Characterization of CD8(+) effector T cell responses in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine

Salmonella enterica serovar Typhi (S. typhi) strain Ty21a remains the only licensed attenuated typhoid vaccine. Despite years of research, the identity of the protective immunological mechanisms elicited by immunization with the Ty21a typhoid vaccine remains elusive. The present study was designed to characterize effector T cell responses in volunteers immunized with S. typhi strain Ty21a typhoid vaccine. We determined whether immunization with Ty21a induced specific CTL able to lyse S. typhi-infected cells and secrete IFN-gamma, a key effector molecule against intracellular pathogens. We measured the functional activity of these CTL by a (51)Cr-release assay using 8-day restimulated PBMC from Ty21a vaccinees as effector cells and S. Typhi-infected autologous PHA-activated PBMC as target cells. Most vaccinees exhibited consistently increased CD8-mediated lysis of targets by postimmunization PBMC when compared with preimmunization levels. We also developed an IFN-gamma ELISPOT assay to quantify the frequency of IFN-gamma spot-forming cells (SFC) in PBMC from Ty21a vaccinees using an ex vivo system. Significant increases in the frequency of IFN-gamma SFC following immunization (mean +/- SD, 393 +/- 172; range 185-548 SFC/10(6) PBMC; p = 0.010), as compared with preimmunization levels, were observed. IFN-gamma was secreted predominantly by CD8(+) T cells. A strong correlation was recorded between the cytolytic activity of CTL lines and the frequency of IFN-gamma SFC (r(2) = 0.910, p < 0.001). In conclusion, this work constitutes the first evidence that immunization of volunteers with Ty21a elicits specific CD8(+) CTL and provides an estimate of the frequency of CD8(+) IFN-gamma-secreting cells induced by vaccination.     

The live oral typhoid vaccine Ty21a is a rpoS mutant and is susceptible to various environmental stresses

Abstract The rpoS (katF) gene, which encodes a RNA polymerase σ factor ( σ ^s ), regulates the virulence of Salmonella typhimurium in mice. In the present study, we show that rpoS mutants can be frequently found among laboratory strains of Salmonella . In addition, a rpoS mutation was identified in the S. typhi live oral vaccine Ty21a. Introduction of a wild-type rpoS gene in Ty21a allowed the bacteria to survive better under starvation conditions and increased their resistance to other stresses. These results contribute to a better understanding of the genetic background of the live typhoid oral vaccine Ty21a and suggest that the rpoS mutation may contribute to the safety of this strain in humans.     

Surface-displayed viral antigens on Salmonella carrier vaccine

We have developed a recombinant live oral vaccine using the ice-nucleation protein (Inp) from Pseudomonas syringae to display viral antigens on the surface of Salmonella spp. Fusion proteins containing viral antigens were expressed in the oral vaccine strain, Salmonella typhi Ty21a. Surface localization was verified by immunoblotting and fluorescence-activated cell sorting. The immunogenicity of surface-displayed viral antigens on the recombinant live vaccine strain was assessed in mice inoculated intranasally and intraperitoneally. Inoculation resulted in significantly higher serum antibody level than those induced by viral antigens expressed intracellularly. Thus, this multivalent mucosal live vaccine may provide an effective means for inducing mucosal or systemic immune responses against multiple viral antigens.     

IgA-driven T cell-mediated anti-bacterial immunity in man after live oral Ty 21a vaccine

Cellular immunity against Salmonella typhi was observed by using a direct anti-bacterial in vitro assay in volunteers orally vaccinated with the live S. typhi mutant strain Ty 21a. With this experimental approach, it was demonstrated that Ty 21a vaccine also induces cellular immunity against S. paratyphi A and B. Interestingly, the mechanism involved in cellular immunity against bacteria seems to be of an antibody-dependent cellular cytotoxicity (ADCC) type, with IgA acting as the humoral arm and CD4+ T lymphocytes as the cellular one. In accordance with the increase in IgA-driven ADCC against S. typhi, a major rise in IgA against O and H antigens was observed in the serum of vaccinees in parallel to an increase in IgG of identical specificity. Furthermore, a Ty 21 vaccine induced cellular activity against flagellar antigens. These results indicate that IgA-ADCC by T lymphocytes against bacteria can originate from local stimulation of the gut mucosal immune system. This cellular defense mechanism might be at the origin of the protection induced by Ty 21a vaccine.     

Modulation of a specific humoral immune response and changes in intestinal flora mediated through fermented milk intake

Abstract This study was undertaken to elucidate whether eating a fermented milk containing Lactobacillus acidophilus La1 and bifidobacteria could induce changes in intestinal flora and modulate the immune response in man. Volunteers consumed a fermented milk containing L. acidophilus La1 and bifidobacteria over a period of three weeks during which an attenuated Salmonella typhi Ty21a was administered to mimic an enteropathogenic infection. A control group ate no fermented foods but received the S. typhi Ty21a. Faecal flora analyses showed an increase in L. acidophilus and bifidobacterial counts during fermented milk intake. The specific serum IgA titre rise to S. typhi Ty21a in the test group was > 4-fold and significantly higher ( P = 0.04) than in the control group. An increase in total serum IgA was also observed. These results indicate that lactic acid bacteria which can persist in the gastrointestinal tract can act as adjuvants to the humoral immune response.     

肠毒素源性定居因子CS6在减素伤寒沙门氏菌中表达及免疫原性的研究

将编码肠毒素源性大肠杆菌定居因子抗原ＣＳ６基因克隆到ｐＸＬ６７０,转化ａｓｄ基因突变的Ｅ．ｃｏｌｉ Ｘ６０９７,获得重组质粒ｐＳＳ６４,再将后者转化至减毒的△ａｒｏＡ、△ａｒｏＣ、△ａｓｄ伤寒沙门氏菌,构建了无药物抗性且稳定的大肠杆菌和伤寒双价菌苗候选株。小鼠腹腔免疫和攻击实验表明,该菌株对伤寒沙门氏菌毒株的攻击具有良好的保护作用。家兔免疫实验证明,该菌株能产生抗ＣＳ６和伤寒菌Ｖｉ抗原的血清抗体。     

The Vi antigen of Salmonella typhi: molecular analysis of the viaB locus.

Strains of Salmonella typhi isolated from the blood of patients with typhoid fever invariably express a capsular polysaccharide, termed the Vi antigen. Vi antigen expression is controlled by two separate chromosomal loci, viaA and viaB. The viaA locus is commonly found in enteric bacteria. In contrast, the viaB locus appears to be specific to Vi-expressing strains of Salmonella and Citrobacter. Here the cloning, expression and analysis of viaB determinants from S. typhi Ty2 is described. Whole-cell DNA from strain Ty2 was size-fractionated and cloned into the pLA2917 cosmid vector. A recombinant cosmid, pVT1, conferring a Vi-positive phenotype upon Escherichia coli and upon the Vi-non-expressing strain Ty21a of S. typhi, was characterized and used for further studies. Transposon Tn5 insertion mutagenesis demonstrated that the Vi-antigen-encoding region on pVT1 consisted of a 15 kb fragment. A subclone, designated pVT3, which contained an 18 kb insert, was sufficient to confer Vi antigen expression upon E. coli and S. typhi Ty21a. Results of recombination experiments indicated that this DNA sequence was the viaB locus of S. typhi Ty2. In E. coli SE5000 maxicells, the viaB determinants encoded at least eight polypeptides, with molecular masses of 80, 65, 59, 48, 44, 39, 35 and 28 kDa. Functional characterization of viaB mutations in S. typhi Ty2 suggested that the 80 and 65 kDa proteins were required for cell-surface localization of the Vi antigen.     

Asymmetric pore occupancy in crystal structure of OmpF porin from Salmonella typhi

OmpF is a major general diffusion porin of Salmonella typhi, a Gram-negative bacterium, which is an obligatory human pathogen causing typhoid. The structure of S. typhi Ty21a OmpF (PDB Id: 3NSG) determined at 2.8 ? resolution by X-ray crystallography shows a 16-stranded β-barrel with three β-barrel monomers associated to form a trimer. The packing observed in S. typhi Ty21a rfOmpF crystals has not been observed earlier in other porin structures. The variations seen in the loop regions provide a starting point for using the S. typhi OmpF for structure-based multi-valent vaccine design. Along one side of the S. typhi Ty21a OmpF pore there exists a staircase arrangement of basic residues (20R, 60R, 62K, 65R, 77R, 130R and 16K), which also contribute, to the electrostatic potential in the pore. This structure suggests the presence of asymmetric electrostatics in the porin oligomer. Moreover, antibiotic translocation, permeability and reduced uptake in the case of mutants can be understood based on the structure paving the way for designing new antibiotics.     

Migration of Salmonella typhi through Intestinal Epithelial Monolayers: An In Vitro Study

This study characterizes the transmigration of enteroinvasive Salmonella typhi in vitro, using a human intestinal epithelial cell line as a model of small intestinal epithelium. C2BBe cells, a subclone of CACO-2 with a highly differentiated enterocytic phenotype, were grown to maturity on Transwell filters. S. typhi Ty2 and the vaccine strain, Ty21a, the S. typhi mutant X7344 and parent strain SB130, and S. typhimurium 5771 in logarithmic phase were introduced to the upper chamber of the filter units. Numbers of bacteria in the lower chamber, TER and permeability of the monolayer to mannitol were measured over time. Monolayers were examined by light, electron and confocal microscopy to determine the pathway of bacterial transmigration, and intracellular bacteria were estimated by gentamicin assay. Epithelial cell injury was quantified by light microscopy. S. typhi transmigrated earlier and in larger numbers than S. typhimurium, inducing marked changes in electrical resistance and permeability. Unlike S. typhimurium, S. typhi selected epithelial cells in small number and caused their death and extrusion from the monolayers leaving holes through which S. typhi transmigrated. Ty2 consistently transmigrated in larger numbers and with more injury to monolayers than Ty21a. S. typhi crosses the monolayers of C2BBe cells by a paracellular route in contrast to the transcellular pathway described for other Salmonellae. This may be related to the unique pathophysiology of S. typhi infection and the restricted host specificity of this pathogen. In these assays the vaccine strain, Ty21a, is slightly less invasive than its parent, though more invasive than S. typhimurium.     

Modulation of humoral immune response through probiotic intake

Thirty healthy volunteers were randomised into three different treatment groups and consumed Lactobacillus GG, Lactococcus lactis or placebo (ethyl cellulose) for 7 days. On days 1, 3 and 5, an attenuated Salmonella typhi Ty21a oral vaccine was given to all subjects to mimic an enteropathogenic infection. All subjects responded well to the vaccine, but no significant differences were observed in numbers of IgA-, IgG- and IgM-secreting cells among the different groups. There was a trend towards a greater increase in specific IgA among the subjects receiving the vaccine in combination with Lactobacillus GG. Those receiving L. lactis with their vaccine evinced significantly higher CR3 receptor expression on neutrophils than those receiving either the placebo or Lactobacillus GG. These results indicate that probiotics may influence differently the immune response to oral S. typhi vaccine and that the immunomodulatory effect of probiotics is strain-dependent.     

利用λRed重组系统敲除伤寒沙门氏菌rfaH基因

目的：利用λRed重组系统敲除伤寒沙门氏菌的rfaH基因。方法：以伤寒沙门氏菌（Salmonella typhi Ty2,S.ty2）基因组为模板扩增得到的同源臂,与两端带有FRT位点的卡那霉素抗性基因片段共同构建同源重组载体;以重组载体为模板扩增打靶片段,将其转化S.ty2;在抗生素压力和λRed重组系统帮助下,打靶片段和菌体基因组发生同源重组,通过卡那抗性筛选得到带有抗性标记的重组菌;转入重组酶表达质粒pCP20以去除抗性标记,得到保留单一FRT位点的突变菌株;通过PCR鉴定重组菌,并经透射电子显微镜分析表型。结果：在S.ty2中敲除了rfaH基因,经PCR扩增和序列测定正确;初步的表型分析表明突变体的鞭毛合成显著减少。结论：获得了S.ty2突变株,为将沙门氏菌进一步减毒成为疫苗表达载体奠定了基础。     

Evaluation of phoP and rpoS mutants of Salmonella enterica serovar Typhi as attenuated typhoid vaccine candidates: virulence and protective immune responses in intranasally immunized mice

The attenuation and immunoenhancing effects of rpoS and phoP Salmonella enterica serovar strain Typhi (Salmonella typhi) mutants have not been compared. Here, three S. typhi deletion mutants (phoP, rpoS, and rpoS-phoP double mutant) are constructed and these mutants are characterized with respect to invasiveness, virulence, and protective immune response compared with wild-type Ty2. It was found that phoP and phoP-rpoS deletion mutants are less invasive to HT-29 cells than the wild-type Ty2 and the rpoS single-deleted strain. The LD(50) of immunized mice was higher for phoP than for rpoS mutants, and the highest for the phoP-rpoS double mutant. In addition, all S. typhi mutants showed an increase in the specific serum IgG levels and T-cell-mediated immunity, and showed equal protection abilities against a wild-type Ty2 challenge after two rounds of immunization in BALB/c mice. It is concluded that phoP genes appear to play a more important role than rpoS genes in both cellular invasion and virulence of S. typhi, but not in immunogenicity in mice. Furthermore, the data indicate that the phoP-rpoS double mutant may show promise as a candidate for an attenuated typhoid vaccine.     

Invasiveness of Salmonella typhi strains in HeLa S3 monolayer cells

The internalization and intracellular multiplication, i.e., the invasiveness, of Salmonella typhi strains recently isolated from typhoid fever patients were confirmed in HeLa cell monolayers. When stained with Giemsa solution, intracellular bacteria were 0.6 X 1.2 micron in size and stained purple, whereas extracellular bacteria associated or not with the HeLa cell surface were 1.0 X 3.0 micron and stained deep blue. Strain GIFU 10007 was internalized into 23% of the HeLa cells within 10 min after inoculation. About 90% of the HeLa cells were infected after 24 hr incubation in kanamycin (KM)-containing medium. Intracellular multiplication of the challenge organism was verified by a large number of intracellular bacteria after 24 hr incubation in KM-containing medium by both light-microscopy of the Giemsa stained preparation and viable counts of intracellular bacteria. The viable counts of strain 10007 showed an increase of more than 40-fold within 24 hr after inoculation, whereas in the four other less or non-infective strains, recovery of viable bacteria was poor or nil. Strains which were highly invasive usually failed to show strong adhesion. The contribution of Vi antigen to the internalization of challenge organisms was not proved. Infective strains, when killed by formalin were still adhesive, but were not internalized. The same strains, when killed by boiling, were neither adhesive nor internalized. From these findings it was concluded that the internalization and multiplication of infective S. typhi strains in cultured HeLa cells should be regarded as an invasion rather than phagocytosis by host cells, and such invasiveness could be an indicator to estimate the virulence of S. typhi strains.     

Modulation of human humoral immune response through orally administered bovine colostrum

Eighteen healthy volunteers were randomized into two treatment groups and consumed liquid prepackaged bovine colostrum whey and placebo for 7 days. On days 1, 3 and 5, an attenuated Salmonella typhi Ty21a oral vaccine was given to all subjects to mimic an enteropathogenic infection. The circulating antibody secreting cells and the expression of phagocytosis receptors of the subjects before and after oral immunization were measured with the ELISPOT assay and flow cytometry. All subjects responded well to the vaccine. No significant differences were observed in ELISPOT values for IgA, IgG, IgM, Fcgamma and CR receptor expression on neutrophils and monocytes between the two groups. There was a trend towards greater increase in specific IgA among the subjects receiving their vaccine with bovine colostrum. These results suggest that bovine colostrum may possess some potential to enhance human special immune responses.     

Polysaccharides and their common proteinic carrier in the Vi antigens of Citrobacter ballerup and Salmonella typhi Ty2

Immunochemical analysis of Citrobacter ballerup and Salmonella typhi Ty2 showed that the strains share native and heat-resistant proteins that are, apparently, the carriers of a common polysaccharidic determinant present in their respective somatic antigens. After the classic acetic acid hydrolysis, the somatic antigen of C. ballerup reacted, in agar gel, against the homologous antiserum by two precipitation lines, one of which also precipitated against the anti S, typhi Ty2 serum; the hydrolysis of the S. typhi Ty2 somatic antigen demonstrated that, in addition to the 'O' polysaccharide, reacting against all the S. typhi antisera, it contains a polysaccharide that precipitated against the anti-C. ballerup serum. The elusiveness in the agglutinability of only freshly isolated bacterial authorizes some doubt concerning the responsibility of the antipolysaccharide antibodies in the agglutinating Vi sera; in order to induce anitpolysaccharides hyperimmunizations are needed while antiproteins are easily induced by short immunizations.     

Purification of integral outer-membrane protein OmpC, a surface antigen from Salmonella typhi for structure-function studies: a method applicable to enterobacterial major outer-membrane protein

Extraction of the outer-membrane porin, OmpC, from Salmonella typhi Ty21a was done by using a modified salt-extraction procedure. It was possible to extract only the major outer-membrane protein (OMP) from the crude membrane using this method. Aberrant lipopolysaccharide (LPS) production in the galE mutant Ty21a has resulted in more isoforms of OmpC and subsequently led to anomalous mobility in SDS-PAGE. The purity of the preparation was confirmed by denaturing urea SDS-PAGE and N-terminal sequencing. The major OMP extracts had LPS of both bound and free forms. The free form of LPS could be removed by gel filtration and the bound form, largely, was removed using ion-exchange chromatography and by passing through ultrafiltration devices. This method has been used to extract the native trimer of OmpC, the major OMP, in a large scale, for structure-function studies. S. typhi Ty21a OmpC preparation yielded reproducible diffraction-quality crystals. Extracts of porin from wild-type Escherichia coli HB101, grown under high osmolarity conditions, showed a single species of OMP on SDS-PAGE. This suggests the possible application of the method to other gram-negative bacterial porins.     

Trypanosoma cruzi: expression of antigens on the membrane surface of parasitized cells

A direct immunofluorescent antibody test with an anti-Trypanosoma cruzi F(ab')2 conjugate was used to demonstrate antigens of T. cruzi on the membrane surface of intact live or fixed macrophages and L929 mouse fibroblasts infected with the organism. Antigens were demonstrated in 5 to 50% of infected cells, and their presence was not directly related to the number of intracellular organisms. Cells with as few as four intracellular amastigotes had demonstrable surface antigens, whereas some cells with as many as twelve or more organisms did not. Capping of antigen-antibody complexes was noted to begin a few minutes after the addition of the anti-T cruzi F(ab')2 conjugate; by 30 min, most of the parasitized cells had eliminated the complexes, and no surface antigen of parasitic nature could be demonstrated. Although capping may have caused a negative result in a previously positive cell, other mechanisms may be involved, because antigens were not demonstrated in some heavily parasitized cells examined immediately after completion of the test. Treatment of the infected cells with trypsin or chymotrypsin resulted in the absence of demonstrable parasite antigens on the cell membrane surface. However, the antigens were again demonstrated 12 hr after the enzymes were removed. The reappearance of parasite antigens on the surface of infected cells was prevented by treatment of the monolayers with puromycin or tunicamycin. A T cell-enriched population of spleen lymphocytes from mice chronically infected with T. cruzi recognized the membrane-bound antigens and proceeded to destroy the host cell and the intracellular organisms. In this process, noninfected cells were also destroyed, possibly because they were coated with antigens released from intact infected cells or from infected cells that had been lysed by the action of the sensitized lymphocytes or their products.     

Lysis of cells infected with typhus group rickettsiae by a human cytotoxic T cell clone

Cytolytic human T cell clones generated in response to the intracellular bacterium Rickettsia typhi were characterized. Growing clones were tested for their ability to proliferate specifically in response to antigens derived from typhus group rickettsiae or to lyse targets infected with R. typhi or Rickettsia prowazekii. Two clones were able to lyse targets infected with typhus group rickettsiae. One of these clones was more fully characterized because of its rapid growth characteristics. This cytolytic clone was capable of lysing an autologous infected target as well as a target matched for class I and II histocompatibility leukocyte antigens (HLA). It was not capable, however, of lysing either a target mismatched for both class I and II HLA or a target partially matched for class I HLA. In addition, the clone exhibited specificity in that it was able to lyse an autologous target infected with typhus group rickettsiae, but did not lyse an autologous target infected with an antigenically distinct rickettsial species, Rickettsia tsutsugamushi. These results demonstrate, for the first time, that cells infected with intracellular bacteria can be lysed by human cytotoxic T lymphocytes.     

Identification and validation of T-cell epitopes in outer membrane protein (OMP) of Salmonella typhi

This study aims to design epitope-based peptides for the utility of vaccine development by targeting outer membrane protein F (Omp F), because two available licensed vaccines, live oral Ty21a and injectable polysaccharide, are 50% to 80% protective with a higher rate of side effects. Conventional vaccines take longer time for development and have less differentiation power between vaccinated and infected cells. On the other hand, Peptide-based vaccines present few advantages over other vaccines, such as stability of peptide, ease to manufacture, better storage, avoidance of infectious agents during manufacture, and different molecules can be linked with peptides to enhance their immunogenicity. Omp F is highly conserved and facilitates attachment and fusion of Salmonella typhi with host cells. Using various databases and tools, immune parameters of conserved sequences from Omp F of different isolates of Salmonella typhi were tested to predict probable epitopes. Binding analysis of the peptides with MHC molecules, epitopes conservancy, population coverage, and linear B cell epitope prediction were analyzed. Among all those predicted peptides, ESYTDMAPY epitope interacted with six MHC alleles and it shows highest amount of interaction compared to others. The cumulative population coverage for these epitopes as vaccine candidates was approximately 70%. Structural analysis suggested that epitope ESYTDMAPY fitted well into the epitope-binding groove of HLA-C*12:03, as this HLA molecule was common which interact with each and every predicted epitopes. So, this potential epitope may be linked with other molecules to enhance its immunogenicity and used for vaccine development.     

Burkholderia pseudomallei interferes with inducible nitric oxide synthase (iNOS) production: a possible mechanism of evading macrophage killing

Burkholderia pseudomallei is a causative agent of melioidosis, a life threatening disease which affects humans and animals in tropical and subtropical areas. This bacterium is known to survive and multiply inside cells such as macrophages. The mechanism of host defense against this bacterium is still unknown. In this study, we demonstrated that B. pseudomallei exhibited unique macrophage activation activity compared with Escherichia coli and Salmonella typhi. The mouse macrophage cell line (RAW 264.7) infected with B. pseudomallei at MOI of 0.1:1, 1:1 and 10:1 did not express a detectable level of inducible nitric oxide synthase (iNOS). Moreover, the B. pseudomallei infected cells released TNF-alpha only when they were infected with high MOI (10:1). Unlike the cells infected with B. pseudomallei, the cells infected with E. coli, and S. typhi expressed iNOS even at MOI of 0.1:1. These infected cells also released a significantly higher level of TNF-alpha at the low MOI ratio. The cells that were preactivated with IFN-gamma prior to being infected with B. pseudomallei exhibited an enhanced production of iNOS and TNF-alpha release. The increased macrophage activation activity in the presence of IFN-gamma also correlated with the restriction of the intracellular bacteria survival. Moreover, IFN-gamma also prevented cell fusion and multinucleated cell formation induced by B. pseudomallei, a phenomenon recently described by our group. Altogether, these results indicate that internalization of B. pseudomallei failed to trigger substantial macrophage activation, a phenomenon which could prolong their survival inside the phagocytic cells and facilitate a direct cell to cell spreading of B. pseudomallei to neighboring cells.