Chemokine-adjuvanted electroporated DNA vaccine induces substantial protection from simian immunodeficiency virus vaginal challenge.

There have been encouraging results for the development of an effective HIV vaccine. However, many questions remain regarding the quality of immune responses and the role of mucosal antibodies. We addressed some of these issues by using a simian immunodeficiency virus (SIV) DNA vaccine adjuvanted with plasmid-expressed mucosal chemokines combined with an intravaginal SIV challenge in rhesus macaque (RhM) model. We previously reported on the ability of CCR9 and CCR10 ligand (L) adjuvants to enhance mucosal and systemic IgA and IgG responses in small animals. In this study, RhMs were intramuscularly immunized five times with either DNA or DNA plus chemokine adjuvant delivered by electroporation followed by challenge with SIVsmE660. Sixty-eight percent of all vaccinated animals (P<0.01) remained either uninfected or had aborted infection compared with only 14% in the vaccine naïve group. The highest protection was observed in the CCR10L chemokines group, where six of nine animals had aborted infection and two remained uninfected, leading to 89% protection (P<0.001). The induction of mucosal SIV-specific antibodies and neutralization titers correlated with trends in protection. These results indicate the need to further investigate the contribution of chemokine adjuvants to modulate immune responses and the role of mucosal antibodies in SIV/HIV protection.

Humoral immune response to keyhole limpet haemocyanin, the protein carrier in cancer vaccines.

Keyhole limpet haemocyanin (KLH) appears to be a promising protein carrier for tumor antigens in numerous cancer vaccine candidates. The humoral immune response to KLH was characterized at the single-cell level with ELISPOT combined with separations of cell populations according to their expression of homing receptors (HRs). The analysis of HR expressions is expected to reveal the targeting of the immune response in the body. Eight orally primed and four nonprimed volunteers received KLH-vaccine subcutaneously. Circulating KLH-specific plasmablasts were found in all volunteers, 60 KLH-specific plasmablasts/10(6) PBMC in the nonprimed and 136/10(6) in the primed group. The proportion of L-selectin(+) plasmablasts proved high and integrin α(4)ß(7) (+) low. KLH serving as protein carrier in several vaccines, the homing profile of KLH-specific response may be applicable to the cancer antigen parts in the same vaccines. The present data reflect a systemic homing profile, which appears advantageous for the targeting of immune response to cancer vaccines.

[Treatment of IgA nephropathy]. / Lécba IgA nefropatie.

IgA nephropathy is the most common cause of chronic renal failure among primary glomerulonephritides. During the last decade, there was a remarkable progress in understanding its pathogenesis. A number of therapeutic trials has been published that shed light on its treatment. ACEI and AT1R antagonists (sartans) or their combination represent the cornerstone of therapy of IgA nephropathy. However, this treatment is not given to patients having optimal blood pressure, normal glomerular filtration rate, proteinuria less than 0.3 g/24 h, mild abnormalities in renal biopsy, and stationary course of the disease. The medication is administered in a maximal tolerated dose to patients with active, progressing disease. ACEI and AT1R antagonists are also drugs of the first choice in patients with proteinuric IgA nephropathy. However, if proteinuria does not decrease significantly within 3 months from the beginning of this treatment, administration of glucocorticosteroids is recommended. On the basis of prospective, controlled clinical trials and metaanalyses of other therapeutic studies, it has been concluded that glucocorticosteroids decrease proteinuria and slow down the decline of renal function. A complete remission of proteinuria is the aim of the treatment. The effectiveness of cyclophosphamide in active forms of IgA nephropathy, described in some studies, was not confirmed by metaanalyses. Nevertheless, cyclophosphamide may be effective in some patients with rapidly deteriorating renal function and active morphological findings with cellular extracapillary proliferation.

Role of aberrant glycosylation of IgA1 molecules in the pathogenesis of IgA nephropathy.

Studies of the properties of immune complexes (IC) in the circulation, urine, and mesangium of IgA nephropathy (IgAN) patients have provided data relevant to the pathogenesis of this disease. IC contain predominantly polymeric IgA1 molecules which are deficient in galactose (Gal) residues on O-linked glycan chains in the hinge region (HR) of their heavy (H) chains. As a result of this aberrancy, a novel antigenic determinant(s) involving N-acetylgalactosamine (GalNAc) and perhaps sialic acid (SA) of O-linked glycans is generated and recognized by naturally occurring GalNAc-specific antibodies. Thus, IC in IgAN consist of Gal-deficient IgA1 molecules as an antigen, and GalNAc-specific IgG and/or IgA1 as an antibody. IgG antibodies to Gal-deficient IgA1 are probably induced by cross-reactive microbial antigens; they are present at variable levels not only in humans with or without IgAN but also in many phylogenetically diverse vertebrate species. Incubation of human mesangial cells with IC from sera of IgAN patients indicated that stimulation of cellular proliferative activity was restricted to the large (>800 kDa) complexes. These findings suggest that experimental approaches that prevent the formation of large Gal-deficient IgA1-IgG IC may be applied ultimately in an immunologically mediated therapy.

Patients with IgA nephropathy have increased serum galactose-deficient IgA1 levels.

Immunoglobulin A (IgA) nephropathy is the most prevalent form of glomerulonephritis worldwide. A renal biopsy is required for an accurate diagnosis, as no convenient biomarker is currently available. We developed a serological test based upon the observation that this nephropathy is characterized by undergalactosylated IgA1 in the circulation and in mesangial immune deposits. In the absence of galactose, the terminal saccharide of O-linked chains in the hinge region of IgA1 is terminal or sialylated N-acetylgalactosamine. A lectin from Helix aspersa, recognizing N-acetylgalactosamine, was used to develop an enzyme-linked immunosorbent assay that measures galactose-deficient IgA1 in serum. The median serum lectin-binding IgA1 level was significantly higher for 153 Caucasian adult patients with IgA nephropathy without progression to end-stage renal disease as compared with that for 150 healthy Caucasian adult controls. As the lectin-binding IgA1 levels for the controls were not normally distributed, the 90th percentile was used for determination of significant elevation. Using a value of 1076 U/ml as the upper limit of normal, 117 of the 153 patients with IgA nephropathy had an elevated serum lectin-binding IgA1 level. The sensitivity as a diagnostic test was 76.5%, with specificity 94%; the positive predictive value was 88.6% and the negative predictive value was 78.9%. We conclude that this lectin-binding assay may have potential as a noninvasive diagnostic test for IgA nephropathy.

[Immunoglobulin A and renal diseases]. / Imunoglobulin A a choroby ledvin.

Immunoglobulin A (IgA) is a dominant immunoglobulin of the mucosal surfaces, but it is also present in plasma. In men and in hominoid primates it occurs in two subclasses: IgA1 and IgA2. Circulating IgA is mostly IgA1 monomer, secretory IgA is mostly dimer or tetramer with varying content of IgA1 and IgA2 on individual mucosal surfaces. Its main physiological function is a defence of the mucosal surfaces against infection. It binds either specifically to bacterial antigens or through its O-linked glycosidic chains, it binds to the lectins of bacterial cells and thus protects mucosal surfaces against bacterial adhesion and infection. On each of its heavy chain, IgA1 has at least two N-glycosidically bound oligosaccharides and 3 to 5 O-linked side-chains. The occurrence of O-glycosidically bound glycans on other circulating immunoglobulins is rare. An aberrant composition of these glycans may be an antigenic determinant for naturally occurring circulating antibodies. The resulting IgA-containing immune complexes, which are deposited in the glomeruli, may be the cause of IgA nephropathy. IgA glomerular deposits are also frequently present in many other primary and systemic glomerulonephritides.

Heterogeneity of IgG glycosylation in adult periodontal disease.

Periodontal disease is a chronic inflammatory disease of bacterial etiology. In many other chronic inflammatory diseases, IgG glycans are galactose-deficient and thus capable of complement activation through the lectin pathway. In this study, we examined whether IgG in serum and gingival crevicular fluid, and IgG locally produced by plasma cells in gingiva of periodontal disease patients, display altered glycosylation. We developed a lectin-ELISA to measure levels of galactose-deficient IgG in the fluids and immunofluorescence staining to detect galactose-deficient IgG-producing cells in gingiva. Our results indicated higher levels of galactose-deficient IgG in sera and gingival crevicular fluid from periodontal disease patients, compared with levels in healthy controls. Furthermore, gingivae from periodontal disease patients exhibited infiltration of IgG-producing plasma cells; many of them contained galactose-deficient IgG in the cytoplasm. Analysis of our data suggests that IgG secreted by B-cells was aberrantly glycosylated, which resulted in the production of pro-inflammatory galactose-deficient IgG.

Interaction of mucosal microbiota with the innate immune system.

Organisms live in continuos interaction with their environment; this interaction is of vital importance but at the same time can be life threatening. The largest and most important interface between the organism and its environment is represented by surfaces covered with epithelial cells. Of these surfaces, mucosae comprise in humans approximately 300 m2, and the skin covers approximately 1.8 m2 surface of the human body. Mucosal tissues contain two effector arms of the immune system, innate and adaptive, which operate in synergy. Interaction with commensal bacteria, which outnumber the nucleated cells of our body, occurs physiologically on epithelial surfaces; this interaction could pose the risk of inflammation. The mucosal immune system has developed a complex network of regulatory signalling cascades that is a prerequisite for proper activation but also for a timely inactivation of the pathway. As demonstrated in gnotobiotic animal models of human diseases, impaired regulation of mucosal responses to commensal bacteria plays an important role in the development of several inflammatory and autoimmune diseases.

[IgA nephropathy. Significance of immunoglobulin A glycosylation in pathogenesis and clinical presentation]. / IgA nefropatie. Význam glykozylace imunoglobulin A pro patogenezi a její klinický obraz.

Human immunoglobulin A is represented by two structurally and functionally distinct subclasses: IgA1 and IgA2. IgA1, which is almost exclusively present in the mesangial deposits in IgA nephropathy patients, contains in its hinge region three to five O-lined carbohydrate chains. A fraction of IgA1 molecules in the circulation of IgA nephropathy patients exhibits aberrant glycosylation. As a result of changes in glycosylation, the neoepitopes represented by glycans are exposed and recognized by naturally occurring antibodies with antiglycan specifciities, and immune complexes are generated. The deposits of these immune complexes in the glomerular mesangia elicit inflammatory response known as IgA nephropathy. Epidemiological studies have shown that dominant hematuria, either isolated or combined with mild proteinuria, is the most frequent urinary syndrome in glomerulonephritis. The morphologic finding of this syndrome is most frequently IgA nephropathy. Originally considered a benign disease, IgA nephropathy is now recognized as a frequent cause of chronic renal failure. The progression is signalized by increasing proteinuria and hypertension. Therefore, a control of blood pressure and lowering of proteinuria remain the corner-stones of the treatment. Angiotensin converting enzyme inhibitors and AT1 blockers may lower both blood pressure and proteinuria and are now increasingly promoted even for treatment of normotensive patients. Steroids are administered to patients with severe proteinuria. High-doses of fish oil seem to slow down the rate of renal failure.

Induction of specific immune responses in the genital tract of women after oral or rectal immunization and rectal boosting with Salmonella typhi Ty 21a vaccine.

The purpose of this study was to determine the efficacy of intestinal tract immunization in the induction of specific antibodies in human female genital tract secretions. Live attenuated typhoid vaccine Ty 21a was administered to three groups of healthy female volunteers, who were not using hormonal contraceptives. Group 1 included 15 women vaccinated orally. Group 2 included seven of the same women, who were vaccinated rectally 6 months later. Group 3 included 11 volunteers, who were vaccinated rectally. Salmonella-specific antibodies of IgG and IgA were measured in vaginal lavage and cervical mucus after oral or rectal primary vaccination. Salmonella-specific antibodies measured 1 month after rectal booster vaccination demonstrated significant increases in vaginal fluids and cervical mucus and were dominated by IgA. These results indicate that specific antibodies in the human female genital tract induced by primary vaccination can be enhanced by subsequent rectal administration of vaccines.

Urethral cytokine and immune responses in Chlamydia trachomatis-infected males.

Penile urethral swabs collected from PCR-confirmed Chlamydia trachomatis-infected, C. trachomatis-uninfected, and non-C. trachomatis-infected, nongonococcal urethritis-infected males were analyzed for cytokine, total immunoglobulin (Ig), and specific antibody levels by enzyme-linked immunosorbent assay. Differential cellular components of the swab transport medium were also enumerated for the same groups. Although low, the levels of C. trachomatis-specific IgA and IgG antibodies and interleukin 8 cytokine were significantly higher in C. trachomatis-infected individuals. There were no significant differences in the levels of seven additional cytokines evaluated.

Mechanisms of immune tolerance to food antigens in humans.

Immune responses and the mechanisms of tolerance to the common dietary antigens bovine gamma globulin (BGG), ovalbumin (OVA), and soybean protein were evaluated in normal human volunteers. Humoral and T cell proliferative responses to these antigens were measurable but low, consistent with immune tolerance. There were limited correlations between responses in the systemic and mucosal compartments, and in general the responses to one dietary antigen could not predict the response to another. T cell proliferation to dietary antigens increased significantly by addition of recombinant human interleukin-2 (rhuIL-2). Peripheral blood mononuclear cells stimulated with BGG or OVA expressed IL-2Ralpha chain but not IL-2 mRNA, consistent with T cell anergy. Incubation with exogenous IL-2 alone did not restore T cell proliferation to BGG or OVA. In some individuals T cell proliferation to an unrelated vaccine antigen was suppressed by addition of BGG or OVA, but could be reversed with low doses of rhuIL-2. We conclude that in humans anergy is the major mechanism of tolerance to chronic antigen feeding, and we propose that such anergic, antigen-specific T cells actively contribute to maintenance of homeostasis in the intestine in the face of massive antigen challenge.

Biodegradable block copolymers for delivery of proteins and water-insoluble drugs.

Release of several drugs from new ABA-type biodegradable thermal gels, ReGel, including proteins and conventional molecules, are presented. These are biodegradable, biocompatible polymers that demonstrate reverse thermal gelation properties. Organic solvents are not used in the synthesis, purification, or formulation of these polymers. The unique characteristics of ReGel hinge on the following two key properties: (1) ReGel is a water soluble, biodegradable polymer at temperatures below the gel transition temperature; (2) ReGel forms a water-insoluble gel once injected. This is consistent with a hydrophobically bonded gel state where all interactions are physical, with no covalent crosslinking. An increase in viscosity of approximately 4 orders of magnitude accompanies the sol--gel transition. The gel forms a controlled release drug depot with delivery times ranging from 1 to 6 weeks. ReGel's inherent ability to solubilize (400 to >2000-fold) and stabilize poorly soluble and sensitive drugs, including proteins is a substantial benefit. The gel provided excellent control of the release of paclitaxel for approximately 50 days. Direct intratumoral injection of ReGel/paclitaxel (OncoGel) results in a slow clearance of paclitaxel from the injection site with minimal distribution into any organ. Efficacies equivalent to maximum tolerated systemic dosing were observed at OncoGel doses that were 10-fold lower. Data on protein release (pGH, G-CSF, insulin, rHbsAg) and polymer biocompatibility are discussed.

A Phase I safety and immunogenicity trial of UBI microparticulate monovalent HIV-1 MN oral peptide immunogen with parenteral boost in HIV-1 seronegative human subjects.

Thirty-three HIV-seronegative adults were recruited into a Phase I safety and immunogenicity HIV-1 vaccine trial. The immunogens were as follows: a synthetic, monovalent, octameric HIV-1 MN V3 peptide in aluminum hydroxide (alum) adjuvant administered by intramuscular delivery; and a similar product encapsulated in biodegradable micro-spheres composed of co-polymers of lactic and glycolic acids, administered by the oral route. These were administered in three sequential oral doses, followed by a parenteral boost. No serious adverse experiences were observed. Oral administration of this vaccine, alone or in combination with parenteral boosting, resulted in no significant humoral, cellular, or mucosal immune responses.

In vitro exposure to highly cytopathic HIV-1 X4 strains increases expression of mucosa-associated integrins on CD4(+) T cells.

To determine whether infection with HIV-1 strains of different tropisms would influence expression of the mucosa-associated integrins alpha 4 beta 7 and alpha E beta 7 or the lymph node homing receptor L-selectin on peripheral T lymphocytes, cells were infected with the CXCR4-tropic (X4)/syncytium-inducing (SI) HIV-1(IIIB) strain or with X4/SI or CCR5-tropic (R5)/non-SI (NSI) primary human isolates. Flow cytometric analyses of CD4(+) T cells from cultures infected with HIV-1(IIIB) and one X4/SI primary HIV-1 isolate revealed a significant increase in surface expression of alpha 4 beta 7 and alpha E beta 7 12 days after infection. L-selectin expression was not significantly affected on CD4(+) T cells. However, infection with another X4/SI and two R5/NSI primary HIV-1 isolates did not significantly alter homing receptor expression on CD4(+) T cells. Since a higher degree of CD4 cytopathicity occurred in those cultures having increased integrin expression, these data suggest that significantly altered mucosal homing receptor expression on CD4(+) T cells may result as a "bystander" effect after infection with some cytopathic isolates of HIV-1.

Heterosubtypic immunity to influenza A virus infection requires B cells but not CD8+ cytotoxic T lymphocytes.

Heterosubtypic immunity (HSI), defined as protective cross-reactivity to lethal infection with influenza A virus of a serotype different from the virus initially encountered, is thought to be mediated by cross-reactive cytotoxic T lymphocytes (CTL). This study provides direct evidence for the role of effector CTL versus B cells in HSI in mice with a targeted disruption in the alpha chain of CD8 molecule (CD8(+) T cell deficient) or the immunoglobulin mu heavy chain (B cell deficient), respectively. CD8(+) T cell-deficient mice developed complete HSI. These mice displayed normal humoral immune responses, as determined by titers of subtype cross-reactive antibodies and virus-neutralizing antibodies specific for the immunizing influenza strain. In contrast, HSI was not observed in B cell-deficient mice, although these mice could mount cross-reactive CTL responses. These results show that B cells are required for HSI and provide new insight into the mechanisms of HSI, with significant implications in vaccine development.

Homeostasis of the mucosal immune system: human milk and lactation.

External secretions, including milk, contain large amounts of immunoglobulins (Ig) and innate factors of humoral immunity The Ig isotype distribution, their origin, transport, and absorbability from the gut lumen differ greatly among various species. Milk of primates contains mainly secretory IgA (sIgA) which has unique biological properties. The functional advantages of sIgA compared with other Ig isotypes include its resistance to proteolysis, presence of 4 to 8 antigen-binding sites, carbohydrate moiety-mediated antiadherence properties, and inhibition of complement activation. Although IgA exhibits its protective effect mainly on mucosal surfaces, internalization by cells expressing various IgA receptors suggests additional protective functions.

Expression of the recombinant human immunoglobulin J chain in Escherichia coli.

Selective transport of polymeric (p) immunoglobulins (Ig) of IgA and IgM isotypes into external secretions by pIg receptor-mediated mechanism depends on the incorporation of joining (J) chain into the polymers. Until now, availability of a free J chain for immunological and biophysical studies has been limited to preparations of denatured J chain forms with moderate yield. Here we report that a recombinant J chain (rJ) can be over-expressed as a soluble fusion protein with thioredoxin using a modified vector pET32 in Escherichia coli. An intact J chain was released by digestion with IgA1 protease from Neisseria gonorrhoeae and isolated in a good yield with immunological and biochemical properties similar to those of J chain obtained by chemical cleavage from pIgA.

Gamma interferon is not required for mucosal cytotoxic T-lymphocyte responses or heterosubtypic immunity to influenza A virus infection in mice.

Heterosubtypic immunity (HSI) is defined as cross-protection against influenza virus of a different serotype than the virus initially encountered and is thought to be mediated by influenza virus-specific cytotoxic T lymphocytes (CTL). Since gamma interferon (IFN-gamma) stimulates cytotoxic cells, including antigen-specific CTL which may control virus replication by secretion of antiviral cytokines such as tumor necrosis factor alpha and IFN-gamma, we have investigated the mechanism of HSI by analyzing the role of IFN-gamma for HSI in IFN-gamma gene-deleted (IFN-gamma(-/-)) mice. It has been reported that IFN-gamma is not required for recovery from primary infection with influenza virus but is important for HSI. Here, we conclusively show that IFN-gamma is not required for induction of secondary influenza virus-specific CTL responses in mediastinal lymph nodes and HSI to lethal influenza A virus infection. Although T helper 2 (Th2)-type cytokines were upregulated in the lungs of IFN-gamma(-/-) mice after virus challenge, either Th1- or Th2-biased responses could provide heterosubtypic protection. Furthermore, titers of serum-neutralizing and cross-reactive antibodies to conserved nucleoprotein in IFN-gamma(-/-) mice did not differ significantly from those in immunocompetent mice. These results indicate that lack of IFN-gamma does not impair cross-reactive virus-specific immune responses and HSI to lethal infection with influenza virus. Our findings provide new insight for the mechanisms of HSI and should be valuable in the development of protective mucosal vaccines against variant virus strains, such as influenza and human immunodeficiency virus.

Role of nuclear factor-kappaB in the expression by tumor necrosis factor-alpha of the human polymeric immunoglobulin receptor (plgR) gene.

We analyzed the mechanism of human polymeric immunoglobulin receptor (pIgR) gene upregulation by tumor necrosis factor (TNF)-alpha. Northern blot analysis showed that the expression of pIgR mRNA was enhanced by TNF-alpha stimulation. This activation was completely inhibited by RNA polymerase or protein synthesis inhibitors, suggesting that the regulation of pIgR gene expression depends on de novo RNA and protein synthesis. Furthermore, the stimulation of pIgR mRNA by TNF-alpha was decreased by pyrrolidinedithiocarbamate and L-1-4'-tosylamino-phenylethyl-chloromethyl ketone, which are known nuclear factor (NF)-kappaB inhibitors. For further analysis of gene regulation, we cloned and sequenced the 1.5-kb 5'-flanking region of the pIgR gene. In the upstream region, we found two NF-kappaB-binding motifs (named kappaB1 and kappaB2 from the 5' region). An electrophoretic mobility shift assay indicated that two components of the NF-kappaB/Re1 family, p50 and p65, bound with higher affinity to the KB2 element than to the kappaB1 element. We also analyzed plgR gene expression using reporter plasmids expressing the firefly luciferase gene. Stimulation by TNF-alpha significantly activated the pIgR gene promoter, as a 775-bp upstream region of the pIgR gene increased luciferase gene expression in cells treated with TNF-alpha. The activation of promoter activity by TNF-alpha was abolished when a mutation was inserted into kappaB1 or kappaB2. These data indicated that pIgR gene expression induced by TNF-alpha is transcriptionally regulated via activation of NF-kappaB. In addition, there is a possibility that another factor may act in concert with NF-kappaB.