. Quantitative H. pylori culture of stomach tissue demonstrated a >97% reduction in bacterial burden in mice immunized by all routes (P < 0.05). Induction of antiurease immunoglobulin A (IgA) levels in gastric luminal secretions after p.o. immunization was greater than after i.n. administration (means, 6.0 and 1.02 ng/ml, respectively) and was dependent upon challenge with H. pylori. However, immunization by the rectal route resulted in the generation of the highest levels of gastric antiurease IgA (mean, 40.89 ng/ml), which was detectable prior to challenge with H. pylori. Immunohistochemical staining of stomach tissue for cells secreting urease-specific antibody and CD4⁺ T cells showed levels of recruitment to be dependent upon challenge with H. pylori and equivalent for all routes. These results identify both the rectum and nasal passages as suitable inductive sites for urease immunization.     

Sublingual Immunization Protects against Helicobacter pylori Infection and Induces T and B Cell Responses in the Stomach

Sublingual (SL) immunization has been described as an effective novel way to induce mucosal immune responses in the respiratory and genital tracts. We examined the potential of SL immunization against Helicobacter pylori to stimulate immune responses in the gastrointestinal mucosa and protect against H. pylori infection. Mice received two SL immunizations with H. pylori lysate antigens and cholera toxin as an adjuvant, and after challenge with live H. pylori bacteria, their immune responses and protection were evaluated, as were immune responses prior to challenge. SL immunization induced enhanced proliferative responses to H. pylori antigens in cervicomandibular lymph nodes and provided at least the same level of immune responses and protection as corresponding intragastric immunization. Protection in SL-immunized mice was associated with strong H. pylori-specific serum IgG and IgA antibody responses in the stomach and intestine, with strong proliferation and gamma interferon (IFN-γ) and interleukin-17 (IL-17) production by spleen and mesenteric lymph node T cells stimulated with H. pylori antigens in vitro, and with increased IFN-γ and IL-17 gene expression in the stomach compared to levels in infected unimmunized mice. Immunohistochemical studies showed enhanced infiltration of CD4⁺ T cells and CD19⁺ B cells into the H. pylori-infected stomach mucosa of SL-immunized but not unimmunized H. pylori-infected mice, which coincided with increased expression of the mucosal addressin cell adhesion molecule (MAdCAM-1) and T and B cell-attracting chemokines CXCL10 and CCL28. We conclude that, in mice, SL immunization can effectively induce protection against H. pylori infection in association with strong T and B cell infiltration into the stomach.At least half of the world''s population is infected with Helicobacter pylori, one of the few microorganisms known to be able to colonize the human stomach. In 10 to 15% of infected individuals, chronic H. pylori infection causes duodenal ulcers, and infection with H. pylori has been shown to be a strong risk factor for the development of gastric adenocarcinoma and malignant mucosa-associated lymphomas (3, 17, 20). Although treatment with a combination of antibiotics and a proton pump inhibitor is usually effective in individual cases, limited treatment compliance, rapidly emerging antibiotic resistance, and frequent reinfection with H. pylori in countries where it is highly endemic make vaccination an increasingly attractive alternative or complement to standard therapy.Vaccination, given either preventively or therapeutically, is especially needed in countries with a high incidence of gastric cancer (20), reinfection (22), or antibiotic resistance. However, clinical trials of various oral or parenteral H. pylori vaccine candidates have not shown much promise to date, pointing to the need for identifying improved antigen-adjuvant formulations and/or alternative routes of immunization in the quest for an effective vaccine against H. pylori (33).The importance of cell-mediated mucosal immunity in protection against experimental H. pylori infection after vaccination is well established (1, 9, 10, 23, 35). In most studies, intragastric (IG) immunization has been used to achieve efficient stimulation of the gastrointestinal immune response. However, this route usually requires large amounts of antigen for efficient immunization, and the environment in the stomach and intestine may have adverse effects on the antigens and adjuvants used. Intranasal immunization against H. pylori has also been used in mice, but studies in humans have indicated that the nasal route of immunization is ineffective in stimulating immune responses in the intestine or stomach (12). In addition, intranasal immunization is associated with a risk of translocation of some types of antigens or adjuvants to the olfactory bulb of the brain, restricting its applicability in humans (31, 34).Sublingual (SL) immunization has recently emerged as an attractive novel approach for mucosal vaccination against pathogens (7, 8, 31). In a model of influenza virus infection, SL immunization with live or adjuvanted killed virus induced immune responses and protection against aerosol challenge with live virus. In contrast to intranasal immunization, SL immunization had no evidence of vaccine or adjuvant entering the brain (31). In another study, SL immunization was found to induce vaccine-specific antibody and T cell responses in the genital tract and, after SL immunization with human papillomavirus (HPV)-like particles, protection against genital HPV infection, indicating the potential of SL immunization to stimulate immune responses also in nonrespiratory mucosal tissues (7).In the present study, we examined whether SL immunization in mice can induce a mucosal immune response in the gastrointestinal tract. More specifically, we addressed the potential of SL immunization with H. pylori antigen and cholera toxin (CT) adjuvant to stimulate T and B cell responses in the stomach and protect against H. pylori infection. Our findings demonstrate that SL immunization induces strong systemic and stomach mucosal antibody and T cell responses and a high level of protection against H. pylori challenge. After SL immunization and H. pylori challenge, the stomach mucosa showed infiltration of both CD4⁺ T cells and CD19⁺ B cells and increased expression of gamma interferon (IFN-γ) and interleukin-17 (IL-17) compared to unimmunized infected mice. This was associated with increased expression of both the mucosal addressin cell adhesion molecule (MAdCAM-1) integrin and chemokines CXCL10 (10-kDa IFN-γ-induced protein) and CCL28 (mucosa-associated epithelial chemokine) in the immunized mice, which probably facilitated the migration of immunization-induced CD4⁺ T cells and CD19⁺ B cells into the stomach mucosa. Our results indicate that SL immunization against H. pylori effectively induces a strong immune response in the gastrointestinal tract mucosa and protects against infection, providing an attractive novel way of vaccinating against H. pylori infection.     

Mice Are Protected from Helicobacter pylori Infection by Nasal Immunization with Attenuated Salmonella typhimurium phoPc Expressing Urease A and B Subunits

Live Salmonella typhimurium phoP^c bacteria were tested as mucosal vaccine vectors to deliver Helicobacter pylori antigens. The genes encoding the A and B subunits of H. pylori urease were introduced into S. typhimurium phoP^c and expressed under the control of a constitutive tac promoter (tac-ureAB) or a two-phase T7 expression system (cT7-ureAB). Both recombinant Salmonella strains expressed the two urease subunits in vitro and were used to nasally immunize BALB/c mice. The plasmid carrying cT7-ureAB was stably inherited by bacteria growing or persisting in the spleen, lungs, mesenteric or cervical lymph nodes, and Peyer’s patches of immunized mice, while the plasmid carrying tac-ureAB was rapidly lost. Spleen and Peyer’s patch CD4⁺ lymphocytes from mice immunized with S. typhimurium phoP^c cT7-ureAB proliferated in vitro in response to urease, whereas cells from mice given S. typhimurium phoP^c alone did not. Splenic CD4⁺ cells from mice immunized with phoP^c cT7-ureAB secreted gamma interferon and interleukin 10, while Peyer’s patch CD4⁺ cells did not secrete either cytokine. Specific H. pylori anti-urease immunoglobulin G1 (IgG1) and IgG2A antibodies were detected following immunization, confirming that both Th1- and Th2-type immune responses were generated by the live vaccine. Sixty percent of the mice (9 of 15) immunized with S. typhimurium phoP^c cT7-ureAB were found to be resistant to infection by H. pylori, while all mice immunized with phoP^c tac-ureAB (15 of 15) or phoP^c (15 of 15) were infected. Our data demonstrate that H. pylori urease delivered nasally by using a vaccine strain of S. typhimurium can trigger Th1- and Th2-type responses and induce protective immunity against Helicobacter infection.     

Effect of Bacterial Flora on Postimmunization Gastritis following Oral Vaccination of Mice with Helicobacter pylori Heat Shock Protein 60

In order to assess the efficacy of oral Helicobacter pylori heat shock protein 60 (HSP60) as a vaccine, protection against H. pylori infection in specific-pathogen-free (SPF) C57BL/6 and germfree (GF) IQI mice was examined. Prophylactic oral vaccination of these two strains of mice with either H. pylori HSP60 or Escherichia coli GroEL inhibited H. pylori colonization by 90 to 95% at 3 weeks postinfection (p.i.). However, these mice were only partially protected because bacterial loads increased in all animals at 10 weeks p.i. Anti-H. pylori HSP60 immunoglobulin G was detected in serum at 3 weeks p.i. in mice vaccinated with either H. pylori HSP60 or GroEL. Significant increases in the gastritis scores were observed only in SPF mice immunized with H. pylori HSP60. These results indicate that oral vaccination with H. pylori HSP60 has partial protective effects on subsequent H. pylori infection but also induces postimmunization gastritis. However, GF mice immunized with H. pylori HSP60 did not suffer from severe gastritis. Therefore, the presence of bacterial flora appears to contribute to the induction of postimmunization gastritis.     

Bioengineered surfaces promote specific protein–glycan mediated binding of the gastric pathogen Helicobacter pylori

Helicobacter pylori colonizes the gastric mucosa of half of the worlds population and persistent infection is related with an increase in the risk of gastric cancer. Adhesion of H. pylori to the gastric epithelium, which is essential for infection, is mediated by bacterial adhesin proteins that recognize specific glycan structures (Gly-R) expressed in the gastric mucosa. The blood group antigen binding adhesin (BabA) recognizes difucosylated antigens such as Lewis B (Le^b), while the sialic acid binding adhesin (SabA) recognizes sialylated glycoproteins and glycolipids, such as sialyl-Lewis x (sLe^x). This work aimed to investigate whether these Gly-Rs (Le^b and sLe^x) can attract and specifically bind H. pylori after immobilization on synthetic surfaces (self-assembled monolayers (SAMs) of alkanethiols on gold). Functional bacterial adhesion assays for (Gly-R)-SAMs were performed using H. pylori strains with different adhesin protein profiles. The results demonstrate that H. pylori binding to surfaces occurs via interaction between its adhesins and cognate (Gly-R)-SAMs and bound H. pylori maintains its characteristic rod-shaped morphology only during conditions of specific adhesin–glycan binding. These results offer new insights into innovative strategies against H. pylori infection based on the scavenging of bacteria from the stomach using specific H. pylori chelating biomaterials.     

The antifungal vaccine derived from the recombinant N terminus of Als3p protects mice against the bacterium Staphylococcus aureus

Vaccination with the recombinant N terminus of the candidal adhesin Als3p (rAls3p-N) protects mice from lethal candidemia. Candidal Als3p also is structurally similar to the microbial surface components recognizing adhesive matrix molecule adhesin, clumping factor, from Staphylococcus aureus. To determine the potential for cross-kingdom vaccination, we immunized mice with rAls3p-N or negative control proteins and challenged them via the tail vein with S. aureus or other gram-positive or gram-negative pathogens. The rAls3p-N vaccine, but neither tetanus toxoid nor a related Als protein (Als5p), improved the survival of vaccinated mice subsequently infected with multiple clinical isolates of S. aureus, including methicillin-resistant strains. The rAls3p-N vaccine was effective against S. aureus when combined with aluminum hydroxide adjuvant. However, the vaccine did not improve the survival of mice infected with other bacterial pathogens. Vaccinated, infected mice mounted moderated type 1 immune responses. T lymphocyte-deficient mice were more susceptible to S. aureus infection, but B lymphocyte-deficient mice were not. Furthermore, T but not B lymphocytes from vaccinated mice mediated protection in adoptive transfer studies. The passive transfer of immune serum was not protective. These data provide the foundation for cross-kingdom vaccine development against S. aureus and Candida, which collectively cause 200,000 bloodstream infections resulting in ≥40,000 to 50,000 deaths annually in the United States alone.     

Baculovirus-mediated expression of a Helicobacter pylori protein-based multiepitope hybrid gene induces a potent B cell response in mice

Helicobacter pylori is a gram-negative bacterium that is present in over half of the world's population. The colonization of the stomach?s gastric mucosa by H. pylori is related to the onset of chronic gastritis, peptic ulcer, and cancer. The estimated deaths from gastric cancer caused by this bacterial infection are in the 15,000–150,000 range. Current treatment for controlling the colonization of H. pylori includes the administration of two to four antibiotics and a gastric ATPase proton pump inhibitor. Nevertheless, the bacterium has shown increased resistance to antibiotics. Despite an extensive list of attempts to develop a vaccine, no approved vaccine against H. pylori is available. Recombinant viruses are a novel alternative for the control of primary pathogenic agents. In this work, we employed a baculovirus that carries a Thp1 transgene coding for nine H. pylori epitopes, some from the literature, and others were selected in silico from the sequence of H. pylori proteins (carbonic anhydrase, urease B subunit, gamma-glutamyl transpeptidase, Lpp20, Cag7, and CagL). We verified the expression of this hybrid multiepitopic protein in HeLa cells. Mice were inoculated with the recombinant baculovirus Bac-Thp1 using various administration routes: intranasal, intragastric, intramuscular, and a combination of intranasal and intragastric. We identified a strong adjuvant-independent IgG-antibody response in the serum of recombinant baculovirus-Thp1 inoculated mice, which was specific for a strain of H. pylori isolated from a human patient. The bacterium-specific IgG-antibodies were present in sera 125 days after the first vaccine administration. Also, H. pylori-specific IgA-antibodies were found in feces at 82 days after the first inoculation. A baculovirus-based vaccine for H. pylori is promising for controlling this pathogen in humans.     

Protection by a Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Vaccine Expressing Shiga Toxin 2 B Subunit against Shiga Toxin-Producing Escherichia coli in Mice

We have developed a novel vaccine against Shiga toxin (Stx)-producing Escherichia coli (STEC) infection using a recombinant Mycobacterium bovis BCG (rBCG) system. Two intraperitoneal vaccinations with rBCG expressing the Stx2 B subunit (Stx2B) resulted in an increase of protective serum IgG and mucosal IgA responses to Stx2B in BALB/c mice. When orally challenged with 10³ CFU of STEC strain B2F1 (O91: H21), the immunized mice survived statistically significantly longer than the nonvaccinated mice. We suggest that intraperitoneal immunization with rBCG expressing Stx2B would be a potential vaccine strategy for STEC.     

Treatment of Helicobacter pylori infection in mice with oral administration of egg yolk-driven anti-UreC immunoglobulin

Background

Helicobacter pylori, the causative agent of gastritis and gastric ulcer, plays a crucial role in development of gastric carcinomas. Antibiotic therapy fails in almost 20% of cases due to development of antibiotic resistance. Development of antibodies against specific H. pylori targets could have significant therapeutic effect. In the present research attempts have been made to study the effect of IgY purified from egg yolk of hens immunized with recombinant UreC in treatment of mice infected with H. pylori.

Materials and methods

Purified IgY-HpUc was used in two forms: powdered and PBS dissolved. 10⁹ bacteria in BHI were orally administered to C57BL6/j mice three times on alternate day intervals. Eight weeks after the last inoculation, the serum was assayed for infection rate by ELISA. The severity of gastritis was analyzed histopathologically. Infected mice were randomly divided into three groups. Groups one and two were treated with dietary IgY-HpUc and IgY-HpUc dissolved in PBS respectively for 28 days. The untreated group served as control.

Results

Serology and histopathology confirmed the establishment of the infection. Indirect ELISA results in the treated animals showed considerable reduction of H. pylori specific antibodies in their sera. Pathological examination of gastric mucosa of infected mice treated with IgY-HpUc showed considerable reduction of inflammation in the stomach tissues. The bacterial presence on mucosal layer of the stomach was considerably reduced.

Conclusions

UreC-induced IgY is specifically successful in inhibition of H. pylori infection and could be an alternative to antibiotic treatment.     

Induction of mucosal immune responses against Helicobacter pylori infection after sublingual and intragastric route of immunization

There is a current lack of effective mucosal vaccines against major gastroenteric pathogens and particularly against Helicobacter pylori, which causes a chronic infection that can lead to peptic ulcers and gastric cancer in a subpopulation of infected individuals. Mucosal CD4⁺ T‐cell responses have been shown to be essential for vaccine‐induced protection against H. pylori infection. The current study addresses the influence of the adjuvant and site of mucosal immunization on early CD4⁺ T‐cell priming to H. pylori antigens. The vaccine formulation consisted of H. pylori lysate antigens and mucosal adjuvants, cholera toxin (CT) or a detoxified double‐mutant heat‐labile enterotoxin from Escherichia coli (dmLT), which were administered by either the sublingual or intragastric route. We report that in vitro, adjuvants CT and dmLT induce up‐regulation of pro‐inflammatory gene expression in purified dendritic cells and enhance the H. pylori‐specific CD4⁺ T‐cell response including interleukin‐17A (IL‐17A), interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) secretion. In vivo, sublingual immunization led to an increased frequency of IL‐17A⁺, IFN‐γ⁺ and TNF‐α⁺ secreting CD4⁺ T cells in the cervical lymph nodes compared with in the mesenteric lymph nodes after intragastric immunization. Subsequently, IL‐17A⁺ cells were visualized in the stomach of sublingually immunized and challenged mice. In summary, our results suggest that addition of an adjuvant to the vaccine clearly activated dendritic cells, which in turn, enhanced CD4⁺ T‐cell cytokines IL‐17A, IFN‐γ and TNF‐α responses, particularly in the cervical lymph nodes after sublingual vaccination.     

Preparation of specific anti-Helicobacter pylori yolk antibodies and their antibacterial effects

Objective: To study immunization procedures and preparation methods of specific IgY antibodies (IgY-Hp, IgY-IB) produced by hens immunized with Helicobacter pylori (Hp) bacterial antigen and recombinant Hp specific antigen IB, detect the inhibition effects on Hp growth and Hp urease activity, and study the effects of oral administration for treating Hp infection. Methods: By using recombinant cholera toxin subunit B (rCTB) as an adjuvant, hens received intramuscular injection immunization for continuous 7 times at an interval of 14 days. Then, the eggs were collected; IgY was purified. Results: On day 49 after hens were immunized, levels of two antibodies all reached 1:12800; after they were purified by Ammonium sulfate precipitation, their purity was over 80%. IgY-Hp could inhibit Hp growth and inhibit Hp urease activity; although in vitro, IgY-IB could not inhibit Hp growth but could inhibit Hp urease activity. The experiments in vivo found that when IgY-Hp or IgY-IB with sucralfate dual oral therapy was used to treat Hp infected mouse model, the cure rate all could reach 83.3%. Conclusion: According to immunization procedure, high titer specific IgY antibody (1:12800) can be obtained in 49 days and its titer remains stable. Oral administration of the specific IgY antibodies in Hp infected mice can reach a cure rate of 83.3%, and the antibodies are expected to become new drugs and therapeutic methods of targeted therapy against Hp infection.     

A Replicating Adenovirus Capsid Display Recombinant Elicits Antibodies against Plasmodium falciparum Sporozoites in Aotus nancymaae Monkeys

Decades of success with live adenovirus vaccines suggest that replication-competent recombinant adenoviruses (rAds) could serve as effective vectors for immunization against other pathogens. To explore the potential of a live rAd vaccine against malaria, we prepared a viable adenovirus 5 (Ad5) recombinant that displays a B-cell epitope from the circumsporozoite protein (CSP) of Plasmodium falciparum on the virion surface. The recombinant induced P. falciparum sporozoite-neutralizing antibodies in mice. Human adenoviruses do not replicate in mice. Therefore, to examine immunogenicity in a system in which, as in humans, the recombinant replicates, we constructed a similar recombinant in an adenovirus mutant that replicates in monkey cells and immunized four Aotus nancymaae monkeys. The recombinant replicated in the monkeys after intratracheal instillation, the first demonstration of replication of human adenoviruses in New World monkeys. Immunization elicited antibodies both to the Plasmodium epitope and the Ad5 vector. Antibodies from all four monkeys recognized CSP on intact parasites, and plasma from one monkey neutralized sporozoites in vitro and conferred partial protection against P. falciparum sporozoite infection after passive transfer to mice. Prior enteric inoculation of two animals with antigenically wild-type adenovirus primed a response to the subsequent intratracheal inoculation, suggesting a route to optimizing performance. A vaccine is not yet available against P. falciparum, which induces the deadliest form of malaria and kills approximately one million children each year. The live capsid display recombinant described here may constitute an early step in a critically needed novel approach to malaria immunization.     

Antibody-Mediated Protection against Infection with Helicobacter pylori in a Suckling Mouse Model of Passive Immunity

Studies of active immunization against Helicobacter pylori indicate that antibodies play a minor role in immunity. There is also evidence, however, that the translocation of antibodies in the stomach may be insufficient to achieve functional antibody levels in the gastric lumen. We have used a suckling mouse model of passive immunity to determine if perorally delivered antibodies can protect against infection with H. pylori. Female C57BL/6 mice were immunized parenterally with formalin-fixed cells of three clinical isolates of H. pylori (3HP) or the mouse-adapted H. pylori strain SS1 before mating. Their pups were challenged with the SS1 strain at 4 days of age and left to suckle before determination of bacterial loads 14 days later. Compared to age-matched controls, pups suckled by 3HP-vaccinated dams were significantly protected against infection (>95% reduction in median bacterial load; P < 0.0001). Pups suckled by SS1-vaccinated dams were also significantly protected in terms of both median bacterial load (>99.5% reduction; P < 0.0001) and the number of culture-negative pups (28% versus 2% for immune and nonimmune cohorts, respectively; P < 0.0001). Similar results were obtained with pups suckled by dams immunized with a urease-deficient mutant of SS1. Fostering experiments demonstrated that protection was entirely attributable to suckling from an immunized dam, and antibody isotype analysis suggested that protection was mediated by the immunoglobulin G fraction of immune milk. Analysis of the bacterial loads in pups sampled before and after weaning confirmed that infection had been prevented in culture-negative animals. These data indicate that antibodies can prevent colonization by H. pylori and suppress the bacterial loads in animals that are colonized.Helicobacter pylori colonizes the gastric mucosa of humans and commandeers host defenses to establish chronic active gastritis while increasing the host''s susceptibility to gastroduodenal ulceration or certain gastric malignancies (37). Although H. pylori induces profound systemic and mucosal immune responses, clearance of infection is infrequent, and there is no protection against reinfection following eradication by antimicrobial chemotherapy (15). Consequently, there are no obvious parameters of natural immunity on which to base effective vaccination strategies.Vaccination studies of animal models have suggested that antibody development is not necessary for protective immunity to H. pylori (19) and may even enhance colonization (5, 6). Conversely, cellular immunity, possibly in concert with innate immune factors, such as defensins (59), elicits protection or eradication by exaggerating the gastric inflammatory response induced by H. pylori, thus interrupting colonization without a need to interact with the bacteria directly (3). The importance of the inflammatory response for protection against H. pylori is supported by the association of postimmunization gastritis with vaccine efficacy (6, 23). Nevertheless, the failure of antibody to limit H. pylori colonization is yet to be fully explained. One reason for this failure may be the relatively low level of antibodies in the gastric lumen due to the apparent inability of the mucosal immune system to translocate sufficient quantities of antibody across the gastric mucosa.Although well characterized in the intestine, relatively little is known about antibody secretion into the stomach. Some studies of H. pylori infection have reported that levels of immunoglobulin A (IgA) in gastric juice are significantly lower than those found in the saliva or intestinal contents (33, 34). Evidence that these low levels of IgA are due to inadequate antibody secretion in the stomach includes the following: (i) H. pylori-specific antibodies in gastric juice of infected individuals are predominantly nonsecretory IgA (10); (ii) equivalent amounts of IgG and IgA in the stomach suggest that IgA may leak across the mucosa rather than being actively secreted (14, 18); and (iii) much of the secretory IgA (sIgA) in the stomach is derived from swallowed saliva (17, 54). In addition, compared to the small intestine, the normal mammalian stomach has barely detectable secretory component (SC), suggesting a limited capacity for translocation of polymeric IgA across the gastric mucosa (8, 33). Moreover, despite considerable upregulation of SC by gamma interferon following the development of gastritis, there is no corresponding increase in the concentration of sIgA in gastric juice (4). Consequently, the concentration of sIgA in the stomach is unlikely to be sufficient to prevent or eradicate colonization by H. pylori.On the other hand, there is evidence that passive immunization with antibodies delivered perorally may reduce the extent of gastric colonization by Helicobacter species. This therapeutic approach has shown some promise in adult mice given monoclonal IgA or hyperimmune bovine colostrum against Helicobacter felis (14, 41) or urease-specific, chicken-derived IgY against H. pylori (44). In addition, reports of delayed acquisition of H. pylori by Gambian infants that corresponded to their mothers’ levels of breast milk IgA specific for H. pylori (58) and the protection of infant mice against full colonization by H. felis while suckling from immunized dams (13) suggest that orally delivered antibodies may be beneficial in controlling gastric Helicobacter infections. Despite these favorable reports, there are no tightly controlled studies that conclusively show prevention of H. pylori infection by orally delivered immune antibodies in the absence of additional factors, such as famotidine (44). Moreover, no studies have investigated the refinement of vaccine preparations for use in the production of anti-H. pylori polyclonal antibody products.In this study, we used a suckling mouse model of infection to investigate whether H. pylori-specific antibodies delivered during lactation to the gastric lumen of infant mice can protect against H. pylori infection. The route and adjuvant used to immunize the dams were selected to evoke an immune response similar to that required for the production of commercial quantities of polyclonal monomeric antibodies, such as from hyperimmune bovine colostrum. The model allowed us for the first time to quantify the contribution of passively acquired H. pylori-specific antibodies to protection against infection and provided an opportunity to examine different vaccine preparations for their ability to elicit these antibodies.     

Immunization against Natural Helicobacter pylori Infection in Nonhuman Primates

Helicobacter pylori infection is widespread in some breeding groups of a rhesus monkey colony (71% H. pylori positive by 1 year), and the rate of seroconversion is also high. As a result, these groups can be used to test the safety and efficacy of an anti-H. pylori vaccine. Nine-month-old female animals were randomized to receive either 8 mg of recombinant urease (rUre) plus 25 μg of Escherichia coli heat-labile enterotoxin (LT) (n = 26) or placebo plus LT (n = 29), given four times at 1-week intervals followed by a booster 1 month later. Ten months after the start of the immunization, the animals were subjected to endoscopy and biopsy samples were obtained. H. pylori negativity was defined as no H. pylori growth by culture and no H. pylori observed at histology. By this criterion, 2 (7%) of 29 animals receiving placebo and 8 (31%) of 26 immunized animals were H. pylori negative (P < 0.035). In addition, antral gastritis score was significantly less in H. pylori-negative immunized monkeys than in H. pylori-positive animals, whether they were given rUre plus LT or placebo plus LT (P < 0.02 or P < 0.01, respectively). Interestingly, antral gastritis was also significantly less in H. pylori-positive animals given rUre plus LT than in H. pylori-positive animals given placebo plus LT (P < 0.02). However, quantitative cultures did not demonstrate significant differences between the two latter groups. It is concluded that oral administration of rUre vaccine plus LT significantly protects nonhuman primates against H. pylori infection while not causing undesirable side effects.     

A Novel Live Vector Group A Streptococcal emm Type 9 Vaccine Delivered Intranasally Protects Mice against Challenge Infection with emm Type 9 Group A Streptococci

The availability of a protective vaccine against Streptococcus pyogenes (group A Streptococcus [GAS]) is a priority for public health worldwide. Here, we have generated six live vaccine strains, each engineered to express an N-terminal M protein peptide from one of six of the most prevalent emm types of GAS (M1, M2, M4, M9, M12, and M28). The vaccine strains are based on a food-grade Lactococcus lactis strain and do not bear any antibiotic resistance. Mice immunized with the vaccine strain expressing the M9 peptide (termed here the L. lactis M9 strain) showed high titers of serum antibodies when delivered intranasally. Mice immunized with the L. lactis M9 strain were protected against infection after intranasal challenge with type 9 streptococci. Several parameters of disease, such as weight loss, body temperature, colony counts in mouth washes, and lung histology, were significantly improved in immunized mice compared to naive control mice. Our results indicate that intranasal delivery of the L. lactis M9 strain live bacterial vaccine induced GAS-specific IgG titers, prevented pharyngeal colonization of GAS, and protected mice from disease upon challenge. The design of this vaccine prototype may provide a lower cost alternative to vaccines comprised of purified recombinant proteins.     

A Euthymic Hairless Mouse Model of Helicobacter pylori Colonization and Adherence to Gastric Epithelial Cells In Vivo

The hairless mouse strain NS:Hr/ICR was examined as a potential small animal model of Helicobacter pylori colonization, adherence to gastric epithelial cells in vivo, and gastritis. Among several small animals tested, NS:Hr/ICR mice proved to be the most highly susceptible to H. pylori infection. Challenge with clinical isolates of H. pylori consisting of either phenotype I or II (VacA and CagA positive and negative, respectively) resulted in colonization by mucus-resident and epithelial cell-adherent bacterial populations. Cell-adherent bacteria resisted 80 cycles of top-speed vortex washing and were recovered only by homogenization of serially washed glandular stomach tissue, indicating intimate association with the mucosal surface. Immunoperoxidase staining of paraffin sections of gastric tissue from infected mice revealed H. pylori antigens localized in the glandular region of the mucosa, with some colonized areas seen in the vicinity of submucosal mononuclear cell infiltration. The latter inflammatory reaction was observed as a function of the H. pylori phenotype (only type I induced inflammation) and the challenge dose (only those mice challenged with 10⁸ CFU or higher showed the reaction). The NS:Hr/ICR strain of mice is a suitable miniature model of H. pylori infection and may prove useful in the quest for an efficacious mode of treatment for this common infection in humans.     

Lactobacillus johnsonii La1 Attenuates Helicobacter pylori-Associated Gastritis and Reduces Levels of Proinflammatory Chemokines in C57BL/6 Mice

In clinical settings, Lactobacillus johnsonii La1 administration has been reported to have a favorable effect on Helicobacter pylori-associated gastritis, although the mechanism remains unclear. We administered, continuously through the water supply, live La1 to H. pylori-infected C57BL/6 mice and followed colonization, the development of H. pylori-associated gastritis in the lamina propria, and the levels of proinflammatory chemokines macrophage inflammatory protein 2 (MIP-2) and keratinocyte-derived cytokine (KC) in the serum and gastric tissue over a period of 3 months. We documented a significant attenuation in both lymphocytic (P = 0.038) and neutrophilic (P = 0.003) inflammatory infiltration in the lamina propria as well as in the circulating levels of anti-H. pylori immunoglobulin G antibodies (P = 0.003), although we did not observe a suppressive effect of La1 on H. pylori colonizing numbers. Other lactobacilli, such as L. amylovorus DCE 471 and L. acidophilus IBB 801, did not attenuate H. pylori-associated gastritis to the same extent. MIP-2 serum levels were distinctly reduced during the early stages of H. pylori infection in the La1-treated animals, as were gastric mucosal levels of MIP-2 and KC. Finally, we also observed a significant reduction (P = 0.046) in H. pylori-induced interleukin-8 secretion by human adenocarcinoma AGS cells in vitro in the presence of neutralized (pH 6.8) La1 spent culture supernatants, without concomitant loss of H. pylori viability. These observations suggest that during the early infection stages, administration of La1 can attenuate H. pylori-induced gastritis in vivo, possibly by reducing proinflammatory chemotactic signals responsible for the recruitment of lymphocytes and neutrophils in the lamina propria.     

Galectin-3 Plays an Important Role in Innate Immunity to Gastric Infection by Helicobacter pylori

We studied the role of galectin-3 (Gal3) in gastric infection by Helicobacter pylori. We first demonstrated that Gal3 was selectively expressed by gastric surface epithelial cells and abundantly secreted into the surface mucus layer. We next inoculated H. pylori Sydney strain 1 into wild-type (WT) and Gal3-deficient mice using a stomach tube. At 2 weeks postinoculation, the bacterial cells were mostly trapped within the surface mucus layer in WT mice. In sharp contrast, they infiltrated deep into the gastric glands in Gal3-deficient mice. Bacterial loads in the gastric tissues were also much higher in Gal3-deficient mice than in WT mice. At 6 months postinoculation, H. pylori had successfully colonized within the gastric glands of both WT and Gal3-deficient mice, although the bacterial loads were still higher in the latter. Furthermore, large lymphoid clusters mostly consisting of B cells were frequently observed in the gastric submucosa of Gal3-deficient mice. In vitro, peritoneal macrophages from Gal3-deficient mice were inefficient in killing engulfed H. pylori. Furthermore, recombinant Gal3 not only induced rapid aggregation of H. pylori but also exerted a potent bactericidal effect on H. pylori as revealed by propidium iodide uptake and a morphological shift from spiral to coccoid form. However, a minor fraction of bacterial cells, probably transient phase variants of Gal3-binding sugar moieties, escaped killing by Gal3. Collectively, our data demonstrate that Gal3 plays an important role in innate immunity to infection and colonization of H. pylori.     

Highly attenuated Bordetella pertussis strain BPZE1 as a potential live vehicle for delivery of heterologous vaccine candidates

Bordetella pertussis, the causative agent of whooping cough, is a promising and attractive candidate for vaccine delivery via the nasal route, provided that suitable attenuation of this pathogen has been obtained. Recently, the highly attenuated B. pertussis BPZE1 strain has been described as a potential live pertussis vaccine for humans. We investigated here the use of BPZE1 as a live vehicle for heterologous vaccine candidates. Previous studies have reported the filamentous hemagglutinin (FHA), a major B. pertussis adhesin, as a carrier to express foreign antigens in B. pertussis. In this study, we also examined the BrkA autotransporter as a surface display system. Three copies of the neutralizing peptide SP70 from enterovirus 71 (EV71) were fused to FHA or in the passenger domain of BrkA, and each chimera was expressed in BPZE1. The FHA-(SP70)₃ and BrkA-(SP70)₃ chimeras were successfully secreted and exposed at the bacterial surface, respectively. Nasal administration of the live recombinant strains triggered a strong and sustained systemic anti-SP70 antibody response in mice, although the titers and neutralizing activities against EV71 were significantly higher in the sera of mice immunized with the BrkA-(SP70)₃-producing strain. These data indicate that the highly attenuated BPZE1 strain is a potential candidate for vaccine delivery via the nasal route with the BrkA autotransporter as an alternative to FHA for the presentation of the heterologous vaccine antigens.