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Salmonella enterica serovar Typhi (S. Typhi) causes substantial morbidity and mortality worldwide, particularly among young children. Humans develop an array of mucosal immune responses following S. Typhi infection. Whereas the cellular mechanisms involved in S. Typhi infection have been intensively studied, very little is known about the early chromatin modifications occurring in the human gut microenvironment that influence downstream immune responses. To address this gap in knowledge, cells isolated from human terminal ileum exposed ex vivo to the wild-type S. Typhi strain were stained with a 33-metal-labeled antibody panel for mass cytometry analyses of the early chromatin modifications modulated by S. Typhi. We measured the cellular levels of 6 classes of histone modifications, and 1 histone variant in 11 major cell subsets (i.e., B, CD3 + T, CD4 + T, CD8 + T, NK, TCR-γδ, Mucosal associated invariant (MAIT), and NKT cells as well as monocytes, macrophages, and epithelial cells). We found that arginine methylation might regulate the early-differentiation of effector-memory CD4+ T-cells following exposure to S. Typhi. We also found S. Typhi-induced post-translational modifications in histone methylation and acetylation associated with epithelial cells, NKT, MAIT, TCR-γδ, Monocytes, and CD8 + T-cells that are related to both gene activation and silencing. [ABSTRACT FROM AUTHOR]

Secretory immunoglobulin A (SIgA) antibodies play an important role in protecting the mucosal surfaces against pathogens and maintaining homeostasis with the commensal microbiota. Because a substantial portion of the gut microbiota is coated with SIgA, we hypothesized that microbiota-SIgA complexes are important for the maintenance of gut homeostasis. Here we investigated the relationship between microbiota-SIgA complexes and inflammatory epithelial cell responses. We used a multi-cellular three-dimensional (3D) organotypical model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes/monocytes, endothelial cells and fibroblasts. We also used human SIgA from human colostrum, and a prominent bacterial member of the first colonizers, Escherichia coli, as a surrogate commensal. We found that free and microbiota-complexed SIgA triggered different epithelial responses. While free SIgA up-regulated mucus production, expression of polymeric immunoglobulin receptor (pIgR) and secretion of interleukin-8 and tumoir necrosis factor-α, microbiota-complexed SIgA mitigated these responses. These results suggest that free and complexed SIgA have different functions as immunoregulatory agents in the gut and that an imbalance between the two may affect gut homeostasis. [ABSTRACT FROM AUTHOR]

Ribonucleic acid extracts (RNA) obtained from the lymph nodes and spleens of guinea pigs, which were immunized with testicular antigen emulsified in Freund's complete adjuvant (FCA), were injected intraperitoneally into normal guinea pigs. The transferred guinea pigs developed a delayed hypersensitivity to sperm antigens and testicular lesions which resembled the lesions obtained in the donor RNA guinea pigs. When the transfer was performed with RNA extracted from guinea pigs immunized with FCA alone or with 'immune' RNA treated with Ribonuclease, neither cellular immunity nor testicular lesions were observed. [ABSTRACT FROM AUTHOR]

Ribonucleic acid (RNA) extracts were obtained from lymph nodes of guinea-pigs that had previously been immunized with a purified testicular antigen emulsified in Freund's complete adjuvant. The RNA extracts were incubated with normal guinea-pig peritoneal exudates cells (NGP-PEC). After treatment, the NGP-PEC cells showed specific inhibition of migration when tested with the specific antigen. No inhibition of migration was observed when cells were tested with an unrelated antigen or when the cells were incubated with RNA obtained from animals immunized with adjuvant alone. Failure of inhibition of migration was also observed when the ‘immune’ RNA was degraded with RNAse. The appearance of this I-RNA in the immunized guinea-pigs correlates with the appearance of delayed hypersensitivity in vivo. [ABSTRACT FROM AUTHOR]

African sleeping sickness is accompanied by a severe immunosuppression. As part of our efforts to examine the mechanisms by which this suppressive state is induced, we studied alterations in human T-lymphocyte function caused by Trypanosoma brucei rhodesiense. To this end, we used an in vitro system in which phytohaemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC) were cultured in a medium containing soluble, non-dialysable parasite products. We were able to demonstrate significant suppression of both lympho-proliferation and interleukin-2 receptor (IL-2R) expression. These effects were found to be dose-dependent and reversible after 48 hr of culture. The suppressive effects of living trypanosomes and the soluble parasite products on lympho- proliferation and interleukin-2 receptor expression were similar in that both precluded the entry of PHA-activated PBMC into the cell cycle. Eighty to ninety-eight per cent of the activated cells remained arrested in the G0/Gla (early G1) phase even 48 hr after stimulation, i.e. when last tested. Parasite-induced expression could not be overcome by the addition of recombinant human 11-2. These results suggest that immunosuppression associated with African trypanosomiasis may result from parasite-induced alteration of very early events during lymphocyte activation, leading to a virtually complete block in cell cycle progression and inhibition of IL-2R expression. [ABSTRACT FROM AUTHOR]

Our research suggests that interferon may have an immunoregulatory role in the initiation phase of immune responses. Recent evidence has demonstrated that lymphokines regulate monocyte cell surface expression of DR antigens and. consequently, the ability of monocytes to activate T lymphocytes in an antigen-specific manner. In this report cloned interferons and a homogeneous cell tine were used to demonstrate that interferon possesses these immunoregulatory functions. Cells of the promyelocytic cell line HL60, when Incubated in vitro with recombinant gamma (IFN-γ) and with alpha interferons (IFN-α), expressed enhanced levels of DR antigen as determined by both cytotoxicity and fluorescence-activated cell sorter analyses. Lower concentrations of IFN-α than IFN-α were needed to induce DR expression, and a higher percentage of monocytes were induced to express DR antigen by IFN-γ than IFN-α. HL60 cells preincubated with lymphocyte-derived lymphokines or IFN-α also stimulated a significantly better in vitro allogeneic response in the mixed leukocyte reaction than untreated HLM cells. Thus, interferon enhanced both the phenotypic expression of DR antigens of HL60 cells and their functional ability to inititiate T-lymphocyte responses to an alloantigen. [ABSTRACT FROM AUTHOR]

An anti-idiotypic serum was prepared by injecting BALB anti-AKR serum into (BALB × AKR)F1 mice. Pretreatment of BALS anti-AKR immune cells with this anti-idiotypic serum plus complement abrogated a leucocyte migration inhibition reaction (LMIR) against AKR extracts but not against purified protein derivative. By itself, the serum induced LMIR in immune cells but not in normal cells. The reaction was strain-specific and anti-Thy 1,2 plus complement sensitive. These results indicate that alloantigen receptors, able to trigger an LMIR in immune cells, have idiotypic determinants similar to those of circulating antibodies of the same specificity. Thus the LMIR is a good model for the study of receptors in cell-mediated immune reactions of primed cells. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi, the causative agent of Chagas' disease, suppresses immune responses during the acute phase and has been shown to induce multiple cellular alterations in activated human T lymphocytes. However, no information is available regarding the effects of this parasite on human B cells. Using an in vitro culture system, in which purified T. cruzi are co-cultured with either peripheral blood mononuclear cells (PBMC) or B-cell-enriched preparations (BCE), we studied whether the organism can induce alterations in DNA synthesis after stimulation with Pansorbin (PS). This response was markedly reduced by the parasite at both suboptimal and optimal PS concentrations, and the extent of the inhibition was augmented as the parasite concentration was increased. Maximal reduction in DNA synthesis was observed when the trypanosomes were incorporated into the cultures at 0 time (i.e. together with PS); the effect was of a much lesser magnitude and undetectable when the parasites were added at 24 and 48 hr. respectively. These results imply that T. cruzi affects a relatively early event during B-cell stimulation. This inference was confirmed by the finding that the proportion of PS-stimulated B cells expressing interleukin-2 (IL-2) receptors was significantly reduced when the parasite was present in the culture. Addition of recombinant human IL-2 did not restore B-cell responsiveness to normal levels. Suppressed B-cell responses were also observed when T. cruzi was separated from the PBMC or the BCE by a cell-impermeable filter, indicating that a soluble factor(s) released by the organism mediated the effect. Accordingly, supernatants of T. cruzi suspensions were found to be suppressive. These results demonstrate for the first time that T. cruzi can affect human B-cell responses and that the mechanism involves inhibition of IL-2 receptor expression. [ABSTRACT FROM AUTHOR]

Commensal microorganisms influence a variety of host functions in the gut, including immune response, glucose homeostasis, metabolic pathways and oxidative stress, among others. This study describes how Salmonella Typhi, the pathogen responsible for typhoid fever, uses similar strategies to escape immune defense responses and survive within its human host. To elucidate the early mechanisms of typhoid fever, we performed studies using healthy human intestinal tissue samples and "mini-guts," organoids grown from intestinal tissue taken from biopsy specimens. We analyzed gene expression changes in human intestinal specimens and bacterial cells both separately and after colonization. Our results showed mechanistic strategies that S. Typhi uses to rearrange the cellular machinery of the host cytoskeleton to successfully invade the intestinal epithelium, promote polarized cytokine release and evade immune system activation by downregulating genes involved in antigen sampling and presentation during infection. This work adds novel information regarding S. Typhi infection pathogenesis in humans, by replicating work shown in traditional cell models, and providing new data that can be applied to future vaccine development strategies., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Dengue has recently been defined by the World Health Organization as a major international public health concern. Although several vaccine candidates are in various stages of development, there is no licensed vaccine available to assist in controlling the further spread of this mosquito borne disease. The need for a reliable animal model for dengue disease increases the risk to vaccine developers as they move their vaccine candidates into large-scale phase III testing. In this paper we describe the cellular immune responses observed in a human challenge model for dengue infection; a model that has the potential to provide efficacy data for potential vaccine candidates in a controlled setting. Serum levels of sIL-2Rα and sTNF-RII were increased in volunteers who developed illness. Supernatants from in vitro stimulated PBMC were tested for cytokines associated with a T(H)1 or T(H)2 T-cell response (IL-2, TNF-α, IFN-γ, IL-4, IL-10, IL-5) and only IFN-γ was associated with protection against fever and/or viremia. Interestingly, IFN-γ levels drop to 0 pg/mL for volunteers who develop illness after challenge suggesting that some mechanism of immunosuppression may play a role in dengue illness. The human challenge model provides an opportunity to test potential vaccine candidates for efficacy prior to large-scale phase III testing, and hints at a possible mechanism for immune suppression by dengue., (Copyright © 2011. Published by Elsevier Ltd.)

We studied the induction of antigen-specific IgA memory B cells (B(M)) in volunteers who received live attenuated Shigella flexneri 2a vaccines. Subjects ingested a single oral dose of 10(7), 10(8) or 10(9) CFU of S. flexneri 2a with deletions in guaBA (CVD 1204) or in guaBA, set and sen (CVD 1208). Antigen-specific serum and stool antibody responses to LPS and Ipa B were measured on days 0, 7, 14, 28 and 42. IgA B(M) cells specific to LPS, Ipa B and total IgA were assessed on days 0 and 28. We show the induction of significant LPS-specific IgA B(M) cells in anti-LPS IgA seroresponders. Positive correlations were found between anti-LPS IgA B(M) cells and anti-LPS IgA in serum and stool; IgA B(M) cell responses to IpaB were also observed. These B(M) cell responses are likely play an important role in modulating the magnitude and longevity of the humoral response., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We evaluated the safety, reactogenicity and immunogenicity of escalating doses of a new Francisella tularensis Live Vaccine Strain (LVS) lot by scarification (SCAR) or subcutaneously (SQ) in humans. Subjects (N=10/group) received one dose of LVS via SCAR at 10(5),10(7) or 10(9)cfu/ml or SQ at 10(2), 10(3),10(4) or 10(5)cfu/ml; 14 subjects received placebo. All doses/routes were well tolerated. When compared to placebo, vaccination with 10(7) SCAR and 10(9) SCAR resulted in significantly higher serologic response frequencies, as measured by ELISA for IgG, IgM, IgA and microagglutination; whereas vaccination with 10(5) SCAR, 10(7) SCAR 10(9) SCAR and 10(5) SQ elicited a significantly higher interferon-gamma response frequency.

We evaluated B memory responses in healthy adult volunteers who received one oral dose of live-attenuated Shigella flexneri 2a vaccine. LPS-specific B(M) cells increased from a median of 0 at baseline to 20 spot forming cells (SFC)/10(6) expanded cells following vaccination (p=0.008). A strong correlation was found between post-vaccination anti-LPS B(M) cell counts and peak serum anti-LPS IgG titers (rs=0.95, p=0.0003). Increases in B(M) specific for IpaB approaching significance were also observed. In sum, oral vaccination with live-attenuated S. flexneri 2a elicits B(M) cells to LPS and IpaB, suggesting that B(M) responses to Shigella antigens should be further studied as a suitable surrogate of protection in shigellosis.

Inflammatory cytokines play an important role in human immune responses to malarial disease. However, the role of these mediators in disease pathogenesis, and the relationship between host protection and injury remains unclear. A total of 248 cases of severe Plasmodium falciparum malaria among children aged 3 months to 14 years residing in Bandiagara, Mali, were matched to cases of uncomplicated malaria and healthy controls. Using modified World Health Organization criteria for defining severe malaria, we identified 100 cases of cerebral malaria (coma, seizure, and obtundation), 17 cases of severe anemia (hemoglobin, <5 g/dl), 18 cases combined cerebral malaria with severe anemia, and 92 cases with hyperparasitemia (asexual trophozoites, >500,000/mm3). Significantly elevated levels (given as geometric mean concentrations in picograms/milliliter) of interleukin-6 (IL-6; 485.2 versus 54.1; P = <0.001), IL-10 (1,099.3 versus 14.1; P = <0.001), tumor necrosis factor alpha (10.1 versus 7.7; P = <0.001), and IL-12(p70) (48.9 versus 31.3; P = 0.004) in serum were found in severe cases versus healthy controls. Significantly elevated levels of IL-6 (485.2 versus 141.0; P = <0.001) and IL-10 (1,099.3 versus 133.9; P = <0.001) were seen in severe malaria cases versus uncomplicated malaria controls. Cerebral malaria was associated with significantly elevated levels of IL-6 (754.5 versus 311.4; P = <0.001) and IL-10 (1,405.6 versus 868.6; P = 0.006) compared to severe malaria cases without cerebral manifestations. Conversely, lower levels of IL-6 (199.2 versus 487.6; P = 0.03) and IL-10 (391.1 versus 1,160.9; P = 0.002) were noted in children with severe anemia compared to severe malaria cases with hemoglobin at >5 g/dl. Hyperparasitemia was associated with significantly lower levels of IL-6 (336.6 versus 602.1; P = 0.002). These results illustrate the complex relationships between inflammatory cytokines and disease in P. falciparum malaria.

Human clinical trials, including experimental challenges of volunteers with pathogenic Salmonella enterica serovar Typhi, small phase I and II trials that monitor the immune responses to vaccines, and large-scale controlled field trials that assess vaccine efficacy under conditions of natural challenge, have helped elucidate the interactions between Salmonella typhi and human hosts.

A promising live attenuated typhoid vaccine candidate strain for mucosal immunization was developed by introducing a deletion in the guaBA locus of pathogenic Salmonella enterica serovar Typhi strain Ty2. The resultant DeltaguaBA mutant, serovar Typhi CVD 915, has a gene encoding resistance to arsenite replacing the deleted sequence within guaBA, thereby providing a marker to readily identify the vaccine strain. CVD 915 was compared in in vitro and in vivo assays with wild-type strain Ty2, licensed live oral typhoid vaccine strain Ty21a, or attenuated serovar Typhi vaccine strain CVD 908-htrA (harboring mutations in aroC, aroD, and htrA). CVD 915 was less invasive than CVD 908-htrA in tissue culture and was more crippled in its ability to proliferate after invasion. In mice inoculated intraperitoneally with serovar Typhi and hog gastric mucin (to estimate the relative degree of attenuation), the 50% lethal dose of CVD 915 (7.7 x 10(7) CFU) was significantly higher than that of wild-type Ty2 (1.4 x 10(2) CFU) and was only slightly lower than that of Ty21a (1.9 x 10(8) CFU). Strong serum O and H antibody responses were recorded in mice inoculated intranasally with CVD 915, which were higher than those elicited by Ty21a and similar to those stimulated by CVD 908-htrA. CVD 915 also elicited potent proliferative responses in splenocytes from immunized mice stimulated with serovar Typhi antigens. Used as a live vector, CVD 915(pTETlpp) elicited high titers of serum immunoglobulin G anti-fragment C. These encouraging preclinical data pave the way for phase 1 clinical trials with CVD 915.

Helicobacter pylori infection of the gastric mucosa can be found in approximately 50% of the world's population and is associated with a range of pathology, including peptic ulcer, atrophic gastritis, and gastric cancer. To explore immunization as a strategy for preventing and treating H. pylori-associated disease, we assessed the safety and immunogenicity in healthy adults of a formalin-inactivated, oral H. pylori whole-cell (HWC) vaccine, administered with or without mutant Escherichia coli heat-labile toxin (LT(R192G)) as a mucosal adjuvant. In a dose-response study, 23 subjects with or without H. pylori infection were vaccinated with either 2.5 x 10(6) HWC, 2.5 x 10(8) HWC, or 2.5 x 10(10) HWC, plus 25 microg of LT(R192G). Thereafter, a randomized study was conducted in which 18 H. pylori-infected subjects were assigned, in a double-blind fashion, to receive either 2.5 x 10(10) HWC plus placebo-adjuvant, placebo-vaccine plus 25 microg of LT(R192G), placebo-vaccine plus placebo-adjuvant, or 2.5 x 10(10) HWC plus 25 microg of LT(R192G). Diarrhea (six subjects), low-grade fever (five subjects), and vomiting (two subjects) were observed, usually after the first dose. Significant rises in geometric mean mucosal (fecal and salivary) anti-HWC immunoglobulin A antibodies occurred among H. pylori-infected and uninfected subjects following inoculation with 2.5 x 10(10) HWC plus 25 microg of LT(R192G). Moreover, among H. pylori-negative volunteers, this regimen induced significant lymphoproliferative responses in 5 of 10 subjects and gamma interferon production responses to H. pylori sonicate in 7 of 10 subjects. There was no evidence that vaccination eradicated H. pylori in infected volunteers. These results suggest that it is possible to stimulate mucosal and systemic immune responses in humans to H. pylori antigens by using an HWC vaccine.

The effects of purified AGC10, a Trypanosoma cruzi membrane glycoprotein, on normal human B lymphocytes were studied in this work. In the presence of AGC10, [3H]-thymidine uptake by human peripheral blood mononuclear cells stimulated with the B cell-specific mitogen SACI (killed Staphylococcus aureus Cowan I) was markedly decreased. This alteration was accompanied by others such as decreased expression of the CD122 and CD132 chains of the IL-2R complex. These inhibitory effects appeared to be somewhat selective, as expression of CD25, another IL-2R chain, was not affected by AGC10 and no significant modification occurred in the expression of the B-cell-specific marker CD19 or CD21. In contrast, AGC10 did reduce the levels of expression of CD86 and CD80, molecules known to play critical roles in B cell interactions with T lymphocytes. Fairly large subpopulations of, but not all, B lymphocytes had their expression of CD122(+), CD132(+), CD86(+) and CD80(+) reduced to undetectable levels in the presence of AGC10. However, the SACI-activated B cells that remained capable of expressing these molecules in the presence of AGC10 did so at normal levels. This was denoted by comparable mean fluorescence intensity values representing the expression of CD122, CD132, CD86 or CD80 molecules on the surface of SACI-stimulated CD19(+) cells cultured without or with AGC10. These results indicated that AGC10, derived from an organism that causes immunosuppression in infected hosts, down-regulates B cell activities and suggested that the relevant mechanism could involve the molecular alterations described above.

Deleting transmembrane alpha-helix motifs from Plasmodium falciparum sporozoite surface protein (SSP-2) allowed its secretion from Salmonella enterica serovar Typhimurium SL3261 and S. enterica serovar Typhi CVD 908-htrA by the Hly type I secretion system. In mice immunized intranasally, serovar Typhimurium constructs secreting SSP-2 stimulated greater gamma interferon splenocyte responses than did nonsecreting constructs (P = 0.04).

Attenuated Salmonella enterica serovar Typhi vaccine strain CVD 908-htrA was used as a vector to deliver fragment C of tetanus toxin as a single-dose oral tetanus vaccine candidate to elicit protective levels of serum tetanus antitoxin. Twenty-one healthy adult volunteers received doses of 1.6 x 10(7) to 8.2 x 10(9) CFU of one of two strains, CVD 908-htrA(pTETnir15) or CVD 908-htrA(pTETlpp), which contained plasmid-encoded fragment C, with sodium bicarbonate, and the safety and immune responses to serovar Typhi antigens and tetanus toxin were assessed. No volunteer had fever or positive blood cultures after vaccination, although diarrhea occurred in 3 volunteers and vomiting in 2 volunteers within 3 weeks after vaccination. Most volunteers excreted the vaccine strain in the first 72 h after vaccination. Three of nine volunteers who received 10(8) CFU or higher doses of the CVD 908-htrA(pTETlpp) construct developed rises in serum antitoxin antibodies. The serum and cellular immune responses to serovar Typhi antigens were less frequent than those previously observed in volunteers who ingested the parent strain CVD 908-htrA. This study demonstrates that fragment C of tetanus toxin delivered orally to volunteers in an S. Typhi vector can elicit protective levels of serum antitoxin., (Copyright 2000 Academic Press.)

One strategy to develop a multi-antigen malaria vaccine is to employ live vectors to carry putative protective Plasmodium falciparum antigens to the immune system. The 19 kDa carboxyl terminus of P. falciparum merozoite surface protein 1 (MSP-1), which is essential for erythrocyte invasion and is a leading antigen for inclusion in a multivalent malaria vaccine, was genetically fused to fragment C of tetanus toxin and expressed within attenuated Salmonella typhi CVD 908. Under conditions in the bacterial cytoplasm, the fragment C-MSP-1 fusion did not form the epidermal growth factor (EGF)-like domains of MSP-1; monoclonal antibodies failed to recognize these conformational domains in immunoblots of non-denatured protein extracted from live vector sonicates. The MSP-1 was nevertheless immunogenic. One month following intranasal immunization of BALB/c mice with the live vector construct, four out of five mice exhibited > or =four-fold rises in anti-MSP-1 by ELISA (GMT=211); a single intranasal booster raised titers further (GMT=1280). Post-immunization sera recognized native MSP-1 on merozoites as determined by indirect immunofluorescence. These data encourage efforts to optimize MSP-1 expression in S. typhi (e.g. as a secreted protein), so that the EGF-like epitopes, presumably necessary for stimulating protective antibodies, can form.

We evaluated the immune responses elicited by attenuated Salmonella enterica serovar Typhi vaccine strain CVD 908-htrA and serovar Typhimurium strain SL3261 alone or as live vectors carrying a plasmid encoding fragment C of tetanus toxin (pTETnir15) in mice immunized intranasally and orogastrically, as well as the in vivo distribution of vaccine organisms following immunization. Higher serologic and proliferative responses against both vector and the foreign antigen were elicited when vaccines were delivered by intranasal route. Whereas both Salmonella strains were detected in the nasal tissue, lungs, and Peyer's patches following intranasal and orogastric immunization, larger numbers of vaccine organisms were recovered from these tissues when the vaccines were delivered intranasally.

Enteropathogenic Escherichia coli (EPEC), a leading cause of diarrhea among infants in developing countries, induces dramatic alterations in host cell architecture that depend on a type III secretion system. EspB, one of the proteins secreted and translocated to the host cytoplasm via this system, is required for numerous alterations in host cell structure and function. To determine the role of EspB in virulence, we conducted a randomized, double-blind trial comparing the ability of wild-type EPEC and an isogenic DeltaespB mutant strain to cause diarrhea in adult volunteers. Diarrhea developed in 9 of 10 volunteers who ingested the wild-type strain but in only 1 of 10 volunteers who ingested the DeltaespB mutant strain. Marked destruction of the microvillous brush border adjacent to adherent organisms was observed in a jejunal biopsy from a volunteer who ingested the wild-type strain but not from two volunteers who ingested the DeltaespB mutant strain. Humoral and cell-mediated immune responses to EPEC antigens were stronger among recipients of the wild-type strain. In addition, four of the volunteers who ingested the wild-type strain had lymphoproliferative responses to EspB. These results demonstrate that EspB is a critical virulence determinant of EPEC infections and suggest that EspB contributes to an immune response.

The DeltaguaBA Shigella flexneri 2a vaccine candidate, CVD 1204, was evaluated as a delivery system for the non-toxic C-terminal of tetanus toxin (fragment C), either as a polypeptide expressed in the bacteria or as a DNA vaccine. CVD 1204 was transformed with plasmid pTETnir15 which encodes the fragment C gene (tetC) under the control of the inducible prokaryotic nir15 promoter or a DNA vaccine plasmid pcDNA3tetC which encodes tetC under the eukaryotic hCMV promoter. Guinea pigs immunised intranasally (i.n.) with either recombinant strain mounted a secretory immune response against S. flexneri 2a Lipopolysaccharide (LPS) and were protected against ocular challenge with wild-type S. flexneri 2a. Both strains were effective in eliciting a serum IgG response against fragment C in guinea pigs following i.n. immunisation. Furthermore, serum from guinea pigs immunised with CVD 1204(pTETnir15) contained tetanus toxin neutralising antibodies. These results demonstrate that this S. flexneri 2a vaccine candidate can serve as a vehicle for the delivery of foreign antigens to the systemic immune system while retaining its capacity to serve as a mucosal Shigella vaccine.

A phase 1 clinical trial was conducted among 35 healthy adult volunteers to evaluate the safety, immunogenicity, and shedding of different doses of CVD 1207, a live attenuated Shigella flexneri 2a vaccine candidate with specific deletion mutations in virG, sen, set, and guaBA. CVD 1207 retains the ability to invade epithelial cells but cannot effectively spread intercellularly after invasion (DeltavirG), does not produce enterotoxin (Deltasen and Deltaset), and has limited proliferation in vivo (DeltaguaBA). In a consecutive fashion, groups of three to seven subjects ingested a single oral dose of CVD 1207 at an inoculum of either 10(6), 10(7), 10(8), 10(9), or 10(10) CFU. CVD 1207 was remarkably well-tolerated at inocula as high as 10(8) CFU. In comparison, one of 12 subjects who received 10(9) CFU experienced mild diarrhea and another experienced a single episode of emesis. One of five subjects who received 10(10) CFU experienced watery diarrhea and emesis. All subjects who ingested doses of 10(8) to 10(10) CFU excreted the vaccine; in 23 of 25, the duration of excretion was

Salmonella enterica serovar Typhi strain CVD 908-htrA is a live attenuated strain which may be useful as an improved oral typhoid vaccine and as a vector for cloned genes of other pathogens. We conducted a phase 2 trial in which 80 healthy adults received one of two dosage levels of CVD 908-htrA in a double-blind, placebo-controlled, crossover study. There were no differences in the rates of side effects among volunteers who received high-dose vaccine (4.5 x 10(8) CFU), lower-dose vaccine (5 x 10(7) CFU), or placebo in the 21 days after vaccination, although recipients of high-dose vaccine (8%) had more frequent diarrhea than placebo recipients (0%) in the first 7 days. Seventy-seven percent and 46% of recipients of high- and lower-dose vaccines, respectively, briefly excreted vaccine organisms in their stools. All blood cultures were negative. Antibody-secreting cells producing antilipopolysaccharide (LPS) immunoglobulin A (IgA) were detected in 100 and 92% of recipients of high- and lower-dose vaccines, respectively. Almost half the volunteers developed serum anti-LPS IgG. Lymphocyte proliferation and gamma interferon production against serovar Typhi antigens occurred in a significant proportion of vaccinees. This phase 2 study supports the further development of CVD 908-htrA as a single-dose vaccine against typhoid fever and as a possible live vector for oral delivery of other vaccine antigens.

Volunteers were orally administered invasive, non-Shiga toxin-producing Shigella dysenteriae 1 to establish a challenge model to assess vaccine efficacy. In stepwise fashion, four separate groups were given 3 x 10(2), 7 x 10(3), 5 x 10(4), or 7 x 10(5) CFU. Using PBMC, proliferative responses and cytokine production were measured to S. dysenteriae whole-cell preparations and to purified recombinant invasion plasmid Ags (Ipa) C and IpaD. Anti-LPS and anti-Ipa Abs and Ab-secreting cells were also evaluated. Preinoculation PBMC produced considerable quantities of IL-10 and IFN-gamma, probably secreted by monocytes and NK cells, respectively, of the innate immune system. Following inoculation, PBMC from 95 and 87% of volunteers exhibited an increased production of IFN-gamma and IL-10, respectively, in response to Shigella Ags. These increases included responses to IpaC and IpaD among those volunteers receiving the lowest inoculum. No IL-4 or IL-5 responses were detected. Whereas there were no Ab or Ab-secreting cell responses in volunteers receiving the lowest inoculum, other dose groups had moderate to strong anti-LPS and anti-Ipa responses. These results suggest that in humans, type 1 responses play an important role in mucosal and systemic immunity to S. dysentariae 1.

Attenuated Salmonella enterica serovar Typhi live vector vaccine strains are highly immunogenic in mice following intranasal but not orogastric inoculation. To elucidate the relationship between organs within which vaccine organisms are found and the induction of specific serum immunoglobulin G (IgG) antibodies, we examined the in vivo distribution of serovar Typhi vaccine strain CVD 908-htrA following intranasal administration. Vaccine organisms were cultured from the nasal lymphoid tissue (NALT), lungs, and Peyer's patches 2 min after intranasal inoculation. Vaccine organisms persisted longer in NALT than in other organs. By decreasing the volume of intranasal inoculum containing 10(9) CFU (from a single 30- or 10-microl dose to four 2.5-microl doses given over the course of 1 h), we were able to significantly reduce the number of vaccine organisms isolated from the lungs (P < 0.05) without reducing the number of vaccine organisms in NALT. Reducing the number of vaccine organisms in the lungs resulted in a significant decrease in the serum tetanus antitoxin response elicited by CVD 908-htrA expressing tetanus toxin fragment C under the control of the redox-responsive nir15 promoter. In contrast, a similar construct expressing tetanus toxin fragment C under control of the constitutive lpp promoter stimulated a strong serum IgG tetanus antitoxin response with both inoculation regimens. The data suggest that following intranasal inoculation, NALT is a sufficient inductive site for elicitation of an immune response against both the live vector and heterologous antigen and, as occurs following oral inoculation of humans, attenuated serovar Typhi vaccine organisms elicit serum IgG responses.

The broad objective of the research presented here is to develop a noncatalytic plasmid maintenance system for the stabilization of multicopy expression plasmids encoding foreign antigens in a Salmonella typhi live-vector vaccine strain such as CVD 908-htrA. We have enhanced the maintenance of expression plasmids at two independent levels. First, we removed dependence upon balanced-lethal maintenance systems that involve catalytic enzymes expressed from multicopy plasmids; we accomplished this through incorporation into expression plasmids of a postsegregational killing system based on the noncatalytic hok-sok plasmid addiction system from the antibiotic resistance factor pR1. We also included at least one naturally occurring plasmid partition function in our expression plasmids, which eliminates random segregation of these plasmids, thereby enhancing their inheritance and stability; to accomplish this, we incorporated either the par locus from pSC101, the parA locus from pR1, or both. We monitored the stability of optimized expression plasmids within CVD 908-htrA by quantitating expression of a variant of green fluorescent protein (GFPuv) by using flow cytometry. In this report, we demonstrate the utility of this novel plasmid maintenance system in enhancing the stability of our expression plasmids and go on to show that as the copy number of stabilized plasmids increases, the toxicity of GFPuv synthesis also increases. The implications of these observations for the rational design of immunogenic and protective bacterial live vector vaccines are discussed.

The cytokine production patterns of human peripheral blood mononuclear cells (PBMC) in response to Salmonella typhi flagella (STF) were examined in culture supernatants of PBMC stimulated with STF. Consistent with previous findings in volunteers vaccinated with aroC aroD deletion mutants of S. typhi, PBMC from volunteers immunized with the licensed live Ty21a S. typhi vaccine secreted gamma interferon following exposure to STF. Stimulation with STF induced rapid de novo synthesis of tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta), followed by IL-6 and IL-10. Trypsin treatment of STF abrogated their effects, while polymyxin B had no effect. Intracellular cytokine measurements of STF-stimulated PBMC revealed the existence of monocyte subpopulations that produce only TNF-alpha, IL-1beta or both cytokines. Moreover, STF markedly decreased the percentage of CD14(+) cells. These data demonstrate that STF are powerful monocyte activators which may have important implications for vaccine development and for understanding the pathogenesis of S. typhi infection.

Attenuated Salmonella typhi strain CVD 915, harboring a deletion in guaBA that interrupts the biosynthesis of guanine nucleotides, was evaluated as a live vector vaccine for delivering foreign antigens utilizing prokaryotic or eukaryotic expression systems. Plasmids pTETnir15 and pcDNA3tetC encoding fragment C (Frag C) of tetanus toxin under the control of prokaryotic or eukaryotic promoters, respectively, were introduced into CVD 915 and administered intranasally to mice. Purified pcDNA3tetC and Frag C were given intramuscularly. High titers of serum IgG1, IgG2a, and IgG2b antibodies against Frag C were elicited by CVD 915(pTETnir15) and CVD 915(pcDNA3tetC). These responses were significantly higher than those induced by pcDNA3tetC. Proliferative responses and IL-2 and IFN-gamma production were observed in splenocytes exposed to S. typhi antigens and Frag C. We conclude that CVD 915 is a highly efficient live vector to carry foreign genes under eukaryotic or prokaryotic control and elicit potent immune responses., (Copyright 1999 Academic Press.)

Vibrio vulnificus is a human pathogen whose virulence has been associated with the expression of capsular polysaccharide (CPS). Multiple CPS types have been described; however, virulence does not appear to correlate with a particular CPS composition. Reversible-phase variation for opaque and translucent colony morphologies is characterized by changes in CPS expression, as suggested by electron microscopy of cells stained nonspecifically with ruthenium red. Isolates with opaque colony morphologies are virulent and appear to be more thickly encapsulated than naturally occurring translucent-phase variants, which have reduced, patchy, or absent CPS. Previously, we have shown that the virulence of translucent-phase variants was intermediate between opaque-phase variants and acapsular transposon mutants, suggesting a correlation between virulence and the amount of CPS expressed. In the present study, CPS expression of phase variants and genetically defined mutants of V. vulnificus M06-24/O was examined by using a CPS-specific monoclonal antibody with an enzyme-linked immunosorbent assay, flow cytometry, and immunoelectron microscopy. Semiquantitative analyses of CPS expression correlated well among these assays, confirming that the translucent-phase variant was intermediate in CPS expression and retained type I CPS-specific epitopes. Cell surface expression of CPS varied with the growth phase, increasing during logarithmic growth and declining in stationary culture. Significantly greater CPS expression (P = 0.026) was observed for cells grown at 30 degrees C than for those at 37 degrees C. These studies confirm that phase variation and virulence in V. vulnificus correlate with the amount of CPS expressed and demonstrate the fluidity of bacterial polysaccharide expression in response to environmental conditions.

The Trypanosoma cruzi membrane glycoprotein AGC10 has been shown to alter some human macrophage functions (De Diego, J. L. et al., J. Immunol. 1997. 159: 4983-4989). We show here that, in the presence of AGC10, [3H]thymidine incorporation by normal human lymphocytes stimulated with anti-CD3 or phytohemagglutinin (PHA) is severely curtailed. This effect was found to involve down-regulation of the expression of both CD25 (IL-2R alpha) and CD122 (IL-2R beta) on the lymphocyte membrane and a marked decrease in the level of up-regulation of the expression of surface CD132 (IL-2R gamma or gamma(c)). These alterations occurred in fairly large proportions of CD4+ and CD8+ lymphocytes. AGC10 also inhibited proliferation and expression of IL-2 receptor chains by activated T lymphocytes virtually depleted of monocytes/macrophages, indicating that these effects do not necessarily require prior modification of monocyte/macrophage function by AGC10. Human lymphocytes stimulated with anti-CD3 or PHA also displayed a markedly decreased capacity to secrete IL-2 and IFN-gamma, suggesting that AGC10 affected at least Th1 cell functions. Cell viability in cultures containing or lacking AGC10 was comparable over a 72-h period, and neither CD25 expression by, nor the viability of, PHA-stimulated Jurkat cells was altered by AGC10, ruling out that the effects of AGC10 are due to cell killing. These results highlight down-regulatory effects on activated T lymphocytes exerted by a membrane molecule from a parasite causing a disease whose acute phase is accompanied by immunosuppression.

A key function of monocytes/macrophages (Mphi) is to present antigens to T cells. However, upon interaction with bacteria, Mphi lose their ability to effectively present soluble antigens. This functional loss was associated with alterations in the expression of adhesion molecules and CD14 and a reduction in the uptake of soluble antigen. Recently, we have demonstrated that Salmonella typhi flagella (STF) markedly decrease CD14 expression and are potent inducers of proinflammatory cytokine production by human peripheral blood mononuclear cells (hPBMC). In order to determine whether S. typhi and soluble STF also alter the ability of Mphi to activate T cells to proliferate to antigens and mitogens, hPBMC were cultured in the presence of tetanus toxoid (TT) or phytohemagglutinin (PHA) and either killed whole-cell S. typhi or purified STF protein. Both whole-cell S. typhi and STF suppressed proliferation to PHA and TT. This decreased proliferation was not a result of increased Mphi production of nitric oxide, prostaglandin E2, or oxygen radicals or the release of interleukin-1beta, tumor necrosis factor alpha, interleukin-6, or interleukin-10 following exposure to STF. However, the ability to take up soluble antigen, as determined by fluorescein isothiocyanate-labeled dextran uptake, was reduced in cells cultured with STF. Moreover, there was a dramatic reduction in the expression of CD54 on Mphi after exposure to STF. These results indicate that whole-cell S. typhi and STF have the ability to alter in vitro proliferation to soluble antigens and mitogens by affecting Mphi function.

Objective: To define the phenotype of cells in the perivascular and vascular infiltrates of Palmerston North (PN) mice and the cytokines that those cells produce., Methods: Immunohistologic analysis, flow cytometric analysis, and reverse transcriptase-polymerase chain reaction (RT-PCR) studies were performed on tissues and cells from female PN mice and age-matched and sex-matched DBA/2 mice., Results: With aging, PN mice developed a female-predominant, lupus-like disease, with a severe systemic mononuclear cell perivasculitis and vasculitis. The perivasculitis involved arteries and veins in kidney, liver, brain, and lung; the vasculitis predominantly involved veins and venules. The perivascular and vascular infiltrates in female PN mice were composed mainly of an unusual cell type that expressed phenotypic markers characteristic of both T cells (Thy1+, CD3+, CD4+, T cell receptor + [TCR+]) and B cells (B220+). In addition, the infiltrates contained a smaller number of conventional CD4+,B220- T cells and macrophages. Very few CD8+ T cells or surface Ig+ B cells were seen. Unlike the Thy1+,B220+ T cells present in MRL/lpr mice, most of which were CD4-,CD8- and TCRalpha/beta+, the majority of the Thy1+,B220+ T cells in the perivascular/vascular infiltrates of PN mice were CD4+ and expressed either TCRalpha/beta or TCRgamma/delta. By immunohistologic staining, the cells in the perivascular and vascular infiltrates in the kidneys of older PN mice were shown to produce interleukin-4 (IL-4), IL-6, and IL-10, but not IL-2, interferon-gamma, transforming growth factor beta, tumor necrosis factor alpha, or IL-1beta. By RT-PCR, the kidneys of older PN mice were found to express high levels of IL-4, IL-6, and IL-10 messenger RNA., Conclusion: The vascular and perivascular infiltrates in PN mice are composed predominantly of an unusual subpopulation of T cells that are Thy1+,B220+,CD4+,CD8-, express either TCRalpha/beta or TCRgamma/delta, and produce mainly type 2 cytokines. The exact role of these cells in the immunopathogenesis of lupus-like disease in PN mice remains to be elucidated.

Many immunosuppressive effects of Trypanosoma cruzi can be reproduced in vitro by a preparation consisting of molecules spontaneously released by this protozoan (termed trypanosomal immunosuppressive factor (TIF)). In this work, we attempted to establish whether TIF-induced inhibition of lymphoproliferation results from preventing lymphocyte activation or impairing a post-activation process. Although [3H]thymidine uptake and expression of CD25 by normal human T lymphocytes stimulated with a phorbol ester were markedly reduced by T. cruzi or TIF, translocation of cytosolic protein kinase C (PKC) to the cell membrane was not affected. Lymphoproliferation induced by ionomycin was also inhibited by T. cruzi or TIF but the typical elevation of intracellular calcium ions [Ca2+]i caused by this calcium ionophore was not altered. The increase in [Ca2+]i induced with anti-CD3 antibody was also unaffected by TIF. TIF did not preclude lymphocytes stimulated with phytohemagglutinin from accumulating normal mRNA levels of NFAT1 (also known as NFATp) and NFATc. NFAT1 and NFATc are components of the NFAT complex that controls transcription of genes coding for several cytokines and whose translocation to the nucleus is dependent upon PKC activation and increased [Ca2+]i. In contrast, the mRNA levels of cyclin D2 and cdk4, which form a holoenzyme complex known to regulate cell progression through the G1 phase, were markedly reduced by TIF. These results indicated that TIF did not inhibit lymphocyte activation leading to early secondary signaling but curtailed a mechanism controlling cell progression through G1 and necessary for reaching S phase.

A single-dose, oral Salmonella typhi vaccine strain has been sought as a carrier or vector of cloned genes encoding protective antigens of other pathogens. Such a hybrid vaccine, administered orally, would stimulate immune responses both at the mucosal surface and in the systemic compartment and would potentially provide protection against multiple pathogens. S. typhi CVD 908 and CVD 906, which harbor deletions in aroC and aroD, were further engineered by deletion in htrA to produce strains CVD 908-htrA and CVD 906-htrA, which are unable to sustain growth and are severely impaired in their ability to survive in host tissues. These strains were fed to humans at doses of 5 x 10(7) to 5 x 10(9) CFU with buffer, and safety and immune responses were assessed. CVD 908-htrA and CVD 906-htrA were well tolerated in volunteers; mild diarrhea in 3 of 36 volunteers and mild fever in 1 volunteer were the only notable adverse responses. The vaccine strains were not detected in blood cultures and only transiently detected in stool. Serum immune responses to S. typhi lipopolysaccharide and H antigens were observed in 75 to 100% of volunteers who received 5 x 10(8) to 5 x 10(9) CFU, and cells secreting S. typhi-specific antibodies were found in all volunteers after ingestion of either strain. Sixty-three percent to 83% of volunteers developed lymphoproliferative responses to S. typhi flagellar and particulate antigens after the higher doses. These studies demonstrate the potential of CVD 908-htrA as a live vector for the delivery of heterologous genes, and a clinical trial of such a construct is planned.

Vibrio cholerae can shift to a "rugose" colonial morphology associated with expression of an amorphous exopolysaccharide that promotes cell aggregation. Flow cytometric studies indicated that up to 3% of particles in rugose cultures represented aggregates of >5 bacterial cells. Rugose variants of our test strains displayed resistance to killing by chlorine, with viable cells persisting for >30 min in 2 mg/L free chlorine; strains also showed resistance to killing by complement-mediated serum bactericidal activity. Six volunteers fed 10(6) cfu of a rugose variant of V. cholerae O1 El Tor Inaba N16961 developed symptoms typical of cholera, with a mean diarrheal stool volume of 2.2 L (range, 1.4-4.3). Isolates recovered from the stool of infected volunteers retained the rugose phenotype. The data suggest that rugose strains cause human disease. The role of these strains in the epidemiology of cholera remains to be determined.