Depressed type 1 cytokine synthesis by superantigen-activated CD4+ T cells of women with human papillomavirus-related high-grade squamous intraepithelial lesions.

Carcinoma of the cervix is causally related to infection with the human papillomavirus (HPV), and T cells play a pivotal role in the immune response of the host to rid itself of HPV infection. Therefore, we assessed the T-cell function of women with HPV-related cervical neoplasia against a superantigen, Staphylococcus enterotoxin B (SEB). Each woman provided a cervical brush specimen for HPV DNA testing and Papanicolaou (Pap) smears for the staging of cervical lesions. They also provided a blood specimen for determination of the ability of CD4(+) T and CD8(+) T cells to synthesize Th1 (interleukin-2 [IL-2], gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and Th2 (IL-10) cytokines in response to activation with SEB. Compared with control subjects with self-attested negative Pap smears, women with high-grade squamous intraepithelial lesions (HSIL) had significantly lower percentages of activated CD4(+) T cells that produced IL-2 (P = 0.045), IFN-gamma (P = 0.040), and TNF-alpha (P = 0.015) and a significantly lower percentage of activated CD8(+) T cells that produced IL-2 (P < 0.01). These data indicate that women with HPV-related cervical HSIL show a decrease in Th1 cytokine production by activated CD4(+) T cells and suggested that compromised T-helper functions may negatively impact the function of cytotoxic CD8(+) T cells.

Detailed analysis of sequence changes occurring during vlsE antigenic variation in the mouse model of Borrelia burgdorferi infection.

Lyme disease Borrelia can infect humans and animals for months to years, despite the presence of an active host immune response. The vls antigenic variation system, which expresses the surface-exposed lipoprotein VlsE, plays a major role in B. burgdorferi immune evasion. Gene conversion between vls silent cassettes and the vlsE expression site occurs at high frequency during mammalian infection, resulting in sequence variation in the VlsE product. In this study, we examined vlsE sequence variation in B. burgdorferi B31 during mouse infection by analyzing 1,399 clones isolated from bladder, heart, joint, ear, and skin tissues of mice infected for 4 to 365 days. The median number of codon changes increased progressively in C3H/HeN mice from 4 to 28 days post infection, and no clones retained the parental vlsE sequence at 28 days. In contrast, the decrease in the number of clones with the parental vlsE sequence and the increase in the number of sequence changes occurred more gradually in severe combined immunodeficiency (SCID) mice. Clones containing a stop codon were isolated, indicating that continuous expression of full-length VlsE is not required for survival in vivo; also, these clones continued to undergo vlsE recombination. Analysis of clones with apparent single recombination events indicated that recombinations into vlsE are nonselective with regard to the silent cassette utilized, as well as the length and location of the recombination event. Sequence changes as small as one base pair were common. Fifteen percent of recovered vlsE variants contained "template-independent" sequence changes, which clustered in the variable regions of vlsE. We hypothesize that the increased frequency and complexity of vlsE sequence changes observed in clones recovered from immunocompetent mice (as compared with SCID mice) is due to rapid clearance of relatively invariant clones by variable region-specific anti-VlsE antibody responses.

Application of in vivo induced antigen technology (IVIAT) to Bacillus anthracis.

In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42); the bacteriophage holin gene BA4074; and pagA (pXO1-110). The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets.

Transcriptional regulation of the Borrelia burgdorferi antigenically variable VlsE surface protein.

The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5' DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator.

Antibodies to flagellin indicate reactivity to bacterial antigens in IBS patients

One of the several possible causes of irritable bowel syndrome (IBS) is thought to be low-grade mucosal inflammation. Flagellin, the primary structural component of bacterial flagellae, was shown in inflammatory bowel disease patients to activate the innate and adaptive immunity. It has not yet been conclusively established if IBS patients show reactivity to luminal antigens. In 266 patients [112 IBS, 61 Crohn's disease (CD), 50 ulcerative colitis (UC) and 43 healthy controls (HC)], we measured antibodies to flagellin (FAB, types A4-Fla2 and Fla-X), anti-Saccharomyces cerevisiae antibodies (ASCA) (both ELISA), antipancreas antibodies (PAB) and perinuclear antineutrophil cytoplasmatic antibodies (p-ANCA) (both IF). All IBS patients had normal fecal calprotectin (mean 21 microg mL(-1), SD 6.6) and fulfilled the ROME II criteria. Frequencies of antibodies in patients with IBS, CD, UC and HC, respectively, are as follows (in per cent): antibodies against A4-Fla2: 29/48/8/7; antibodies against Fla-X: 26/52/10/7; ASCA: 6/59/0/2; p-ANCA: 0/10/52/0; and PAB: 0/28/0/0. Antibodies against A4-Fla2 and Fla-X were significantly more frequent in IBS patients than in HC (P = 0.004 and P = 0.009). Antibodies to A4-Fla2 and Fla-X were significantly more frequent in IBS patients with antecedent gastroenteritis compared to non-postinfectious IBS patients (P = 0.002 and P = 0.012). In contrast to ASCA, PAB and p-ANCA, antibodies against A4-Fla2 and Fla-X were found significantly more often in IBS patients, particularly in those with postinfectious IBS, compared to HC. This observation supports the concept that immune reactivity to luminal antigens has a putative role in the development of IBS, at least in a subset of patients.

Discriminating antigen and non-antigen using proteome dissimilarity:bacterial antigens

It has been postulated that immunogenicity results from the overall dissimilarity of pathogenic proteins versus the host proteome. We have sought to use this concept to discriminate between antigens and non-antigens of bacterial origin. Sets of 100 known antigenic and nonantigenic peptide sequences from bacteria were compared to human and mouse proteomes. Both antigenic and non-antigenic sequences lacked human or mouse homologues. Observed distributions were compared using the non-parametric Mann-Whitney test. The statistical null hypothesis was accepted, indicating that antigen and non-antigens did not differ significantly. Likewise, we were unable to determine a threshold able to separate meaningfully antigen from non-antigen. Thus, antigens cannot be predicted from pathogen genomes based solely on their dissimilarity to the human genome.

Haloarchaeal gas vesicle nanoparticles displaying Salmonella antigens as a novel approach to vaccine development

A safe, effective, and inexpensive vaccine against typhoid and other Salmonella diseases is urgently needed. In order to address this need, we are developing a novel vaccine platform employing buoyant, self-adjuvanting gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1, bioengineered to display highly conserved Salmonella enterica antigens. As the initial antigen for testing, we selected SopB, a secreted inosine phosphate effector protein injected by pathogenic S. enterica bacteria during infection into the host cells. Two highly conserved sopB gene segments near the 3'- region, named sopB4 and sopB5, were each fused to the grIpC gene, and resulting SopB-GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and SopB5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of SopB-GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ApmrG-H111-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-y, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Thl response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were also found to be stable at elevated temperatures for extended periods without refrigeration. The results show that bioengineered GVNPs are likely to represent a valuable platform for antigen delivery and development of improved vaccines against Salmonella and other diseases.

Evaluation of live-attenuated Salmonella vaccines expressing Campylobacter antigens for control of C. jejuni in poultry

Campylobacter jejuni is a zoonotic bacterial pathogen of worldwide importance. It is estimated that 460,000 human infections occur in the United Kingdom per annum and these involve acute enteritis and may be complicated by severe systemic sequelae. Such infections are frequently associated with the consumption of contaminated poultry meat and strategies to control C. jejuni in poultry are expected to limit pathogen entry into the food chain and the incidence of human disease. Toward this aim, a total of 840 Light Sussex chickens were used to evaluate a Salmonella enterica serovar Typhimurium ΔaroA vaccine expressing the C. jejuni amino acid binding protein CjaA as a plasmid-borne fusion to the C-terminus of fragment C of tetanus toxin. Chickens were given the vaccine at 1-day-old and two weeks later by oral gavage, then challenged after a further two weeks with C. jejuni. Across six biological replicates, statistically significant reductions in caecal C. jejuni of c. 1.4 log10 colony-forming units/g were observed at three and four weeks post-challenge relative to age-matched unvaccinated birds. Protection was associated with the induction of CjaA-specific serum IgY and biliary IgA. Protection was not observed using a vaccine strain containing the empty plasmid. Vaccination with recombinant CjaA subcutaneously at the same intervals significantly reduced the caecal load of C. jejuni at three and four weeks post-challenge. Taken together these data imply that responses directed against CjaA, rather than competitive or cross-protective effects mediated by the carrier, confer protection. The impact of varying parameters on the efficacy of the S. Typhimurium ΔaroA vaccine expressing TetC-CjaA was also tested. Delaying the age at primary vaccination had little impact on protection or humoral responses to CjaA. The use of the parent strain as carrier or changing the attenuating mutation of the carrier to ΔspaS or ΔssaU enhanced the protective effect, consistent with increased invasion and persistence of the vaccine strains relative to the ΔaroA mutant. Expression in the ΔaroA strain of a TetC fusion to Peb1A, but not TetC fusions to GlnH or ChuA, elicited protection against intestinal colonisation by C. jejuni that was comparable to that observed with the TetC-CjaA fusion. Our data are rendered highly relevant by use of the target host in large numbers and support the potential of CjaA- and Peb1A-based vaccines for control of C. jejuni in poultry. © 2009 Elsevier Ltd. All rights reserved.

Molecular modeling on human CCR5 receptors and complex with CD4 antigens and HIV-1 envelope glycoprotein gp120

AIM: To investigate the interaction between human CCR5 receptors (CCR5) and HIV-1 envelope glycoprotein gp120 (HIV-1 gp120) and HIV-1 receptor CD4 antigens (CD4). METHODS: The structurally con served regions (SCR) of human CCR5 was built by the SYBYL/Biopolymer module using the corresponding transmembrane (TM) domain of bacteriorhodopsin (bR) as the template. The coordinates for amino-ter minal residue sequence, and carboxyl-terminal residue sequence, extracellular and cytoplasmic loops were generated using LOOP SEARCH algorithm. Subsequently the structural model was merged into the complex with HIV-1 gp120 and CD4. RESULTS: Human CCR5 interacted with both an HIV-1 gp120 and CD4. The N-terminal residues (especially Met1 and Gln4) of human CCR5, contacted with CD4 residues, mainly 7Nith one span (56 - 59) of CD4 in electrostatic interaction and hydrogen-bonds. The binding sites of human CCR5 were buried in a hydrophobic center surrounded by a highly basic periphery. On the other hand, direct interatomic contacts were made between ? CCR5 residues and 6 gp120 amino-acid residues, which included van der Waals contacts, hydrophobic interaction, and hydrogen bonds. CONCLUSION: The interaction model should be helpful for rational design of novel anti-HIV drugs.

Differential inducibility of HLA class I and II antigens by r-IFN gamma in type III bare lymphocyte syndrome.

Case Reports

A novel syndrome of severe neutrophil dysfunction: unresponsiveness confined to chemotaxin-induced functions.

We have identified a patient with a number of neutrophil dysfunctions. The patient was a female baby who lived for 8 months. During her life, she developed severe bacterial infections and showed omphalitis, impaired wound healing, and a pronounced leukocytosis. She was not a patient with leukocyte adhesion deficiency, because all leukocyte CD18 complex proteins were expressed at normal levels. Yet, neutrophil polarization and chemotaxis to platelet-activating factor, leukotriene B4, or formyl-methionyl-leucyl-phenylalanine (FMLP) were completely absent. We found a strong defect in actin polymerization in response to chemotactic stimuli, but only a retarded or even normal reaction with other stimuli. This indicates that the cellular dysfunctions were not due to an intrinsic defect in actin metabolism. Instead, the regulation of actin polymerization with chemotactic stimuli seemed to be defective. We concentrated on FMLP-induced responses in the patient's neutrophils. Functions dependent on activation of complement receptor type 3, such as aggregation or adherence to endothelial cells, were normally induced. Binding to serum-coated coverslips was normal in cell number; however, spreading was not observed. Exocytosis from the specific granules was readily induced. In contrast, FMLP failed to induce a respiratory burst activity or degranulation of the azurophil granules. FMLP induced a normal increase in free intracellular Ca2+, but a decreased formation of diglycerides (especially the 1-O-alkyl,2-acyl compounds). Thus, we have described a patient whose neutrophils show a severe defect in functional activation via chemotaxin receptors, resulting in a selective absence of NADPH oxidase activity, exocytosis from the azurophil granules, and actin polymerization. Our findings show that actin polymerization for neutrophil spreading and locomotion is regulated differently from that for phagocytosis. Also, the release of azurophil and specific granule contents is clearly shown to be regulated in a different way.

In situ chemical modification of peptide microarrays: application to the study of the antibody responses to methylated antigens.

Peptide microarrays are useful tools for characterizing the humoral response against methylated antigens. They are usually prepared by printing unmodified and methylated peptides on substrates such as functionalized microscope glass slides. The preferential capture of antibodies by methylated peptides suggests the specific recognition of methylated epitopes. However, unmodified peptide epitopes can be masked due to their interaction with the substrate. The accessibility of unmodified peptides and thus the specificity of the recognition of methylated peptide epitopes can be probed using the in situ methylation procedure described here. Alternately, the in situ methylation of peptide microarrays allows probing the presence of antibodies directed toward methylated epitopes starting from easy-to-make and cost-effective unmodified peptide libraries. In situ methylation was performed using formaldehyde in the presence of sodium cyanoborohydride and nickel chloride. This chemical procedure converts lysine residues into mono- or dimethyl lysines.