Janet Wee

M-Cell Targeting of Whole Killed Bacteria Induces Protective Immunity against Gastrointestinal Pathogens

ABSTRACT As the majority of human pathogens infect via a mucosal surface, delivery of killed vaccines by mucosal routes could potentially improve protection against many such organisms. Our ability to develop effective killed mucosal vaccines is inhibited by a lack of adjuvants that are safe and effective in humans. The Ulex europaeus agglutinin I (UEA-I) lectin specifically binds M cells lining the murine gastrointestinal tract. We explored the potential for M-cell-targeted vaccination of whole, killed Helicobacter pylori, the main causative agent of peptic ulcer disease and gastric cancer, and Campylobacter jejuni, the most common cause of diarrhea. Oral delivery of UEA-I-agglutinated H. pylori or C. jejuni induced a significant increase in both serum and intestinal antibody levels. This elevated response (i) required the use of whole bacteria, as it did not occur with lysate; (ii) was not mediated by formation of particulate clumps, as agglutination with a lectin with a different glycan specificity had no effect; and (iii) was not due to lectin-mediated, nonspecific immunostimulatory activity, as UEA-I codelivery with nonagglutinated bacteria did not enhance the response. Vaccination with UEA-I-agglutinated, killed whole H. pylori induced a protective response against subsequent live challenge that was as effective as that induced by cholera toxin adjuvant. Moreover, vaccination against C. jejuni by this approach resulted in complete protection against challenge in almost all animals. We believe that this is the first demonstration that targeting of whole killed bacteria to mucosal M cells can induce protective immunity without the addition of an immunostimulatory adjuvant.

Combined mucosal and systemic immunity following pulmonary delivery of ISCOMATRIX adjuvanted recombinant antigens.

Deep pulmonary delivery of an influenza ISCOMATRIX vaccine has previously been shown to induce a combined mucosal and systemic antibody response. To explore whether this combined response is influenced by intrinsic properties of the component antigen, we examined the efficacy of deep pulmonary delivery of ISCOMATRIX vaccines containing different recombinant antigens, specifically gB glycoprotein from cytomegalovirus and a fragment of catalase from Helicobacter pylori. Both these vaccines induced antigen-specific mucosal and systemic immunity, as well as antigen-specific proliferative cellular responses. Pulmonary immunisation with ISCOMATRIX vaccines may therefore be a generic way of inducing combined systemic and mucosal immunity.

Protease-Activated Receptor-1 Down-regulates the Murine Inflammatory and Humoral Response to Helicobacter pylori

BACKGROUND & AIMS Helicobacter pylori infection results in a diversity of pathologies, from asymptomatic gastritis to adenocarcinoma. The reason for these diverse outcomes is multifactorial and includes host factors that regulate severity of Helicobacter-induced gastritis. Protease-activated receptors (PAR) are environmental sensors that can detect tissue damage and pathogens. Whereas PAR-2 has proinflammatory activity and PAR-1 can protect the gastric mucosa against chemical damage, neither has previously been examined for their potential roles in regulating Helicobacter pathogenesis. METHODS PAR-1(-/-), PAR-2(-/-), and wild-type mice were infected with H pylori for up to 2 months then colonization levels determined by colony-forming assay, gastritis by histology, and serum antibody levels by enzyme-linked immunosorbent assay. Responsiveness of primary epithelial cells to PAR-1 activation was assessed by calcium mobilization assay. Primary epithelial cells, macrophages, and dendritic cells were cocultured with H pylori and nuclear factor (NF)-kappaB, and cytokine secretion was determined by enzyme-linked immunosorbent assay. RESULTS Two months postinfection, H pylori levels were significantly reduced in PAR-1(-/-) and increased in PAR-2(-/-) mice. This effect on colonization was inversely correlated with inflammation severity. Infection of PAR-1(-/-) mice induced an increased serum antibody response. Primary epithelial cells were activated by a PAR-1-activating peptide. H pylori stimulation of primary epithelial cells, but not macrophages or dendritic cells, from PAR-1(-/-) mice induced increased levels of NF-kappaB and the proinflammatory cytokine macrophage-inflammatory protein (MIP)-2. PAR-1 also down-regulated MIP-2 secretion in response to cag pathogenicity island activity. CONCLUSIONS PAR-1 protects the host against severe Helicobacter-induced gastritis. This may be mediated by suppressing the production of proinflammatory cytokines such as MIP-2.

Long-Term Antibody and Immune Memory Response Induced by Pulmonary Delivery of the Influenza Iscomatrix Vaccine

ABSTRACT Pulmonary delivery of an influenza Iscomatrix adjuvant vaccine induces a strong systemic and mucosal antibody response. Since an influenza vaccine needs to induce immunological memory that lasts at least 1 year for utility in humans, we examined the longevity of the immune response induced by such a pulmonary vaccination, with and without antigen challenge. Sheep were vaccinated in the deep lung with an influenza Iscomatrix vaccine, and serum and lung antibody levels were quantified for up to 1 year. The immune memory response to these vaccinations was determined following antigen challenge via lung delivery of influenza antigen at 6 months and 1 year postvaccination. Pulmonary vaccination of sheep with the influenza Iscomatrix vaccine induced antigen-specific antibodies in both sera and lungs that were detectable until 6 months postimmunization. Importantly, a memory recall response following antigenic challenge was detected at 12 months post-lung vaccination, including the induction of functional antibodies with hemagglutination inhibition activity. Pulmonary delivery of an influenza Iscomatrix vaccine induces a long-lived influenza virus-specific antibody and memory response of suitable length for annual vaccination against influenza.

Local expression of IDO, either alone or in combination with CD40Ig, IL10 or CTLA4Ig, inhibits indirect xenorejection responses.

Abstract:  Background:  To overcome cell‐mediated xenorejection by transgenic expression of immunomodulatory molecules by a graft, it is likely that expression of multiple molecules will be required. Previous studies support the use of the immunomodulatory agents indoleamine 2,3‐dioxygenase (IDO), CD40Ig, interleukin 10 (IL10), and CTLA4Ig for suppression of rejection responses. We examined the effects of local expression of these molecules by a porcine cell line (PIEC) on indirect murine xenorejection responses in vitro and in vivo.

Lentiviral expression of CTLA4Ig inhibits primed xenogeneic lymphocyte proliferation and cytokine responses

Mulley WR, Wee JL, Christiansen D, Milland J, Ierino FL, Sandrin MS. Lentiviral expression of CTLA4Ig inhibits primed xenogeneic lymphocyte proliferation and cytokine responses.
Xenotransplantation 2006; 13: 248–252.

Production and purification of human indoleamine 2,3-dioxygenase (HuIDO) protein in a baculovirus expression system and production and characterization of egg yolk antibody against the purified HuIDO

The human indoleamine 2,3‐dioxygenase (HuIDO) baculoviral construct, for expression of HuIDO protein with a hexa‐histidine and FLAG (DYKDDDDK) tag, was produced using the BacPAK Baculovirus Expression System. HuIDO baculovirus was used to infect Sf21 insect cells to produce functionally active protein in large amounts. Conditions for protein purification by metal affinity chromatography were determined and optimized. Addition of haemin ensured optimal activity of the purified heme‐containing oxygenase. The soluble purified protein was used to immunize a chicken to produce large quantities of polyclonal IgY against HuIDO. The anti‐HuIDO IgY antibody specifically detected HuIDO produced by a range of cell types including transfectants and native HuIDO expression induced in IFN‐γ‐stimulated cells. The antibody detected HuIDO in cell lysates by western blotting and in the cytoplasm of cells by microscopy. The antibody was unable to block the function of the enzyme, indicating that this antibody binds outside the active site of HuIDO.

Did transmission of Helicobacter pylori from humans cause a disease outbreak in a colony of Stripe-faced Dunnarts (Sminthopsis macroura)?

Since the discovery that Helicobacter pylori causes a range of pathologies in the stomachs of infected humans, it has become apparent that Helicobacters are found in a diverse range of animal species where they are frequently associated with disease. In 2003 and 2004, there were two outbreaks of increased mortality associated with gastric bleeding and weight-loss in a captive colony of the Australian marsupial, the Stripe-faced Dunnart (Sminthopsis macroura). The presence of gastric pathology led to an investigation of potential Helicobacter pathogenesis in these animals. Histological examination revealed the presence of gastritis, and PCR analysis confirmed the presence of Helicobacter infection in the stomachs of these marsupials. Surprisingly, sequencing of 16S rRNA from these bacteria identified the species as H. pylori and PCR confirmed the strain to be positive for the important pathogenesis factor, cagA. We therefore describe, for the first time, an apparent reverse zoonotic infection of Stripe-faced Dunnarts with H. pylori. Already prone to pathological effects of stress (as experienced during breeding season), concomitant H. pylori infection appears to be a possible essential but not sufficient co-factor in prototypic gastric bleeding and weight loss in these marsupials. The Stripe-faced Dunnart could represent a new model for investigating Helicobacter-driven gastric pathology. Infections from their human handlers, specifically of H. pylori, may be a potential risk to captive colonies of marsupials.