T cell cytokine polarity as a determinant of immunoglobulin A (IgA) glycosylation and the severity of experimental IgA nephropathy.

Immunoglobulin A (IgA) glycosylation, recognized as an important pathogenic factor in IgA nephropathy (IgAN), is apparently controlled by the polarity of T helper (Th) cytokine responses. To examine the role of cytokine polarity in IgAN, inbred mice were immunized by intraperitoneal priming with inactivated Sendai virus (SeV) emulsified in either complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA), which promote Th1- or Th2-immune response, respectively, and then boosted identically twice orally with aqueous suspensions of inactivated virus. Next, some mice were challenged intranasally with infectious SeV. Mice primed with CFA or IFA had equal reductions in nasal viral titre relative to non-immune controls, and equally increased serum levels of SeV-specific IgA antibody. Mice primed with CFA showed higher SeV-specific IgG than those with IFA. Splenocytes from mice primed with IFA produced copious amounts of interleukin (IL)-4 and IL-5, but little interferon-gamma and IL-2; those primed with CFA had reciprocal cytokine recall responses. Total serum IgA and especially SeV-specific IgA from mice primed with IFA showed a selective defect in sialylation and galactosylation. Although the frequency and intensity of glomerular deposits and haematuria did not differ, glomerulonephritis in mice primed with IFA and challenged with infectious virus was more severe than in those given CFA, as judged by serum creatinine level. We conclude that the polarity of T cell cytokines controls the pattern of IgA glycosylation and exerts direct or indirect effects on functional glomerular responses to immune complex deposition.

The role of nasal tolerance in a model of IgA nephropathy induced in mice by Sendai virus.

Mucosal antigenic exposure is implicated in pathogenesis of IgA nephropathy. Although IgG and/or IgM codeposits may promote disease, protracted mucosal antigenic exposure reduces IgG and IgM antibody, a process termed mucosal tolerance. We immunized mice intranasally with infectious or inactivated Sendai virus for 6 or 14 weeks. Anti-virus IgG remained high in mice given infectious virus for 14 weeks, but decreased after 6 weeks in mice given inactivated virus; IgA antibody remained high in both groups. Upon viral challenge, glomerular IgG and complement deposits and the frequency of hematuria, all equal after 6 weeks of immunization, were lower in mice immunized with inactivated virus for 14 weeks but remained high in mice given infectious virus; glomerular IgA increased over time in both immunized groups. Viremia in a non-tolerized immune host can promote glomerulonephritis with IgG and complement codeposits and glomerular dysfunction. These preliminary experiments may guide future, more mechanistic, investigation.

T cell cytokines determine the severity of experimental IgA nephropathy by regulating IgA glycosylation.

Hyperfunction of Th2 cells and aberrant glycosylation of IgA have been proposed independently as factors in the pathogenesis of IgA nephropathy (IgAN), the most common form of glomerulonephritis. To investigate the relationship between Th2 cytokines and IgA glycosylation in the genesis of IgAN, we induced IgAN in C3HeB and BALB/c mice by oral immunization and intranasal challenge with Sendai virus. Although both strains of mice developed microhaematuria and glomerular IgA immune deposits to similar degrees, only BALB/c mice developed significant renal insufficiency. More profound reductions of terminal galactosylation and sialylation occurred in Sendai virus-specific IgA from BALB/c versus C3HeB mice, and splenocytes from immunized BALB/c mice produced more Th2 and less Th1 cytokines compared to C3HeB mice when stimulated with antigen in vitro. Furthermore, the decreased glycosylation of IgA elicited by Th2 cytokines in vitro was blunted by the addition of IFN-gamma. We conclude that increased production of Th2 cytokines can lead to abnormalities in IgA glycosylation, which in turn promote heightened phlogistic responses to IgA immune complexes lodging in the glomerulus. We suggest that a relative or absolute increase in Th2 cytokine production in response to mucosal infection is a significant pathogenic factor in human IgAN.

Protective anti-Helicobacter immunity is induced with aluminum hydroxide or complete Freund's adjuvant by systemic immunization.

To determine whether systemic immunization against Helicobacter pylori could be achieved with an adjuvant approved for human use, the efficacy of vaccination with Helicobacter antigen in combination with aluminum hydroxide (AlOH) was evaluated in a murine model of Helicobacter infection. Immunization with antigen and AlOH induced interleukin-5-secreting, antigen-specific T cells, and immunization with antigen and complete Freund's adjuvant induced interferon-gamma-secreting, antigen-specific T cells, as determined by ELISPOT assay. Both immune responses conferred protection after challenge with either H. pylori or H. felis, as confirmed by the complete absence of any bacteria, as assessed by both histology and culture of gastric biopsy samples. Protection was antibody independent, as demonstrated with antibody-deficient muMT mice (immunoglobulin-gene knockout mice), and CD4(+) spleen T cells from immunized mice were sufficient to transfer protective immunity to otherwise immunodeficient rag1(-/-) recipients. These results suggest an alternative and potentially more expeditious strategy for development of a human-use H. pylori vaccine.

Helicobacter animal models.

In this unit, protocols for growing Helicobacter organisms on plates or in liquid cultures are presented, followed by protocols for infecting mice with Helicobacter felis and H. pylori and for infecting ferrets with H. mustelae. Also, a procedure is described for adapting an H. pylori isolate to growth in mice. Support protocols describe methods for quantifying numbers of Helicobacter organisms, and how to create a growth curve for Helicobacter cultures. One important technique in investigating Helicobacter infection is assaying the disease processes that occur in the stomach, and a protocol is provided for preparing tissue sections for this purpose. It is also important to confirm that organisms recovered from tissue samples are, in fact, Helicobacter species, and a support protocol describes morphological and biochemical tests for this purpose. Helicobacter bacteria produce large amounts of the enzyme urease, and a support protocol describes how to perform a rapid urease test on animal-tissue biopsies. Assays of Helicobacter-specific immune responses require appropriate antigens, and preparation of both Helicobacter lysates and outer-membrane proteins are detailed for use in these assays.

IgA immunodeficiency leads to inadequate Th cell priming and increased susceptibility to influenza virus infection.

IgA is considered to be the principal Ab involved in defense against pathogens in the mucosal compartment. Using mice with a targeted disruption in IgA gene expression (IgA(-/-) mice), we have examined the precise role of IgA in protective anti-influenza responses after intranasal vaccination. IgA(-/-) mice immunized intranasally with soluble hemagglutinin (hemagglutinin subtype 1) and neuraminidase (neuraminidase subtype 1) vaccine in the absence of adjuvant were found to be more susceptible to influenza virus infection than IgA(+/+) mice (13 vs 75% survival after virus challenge). Inclusion of IL-12 during immunization restored the protective efficacy of the vaccine to that seen in IgA(+/+) animals. IgA(-/-) mice had no detectable IgA expression, but displayed enhanced serum and pulmonary IgM and IgG Ab levels after IL-12 treatment. Assessment of T cell function revealed markedly depressed splenic lymphoproliferative responses to PHA in IgA(-/-) animals compared with IgA(+/+) mice. Furthermore, IgA(-/-) animals displayed impaired T cell priming to the H1N1 subunit vaccine, with concomitant reduction in recall memory responses due to a defect in APC function. Collectively, these results provide evidence that a major role of IgA is to facilitate presentation of Ag to mucosal T cells. IL-12 treatment can overcome IgA deficiency by providing adequate T cell priming during vaccination.

Local and systemic antibody responses in humans with Helicobacter pylori infection.

Immunization can prevent or cure an otherwise chronic helicobacter infection in several animal models despite the chronic nature of natural helicobacter infections. Differences in the antigenic specificity of the antibodies may contribute to the protection observed in these experimental animals. The goal of the present study was to compare the local and systemic antibody responses of humans with chronic Helicobacter pylori infection with those of an individual with spontaneous resolution of infection to find an immunological correlate of protection. Spontaneous resolution of infection was accompanied by a change in immunoblot profiles. Whereas a broad range of H pylori antigens was recognized in chronically infected patients (including the patient who ultimately cleared the infection spontaneously), resolution of infection in the absence of therapeutic agents resulted in the recognition of only several immunodominant antigens. The most dominant antigen was approximately 66 kDa in molecular mass. Immunoblot analysis demonstrated that these antibodies were specific for the structural subunits of the urease enzyme. These studies suggest that the success of antihelicobacter immunization may be due to the ability of vaccination to induce an immune response against antigens that are normally not immunodominant during the course of infection.

Phagocytic antigen processing and effects of microbial products on antigen processing and T-cell responses.

Processing of exogenous antigens and microbes involves contributions by multiple different endocytic and phagocytic compartments. During the processing of soluble antigens, different endocytic compartments have been demonstrated to use distinct antigen-processing mechanisms and to process distinct sets of antigenic epitopes. Processing of particulate and microbial antigens involves phagocytosis and functions contributed by phagocytic compartments. Recent data from our laboratory demonstrate that phagosomes containing antigen-conjugated latex beads are fully competent class II MHC (MHC-II) antigen-processing organelles, which generate peptide:MHC-II complexes. In addition, phagocytosed antigen enters an alternate class I MHC (MHC-I) processing pathway that results in loading of peptides derived from exogenous antigens onto MHC-I molecules, in contrast to the cytosolic antigen source utilized by the conventional MHC-I antigen-processing pathway. Antigen processing and other immune response mechanisms may be activated or inhibited by microbial components to the benefit of either the host or the pathogen. For example, antigen processing and T-cell responses (e.g. Th1 vs Th2 differentiation) are modulated by multiple distinct microbial components, including lipopolysaccharide, cholera toxin, heat labile enterotoxin of Escherichia coli, DNA containing CpG motifs (found in prokaryotic and invertebrate DNA but not mammalian DNA) and components of Mycobacterium tuberculosis.

Animal models for gastric Helicobacter immunology and vaccine studies.

Over the last decade animal models have been used extensively to investigate disease processes and therapy for Helicobacter pylori infections. The H. pylori animal models which have been used in pathogenesis and vaccine studies include the gnotobiotic pig, non-human primates, cats, dogs, and several species of rodents including mice, rats, gerbils and guinea pigs. H. felis infection of mice and H. mustelae infection of ferrets have also been used. Recently, investigators have begun using transgenic mice and gene-targeted 'knock-out' mice to investigate Helicobacter infections. Each of these animal models has distinct advantages and disadvantages which are discussed in this minireview. The choice of an animal model is dictated by factors such as cost and an understanding of how each model will or will not allow fulfillment of experimental objectives.

Replication and budding of simian immunodeficiency virus in polarized epithelial cells.

Simian immunodeficiency virus (SIV) infection of primates provides an important model for infection of humans by HIV. Since mucosal epithelium is likely to be an important portal of entry, we decided to study aspects of the interaction of SIV with epithelial cells. SIV was shown to produce virus efficiently in polarized epithelial cells (Vero C1008) transfected with SIVmac239 proviral DNA. The virus titer in the epithelial cell culture fluid reached 10(3) TCID50/ml at day 3 posttransfection. Initially after transfected epithelial cells were plated on a permeable membrane, virus budded at both the apical and the basolateral domains. However, after the cells formed a tight monolayer, 95-100% of the virus particles budded basolaterally, as assessed by release of p27 antigen into the fluid above and below the monolayer. This finding was confirmed by electron microscopy, which showed that the mature virus budded basolaterally in polarized cells. After introduction of the CD4 gene into Vero cells by a retrovirus vector, polarizable cells were able to be infected by cell-free SIVmac239 virus. The virus titer reached 10(4) TCID50/ml in culture fluid and virions also budded basolaterally, the same as the virus from transfected cells. Two viruses (SIVmac1A11 and SIVmac251) that contain truncated TMgp28 instead of TMgp41 also budded basolaterally. Furthermore, we found that HIV-1 with full-length or truncated TMgp41 also budded basolaterally.

Host response and vaccine development to Helicobacter pylori infection.

Studies in both humans and animals demonstrate that H. pylori is capable of illiciting an innate response that in part is regulated by the genetic makeup of the host. These innate responses includes stimulating immune effector mechanisms at the cellular and biochemical level resulting in the influx of neutrophils into the lamina propria and have even been shown to modify gastric acid secretion. The availability of good animal models of chronic Helicobacter infection has also allowed investigators to begin to examine how the adaptive host immune response prevents and/or exacerbates Helicobacter-induced gastroduodenal disease. The experimental H. felis/mouse model has been utilized by a number of laboratories to investigate mechanisms of host defense against chronic Helicobacter infection. This model and the more recently developed H. pylori rodent model has not only allowed investigators to confirm the feasibility of immunotherapy to prevent and/or cure Helicobacter infection but also to begin to examine how the host immune response prevents and/or exacerbates Helicobacter-induced gastroduodenal disease. Based on these studies a hypothesis is emerging that suggests that protection and/or cure from Helicobacter infection is mediated primarily by an upregulated cellular immune response which may act via an antibody independent mechanism. Paradoxically, following natural infection with H. pylori, a component of the cellular immune response also promotes chronic gastric inflammation without clearance of the organism. The recent development of reliable and reproducible H. pylori/rodent models of disease and the availability of numerous inbred strains, transgenic and knockout animals, will allow investigators to continue to explore the role the host cellular and humoral immune response plays in promoting or preventing this infection.

Qualitative and quantitative analysis of the local and systemic antibody response in mice and humans with Helicobacter immunity and infection.

Immunization can prevent or cure an otherwise chronic gastric Helicobacter infection in several different animal models. The goal of the present study was to compare the titers and specificities of local and systemic antibody responses generated by Helicobacter infection and immunization. Protective immunization results in levels of specific gastric antibody significantly lower than induced by infection. However, antibodies from protectively immunized mice preferentially recognize immunodominant proteins of 10-22 and 30 kDa. Immunoblot analysis of infected mice and humans demonstrated that the serum IgA, but not serum IgG, binding profiles yield an accurate profile of the antigenic specificity of the host's gastric IgA. Therefore, serum IgA may be useful in evaluating the immunodominant antigens at the gastric mucosa of infected persons and possibly in determining the immunogenicity of orally applied Helicobacter vaccines.

Adjuvanticity of the cholera toxin A1-based gene fusion protein, CTA1-DD, is critically dependent on the ADP-ribosyltransferase and Ig-binding activity.

The ADP-ribosylating enterotoxins, cholera toxin (CT) and Escherichia coli heat-labile toxin, are among the most powerful immunogens and adjuvants yet described. An innate problem, however, is their strong toxic effects, largely due to their promiscuous binding to all nucleated cells via their B subunits. Notwithstanding this, their exceptional immunomodulating ability is attracting increasing attention for use in systemic and mucosal vaccines. Whereas others have separated adjuvanticity from toxicity by disrupting the enzymatic activity of the A1 subunit by site-directed mutagenesis, we have constructed a nontoxic molecule that combines the full enzymatic activity of the A1 subunit with a B cell targeting moiety in a gene fusion protein, the CTA1-DD adjuvant. Despite its more selective binding properties, we found comparable adjuvant effects of the novel CTA1-DD adjuvant to that of CT. Here we unequivocally demonstrate, using a panel of mutant CTA1-DD molecules, that the immunomodulating ability of CTA1-DD is dependent on both an intact enzymatic activity and the Ig-binding ability of the DD dimer. Both agents, CT and CTA1-DD, ADP-ribosylate intact B cells. However, contrary to CT, no increase in intracellular cyclic AMP in the targeted cells was detected, suggesting that cyclic AMP may not be important for adjuvanticity. Most remarkably, CTA1-DD achieves similar immunomodulating effects to CT using a ganglioside-GM1 receptor-independent pathway for internalization.

Antibody-independent protective mucosal immunity to gastric helicobacter infection in mice.

Helicobacter pylori infection of the gastric mucosa can result in gastritis and peptic ulcer disease. Although vaccination can induce protective immunity in animal models of Helicobacter infection, the mechanism(s) of protective immunity has not been fully elucidated. This study was designed to determine whether humoral immune responses are required for protective Helicobacter immunity. IgA-deficient or immunoglobulin-deficient mice were orally immunized against Helicobacter felis and then challenged with live H. felis. Both groups were protected at levels comparable to that of wild-type mice. Additionally, inflammation was equivalent in extent and character between wild-type and antibody-deficient mice. Therefore antibody-independent mechanisms of immunity can protect mice against gastric Helicobacter infection.

Recombinant cholera toxin B subunit is not an effective mucosal adjuvant for oral immunization of mice against Helicobacter felis.

Cholera toxin is a potent oral mucosal adjuvant for enteric immunization. Several studies suggest that commercial cholera toxin B subunit (cCTB; purified from holotoxin) may be an effective non-toxic alternative for oral immunization. The present study was performed, using an infectious disease model, to determine if the oral mucosal adjuvanticity of CTB is dependent on contaminating holotoxin. Mice were orally immunized with Helicobacter felis sonicate and either cholera holotoxin, cCTB or recombinant cholera toxin B subunit (rCTB). Serum immunoglobulin G (IgG) and intestinal immunoglobulin A (IgA) antibody responses were determined and the mice were challenged with live H. felis to determine the degree of protective immunity induced. All orally immunized mice responded with serum IgG antibody titres regardless of the adjuvant used. However, only mice immunized with either holotoxin or the cCTB responded with an intestinal mucosal IgA response. Consistent with the production of mucosal antibodies, mice immunized with either holotoxin or cCTB as adjuvants were protected from challenge while mice receiving H. felis sonicate and rCTB all became infected. cCTB induced the accumulation of cAMP in mouse thymocytes at a level equal to 0.1% of that induced by holotoxin, whereas rCTB was devoid of any activity. These results indicate that CTB possesses no intrinsic mucosal adjuvant activity when administered orally. Therefore, when used as an oral adjuvant, CTB should also include small, non-toxic doses of cholera toxin.

Inhibition of class II major histocompatibility complex antigen processing by Escherichia coli heat-labile enterotoxin requires an enzymatically active A subunit.

Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT) were found to inhibit intracellular antigen processing. Processing was not inhibited by mutant LT with attenuated ADP-ribosyltransferase activity, CT B or LT B subunit, which enhanced presentation of preexisting cell surface peptide-class II major histocompatibility complex complexes. Inhibition of antigen processing correlated with A subunit ADP-ribosyltransferase activity.