The identification of novel immunogenic antigens as potential Shigella vaccine components.

BACKGROUND: Shigella is a major diarrheal pathogen for which there is presently no vaccine. Whole genome sequencing provides the ability to predict and derive novel antigens for use as vaccines. Here, we aimed to identify novel immunogenic Shigella antigens that could serve as Shigella vaccine candidates, either alone, or when conjugated to Shigella O-antigen. METHODS: Using a reverse vaccinology approach, where genomic analysis informed the Shigella immunome via an antigen microarray, we aimed to identify novel immunogenic Shigella antigens. A core genome analysis of Shigella species, pathogenic and non-pathogenic Escherichia coli, led to the selection of 234 predicted immunogenic Shigella antigens. These antigens were expressed and probed with acute and convalescent serum from microbiologically confirmed Shigella infections. RESULTS: Several Shigella antigens displayed IgG and IgA seroconversion, with no difference in sero-reactivity across by sex or age. IgG sero-reactivity to key Shigella antigens was observed at birth, indicating transplacental antibody transfer. Six antigens (FepA, EmrK, FhuA, MdtA, NlpB, and CjrA) were identified in in vivo testing as capable of producing binding IgG and complement-mediated bactericidal antibody. CONCLUSIONS: These findings provide six novel immunogenic Shigella proteins that could serve as candidate vaccine antigens, species-specific carrier proteins, or targeted adjuvants.

Shigella antigen-specific B memory cells are associated with decreased disease severity in subjects challenged with wild-type Shigella flexneri 2a.

The role of Shigella-specific B memory (BM) in protection has not been evaluated in human challenge studies. We utilized cryopreserved pre- and post-challenge peripheral blood mononuclear cells and sera from wild-type Shigella flexneri 2a (wt-2457T) challenges. Challenged volunteers were either naïve or subjects who had previously ingested wt-2457T or been immunized with hybrid Escherichia coli-Shigella live oral candidate vaccine (EcSf2a-2). BM and antibody titers were measured against lipopolysaccharide (LPS) and recombinant invasion plasmid antigen B (IpaB); results were correlated with disease severity following challenge. Pre-challenge IgA IpaB-BM and post-challenge IgA LPS-BM in the previously exposed subjects negatively correlated with disease severity upon challenge. Similar results were observed with pre-challenge IgG anti-LPS and anti-IpaB titers in vaccinated volunteers. Inverse correlations between magnitude of pre-challenge IgG antibodies to LPS and IpaB, as well as IgA IpaB-BM and post-challenge IgA LPS-BM with disease severity suggest a role for antigen-specific BM in protection.

A multi-center randomized trial to assess the efficacy of gatifloxacin versus ciprofloxacin for the treatment of shigellosis in Vietnamese children.

BACKGROUND: The bacterial genus Shigella is the leading cause of dysentery. There have been significant increases in the proportion of Shigella isolated that demonstrate resistance to nalidixic acid. While nalidixic acid is no longer considered as a therapeutic agent for shigellosis, the fluoroquinolone ciprofloxacin is the current recommendation of the World Health Organization. Resistance to nalidixic acid is a marker of reduced susceptibility to older generation fluoroquinolones, such as ciprofloxacin. We aimed to assess the efficacy of gatifloxacin versus ciprofloxacin in the treatment of uncomplicated shigellosis in children. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a randomized, open-label, controlled trial with two parallel arms at two hospitals in southern Vietnam. The study was designed as a superiority trial and children with dysentery meeting the inclusion criteria were invited to participate. Participants received either gatifloxacin (10 mg/kg/day) in a single daily dose for 3 days or ciprofloxacin (30 mg/kg/day) in two divided doses for 3 days. The primary outcome measure was treatment failure; secondary outcome measures were time to the cessation of individual symptoms. Four hundred and ninety four patients were randomized to receive either gatifloxacin (n=249) or ciprofloxacin (n=245), of which 107 had a positive Shigella stool culture. We could not demonstrate superiority of gatifloxacin and observed similar clinical failure rate in both groups (gatifloxacin; 12.0% and ciprofloxacin; 11.0%, p=0.72). The median (inter-quartile range) time from illness onset to cessation of all symptoms was 95 (66-126) hours for gatifloxacin recipients and 93 (68-120) hours for the ciprofloxacin recipients (Hazard Ratio [95%CI]=0.98 [0.82-1.17], p=0.83). CONCLUSIONS: We conclude that in Vietnam, where nalidixic acid resistant Shigellae are highly prevalent, ciprofloxacin and gatifloxacin are similarly effective for the treatment of acute shigellosis.

Selective deletion of CD8(+) cells upregulated by caspases-1 via IL-18 in mice immunized with major outer membrane protein of Shigella dysenteriae 1 following infection.

INTRODUCTION: Mucosal lymphoid changes were observed in cryopreserved rectal tissues obtained from BALB/c mice infected with Shigella dysenteriae 1, immunized with 57-kDa major antigenic outer membrane protein, and infection after immunization. DISCUSSION: Our data suggested that caspase-3 is downregulated in CD4(+) cells of immunized BALB/c mice following infection with substantial increased expression of interleukin (IL)-2 and interferon (IFN)-gamma, while caspase-1 is upregulated in CD8(+) cells with decreased expression of IL-4 and IL-10. This indicated an involvement of Fas-mediated lytic pathway for selective deletion of CD8(+) cells out of CD3(+) T cells. IL-18 promotes inflammation and induces IFN-gamma and tumor necrosis factor (TNF)-alpha as the expression of IFN-gamma and TNF-alpha cytokines was evident in this study. It is assumed that the role of caspase-1 in inducing the CD4+ T cell activity increased with IL-18 rather than CD8+ suppressor cell activity. Bcl-2 is capable of inhibiting the Fas/Fas-L-mediated cell death for helper cells. Overall, the findings indicate that majority of the apoptotic cells were CD8(+) T cells in the groups of infection following immunization, and there might be a selective deletion of T lymphocytes mediated by caspase-1 via IL-18.

Safety and colonization of two novel VirG(IcsA)-based live Shigella sonnei vaccine strains in rhesus macaques (Macaca mulatta).

Shigella are gram-negative bacterium that cause bacillary dysentery (shigellosis). Symptoms include diarrhea and discharge of bloody mucoid stools, accompanied by severe abdominal pain, nausea, vomiting, malaise, and fever. Persons traveling to regions with poor sanitation and crowded conditions become particularly susceptible to shigellosis. Currently a vaccine for Shigella has not been licensed in the United States, and the organism quickly becomes resistant to medications. During the past 10 y, several live attenuated oral Shigella vaccines, including the strain WRSS1, have been tested in humans with considerable success. These Phase I vaccines lack the gene for the protein VirG also known as IcsA, which enables the organism to disseminate in the host target tissue. However, 5% to 20% of the vaccinated volunteers developed mild fever and brief diarrhea, and the removal of additional virulence-associated genes from the vaccine strain may reduce or eliminate these side effects. We administered 2 Shigella sonnei vaccines, WRSs2 and WRSs3, along with WRSS1 to compare their rates of colonization and clinical safety in groups of 5 rhesus macaques. The primate model provides the most physiologically relevant animal system to test the validity and efficacy of vaccine candidates. In this pilot study using a gastrointestinal model of infection, the vaccine candidates WRSs2 and WRSs3, which have additional deletions in the enterotoxin and LPS modification genes, provided better safety and comparable immunogenicity to those of WRSS1.

[Proinflammatory cytokines in children with acute enteric infections caused by enterobacteria].

The levels of the proinflammatory cytokines including interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) was investigated in 73 children in the age range from 3 to 17 years with slight and medium-heavy diarrheal illness caused by pathogenic and conditionally pathogenic entrobacteria. Strong positive correlation was found between: the levels of IL-1beta, TNF-alpha and intoxication rate (r=0.65 and r=0.49); height of temperature and duration of fever (r=0.86 and r=0.66); dyspepsia (r=0.48 and r=0.41); diarrhea (r=0.37 and r=0.54) and changes in blood including number of leucocytes (r=0.40 and r=0.52) and level of erythrocyte sedimentation rate (ESR) (r=0.65 and r=0.52). Medium positive relationship between the level of IL-6 and intoxication rate (r=0.40 and r=0.52), height of temperature and duration of fever (r=0.45), changes in blood including ESR (r=0.42) and number of leucocytes (r=0.46) was shown. There was a strong positive correlation between IL-1beta and TNF-alpha (r=0.74), between IL-6 and TNF-alpha (r=0.71) and a medium positive correlation between IL-1beta and IL-6 (r=0.61). Considerable decrease in concentration of all cytokines during early period of convalescent at disease with no complications was revealed. Change in concentration of IL-1beta and TNF-alpha determines both intoxication rate and fever. High level of IL-6 is related with complications and lingering course of disease.

Immunogenicity and characterization of WRSF2G11: a second generation live attenuated Shigella flexneri 2a vaccine strain.

Recent clinical trials involving live attenuated Shigella vaccine strains SC602 and WRSS1 have revealed that deletion of the virG(icsA) gene dramatically reduces virulence in human volunteers. These strains can be given at low oral doses and induce a strong, and in some cases, protective immune responses. However, residual vaccine associated reactogenicity suggests that further attenuation is required. A recent clinical trial indicated that the set and sen enterotoxin genes contribute to the symptoms of fever and diarrhea observed with live Shigella vaccine strains. Based on these findings, a Shigella flexneri 2a vaccine candidate, WRSf2G11, with deletions in the virG(icsA), set and sen genes has been constructed using the lambda red recombinase system. The immunogenicity and protective efficacy of WRSf2G11 compares favorably with SC602 following either intranasal (IN) or ocular (OC) immunization of guinea pigs. Taken together, these data indicate that second generation virG-based Shigella vaccine strains which lack enterotoxin genes, such as WRSf2G11, will likely show lower levels of reactogenicity without hampering the robust immune responses achieved with previous live vaccines.

Immunoproteome analysis of soluble and membrane proteins of Shigella flexneri 2457T.

AIM: To profile the immunogenic proteins of Shigella flexneri (S. flexneri) expressed during human infection using a proteomic approach. METHODS: Soluble and membrane protein extractions of S. flexneri 2457T were separated by two-dimensional gel electrophoresis (2-DE). Proteins were transferred to PVDF membrane and immunoblotted with sera from shigellosis patients. Reactive protein spots were matched to Coomassie stained gels run in parallel, cut out and trypsin digested. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) was used to determine the peptide mass fingerprints, which were searched in the MASCOT database to identify the protein. RESULTS: A total of 8 immunoreactive proteins were successfully identified from the Coomassie stained gels in three repeats. Six of these proteins have not previously been reported as immunogenic in S. flexneri. These proteins could be potential candidates for vaccine or attenuation studies. CONCLUSION: Soluble and membrane proteins of S. flexneri 2457T have been screened by 2-DE and immunoblotting with sera from shigellosis patients. Eight proteins are identified as immunogenic.

Acute renal dysfunction in salmonella gastroenteritis.

GOALS: To determine the incidence of renal function deterioration in adult patients with Salmonella infection. BACKGROUND: Renal impairment has been described during severe Salmonella infection and is mainly due to shock, dehydration, or rhabdomyolysis. However, it is unclear whether less severe Salmonella infection also has an impact on kidney function. STUDY: We retrospectively reviewed over a 2-year period the data of all hospitalized adult patients with microbiologically proven gastrointestinal infection. Different biologic parameters were compared between patients infected with Salmonella and patients with other gastrointestinal infections. RESULTS: One hundred and seven patients with positive stool cultures were identified; 44 of them had proven Salmonella infection. Renal dysfunction, defined as an increase in serum creatinine above 1.5 mg/dL in men and above 1.3 mg/dL in women, was observed in 16 (36%) patients infected by Salmonella but only in 3 (5%) comparators (P<0.0001). Hydration status and creatine kinase levels were not different in patients affected by Salmonella as compared with other pathogens. Kidney function recovered in all but 1 patient. CONCLUSIONS: Salmonella gastroenteritis in adults is frequently accompanied by renal dysfunction that is caused by mechanisms other than dehydration or rhabdomyolysis.

The diagnostic value of serum interleukins 6 and 8 in children with acute gastroenteritis.

OBJECTIVES: Early identification of the pathogen causing acute gastroenteritis in children helps the physicians managing the disease and prevents unnecessary antibiotic treatment. C-reactive protein (CRP), interleukin (IL) 6 and IL-8 play a major role in immune responses and have been studied in a large number of infectious and noninfectious inflammatory diseases. The purpose of this study was to determine the serum IL-6 and IL-8 concentrations early in the course of acute gastroenteritis to see if these cytokines were useful diagnostic markers in differentiating viral from bacterial gastroenteritis. METHODS: Interleukin 6, IL-8 and CRP were measured in 18 patients with bacterial gastroenteritis, 21 patients with viral gastroenteritis and 17 healthy children. RESULTS: Interleukin 6 and CRP concentrations in patients with bacterial gastroenteritis were significantly higher than those in patients with viral gastroenteritis and healthy controls (P < 0.001). IL-8 concentrations in patients with viral and bacterial gastroenteritis were both increased and were not statistically different. IL-6 and IL-8 levels had diagnostic sensitivities of 79% and 50% and specificities of 86% and 67%, respectively. The combination of IL-6 and CRP had a sensitivity of 94%, specificity of 71%, a positive predictive value of 74% and a negative predictive value of 93.75%. CONCLUSIONS: Serum IL-6 may be a useful marker for early differentiation of viral and bacterial gastroenteritis in children, especially in combination with CRP.

[T-lymphocyte subpopulations and tumor necrosis factor-alpha in patients with acute Flexner's dysentery]. / Subpopuliatsii T-limfotsitov i factor nekroza opukholi alpha u bol'nykh ostroi dizenteriei Fleksnera..

In 41 patients aged 24-63 years with acute Flexner's dysentery T-lymphocyte subpopulations CD-3, CD-4, CD-8, IRI and the content of tumor necrosis factor alpha (TNF-alpha) in the blood serum were detected. The suppression of T-cell mediated immunity on account of the presence of CD-4 with decreased immunoregulatory index and an increased content of TNF-alpha were established. Changes in T-cell mediated immunity and TNF-alpha depended on the period and the severity of the diseases.

The factor C3 conversion in human complement by smooth Shigella flexneri lipopolysaccharides.

Shigella flexneri rods play an important role in human intestinal infections. In the presented studies we have shown that O-acetyl and glucose residues, substituted in main GalNAc-Rha chains of lipopolysaccharide (LPS) are important for the bactericidal effect of human serum. By dot-blot, immunoblotting and ELISA with immobilized LPS we have shown correlation of C3 fragments deposition and serum resistance. LPSs isolated from a serum-sensitive strain deposited more C3 fragments than LPSs from serum-resistant Shigella flexneri strains.

[Some aspects of evaluating endogenous intoxication in patients with acute dysentery]. / Nekotorye voprosy otsenki éndogennoi intoksikatsii u bol'nykh ostroi dizenteriei.

AIM: To evaluate endogenic intoxication in patients with acute dysentery (AD) by examination of substances of low and medium molecular mass (LM and MM) in different body media. MATERIAL AND METHODS: 122 patients with acute dysentery were tested for LM and MM substances by M. Ya. Malakhova in plasm, red cells, urine in the course of the disease. RESULTS: Concentration of LM and MM substances in patients with AD was much higher compared to healthy subjects in all the examined fluids at the height of the disease. In decline of clinical symptoms the studied concentrations diminished, in early convalescence the substances were at normal levels in red cells and urine being higher in plasm. Maximal changes of the LM and MM substances' concentrations were noted in a severe course of AD. CONCLUSION: In acute bacterial dysentery concentration of LM and MM substances in blood plasm, red cells and urine depend on the period of the disease course and AD severity. This may indicate the degree of endogenic intoxication in these patients.

[Detection of tissue specific antigen-binding lymphocytes in patients with acute enteric infections]. / Vyiavlenie antigensviazyvaiushchikh limfotsitov tkanevoi spetsifichnosti u bol'nykh ostrymi kishechnymi infektsiiami.

A total of 88 patients with salmonellosis, acute dysentery, alimentary toxicoinfection, acute gastroenterocolitis were examined. The study was aimed at early determination of the involvement of organs and tissues into the inflammatory process and detection of antigen-binding lymphocytes with the use of erythrocytic immunoreagents prepared from tissue antigens of mucous membranes of small and large intestine, duodenum, stomach, gall bladder, as well as liver and pancreas. The study demonstrated that as early as on day 1-3 of the disease the development of the inflammatory process in different organs was accompanied by the appearance of the corresponding tissue specific (organ specific) antigen-binding lymphocytes in all patients. As a rule, patients with different acute enteric diseases significantly differed by the frequency and spectrum of the involvement of such organs and tissues into pathological process.

[Priming-phenomenon of neutrophils in patients with Flexner infection]. / Praiming-fenomen neitrofilov u bol'nykh shigellezom Fleksnera.

AIM: To study reproducibility of priming phenomenon of neutrophils in patients with acute Flexner's dysentery and its realization manifestation. MATERIAL AND METHODS: A chemiluminescent response of peripheral blood neutrophils was studied in patients with acute mild and moderate dysentery in the presence of luminol. Lipopolysaccharide Salmonella typhimurium at a final concentration of 20 ng/ml served as a priming substance of S-chemotype. Neutrophils were stimulated with phorbol myristate acetate in concentration 10(-6) M and isolated on Histopaque double gradient (Sigma reagents, USA). RESULTS: In reproduction of priming-phenomenon in vitro on neutrophils of patients with acute Flexner's dysentery chemiluminescence amplitude increased 1.29-1.69-fold vs control. CONCLUSION: Priming-phenomenon on neutrophils in acute dysentery is reproducible. This confirms modulating properties of lipopolysaccharides in conditions of systemic nonphysiological endotoxinemia. Priming phenomenon may be involved in both maintenance of homeostasis and pathophysiological processes.

Disease-dependent changes in T-cell populations in patients with shigellosis.

In shigellosis, bacterial infection is associated with an extensive inflammation of the rectal mucosa, resulting in bloody dysentery. The role of T-cell-mediated pro-inflammatory mechanisms has been implicated in this process, but the specific role of T-cell subsets is still not well understood. In this study we attempted to identify the changes in T-cell populations in patients with shigellosis during the disease course. The T-cell subset distribution was analyzed by immunohistochemistry in the rectal mucosa and by immuno-flow cytometry in the peripheral blood. Blood and rectal biopsies were studied from patients with Shigella dysenteriae 1 (n= 11) and S. flexneri (n= 11) infection and 20 healthy age-matched controls. We found an expansion of gammadelta+T cells in the rectal mucosa, but a decrease in the percentage of gammadelta+T cells in the blood in acute shigellosis. There was also a preferential increase in CD8+ T cells in the surface epithelium of rectal tissue in patients infected with S. dysenteriae 1, but not in patients infected with S. flexneri. Our findings suggest that the rectal mucosal inflammation in shigellosis is associated with an expansion of T cells, in particular CD8+ and gammadelta+T-cell subsets in the gut mucosa, which may be of importance for the pathogenesis of shigellosis.

[The assessment of the course of an infectious process by the membrane status of the peripheral blood lymphocytes]. / Otsenka techeniia infektsionnogo protsessa po sostoianiiu membran limfotsitov perifericheskoi krovi.

Lipid peroxidation in lymphocyte membranes is studied in patients with acute viral hepatitis B and B + C and in chronic alcoholics with Flexner's dysentery and with uneventful premorbid history. The intensity of lipid peroxidation in lymphocytes was increased, corresponding to the severity and period of infection. The premorbid background and therapy influenced the lymphocyte membrane status, which can serve as an integral indicator of immunological reactivity of the organism.

Protein conjugates of synthetic saccharides elicit higher levels of serum IgG lipopolysaccharide antibodies in mice than do those of the O-specific polysaccharide from Shigella dysenteriae type 1.

Our development of vaccines to prevent shigellosis is based on the hypothesis that a critical (protective) level of serum IgG to the O-specific polysaccharide (O-SP) domain of Shigella lipopolysaccharide (LPS) confers immunity. The O-SP is a hapten and must be conjugated to a protein to induce serum antibodies. The O-SP of Shigella dysenteriae type 1 (approximately 27 tetrasaccharide repeat units), prepared by acid hydrolysis of the LPS, was bound to human serum albumin (HSA) by multiple point attachment (O-SP-HSA): The molar ratio of HSA to O-SP was 1.0. Synthetic saccharides, composed of one or multiples of the O-SP tetrasaccharide, equipped with a spacer at their reducing end, were bound to HSA by a single point attachment: The average molar ratios of the saccharides to HSA ranged from 4 to 24. Serum IgG anti-LPS, elicited in mice by O-SP-HSA or synthetic tetra-, octa-, dodeca-, and hexadecasaccharide fragments, was measured by ELISA. Outbred 6-week-old female mice were injected s.c. three times at biweekly intervals with 2.5 micrograms of saccharide as a conjugate and were bled 7 days after the second and third injections. Excepting the tetramer, conjugates of the octamer, dodecamer and hexadecamer elicited IgG LPS antibodies after the second injection, a statistically significant rise (booster) after the third injection, and higher levels than those vaccinated with O-SP-HSA (P = 0.0001). The highest geometric mean levels of IgG anti-LPS were elicited by the hexadecamer with 9 chains or 9 moles of saccharide/HSA (15.5 ELISA units) followed by the octamer with 20 chains (11.1 ELISA units) and the dodecamer with 10 chains (9.52 ELISA units). Clinical evaluation of these synthetic saccharides bound to a medically useful carrier is planned.

Antibodies to Shiga toxin in the serum of children with Shigella-associated haemolytic uraemic syndrome.

Antibodies to Shiga toxin (Stx) were measured in the sera of 49 children with Shigella dysenteriae serotype 1 infection, of whom 17 had haemolytic uraemic syndrome (HUS) and 32 had no complications (uncomplicated shigellosis, UCS). Children with HUS had lower levels of total IgG and IgM and lower IgM titres to Stx than those with UCS. The number of children with neutralising antibodies was similar in the two groups. Of the children with HUS, 11 had HUS on enrolment and six developed HUS subsequent to enrolment. Antibody titres in children who subsequently developed HUS were compared with those in children with UCS to assess whether differences in antibody titres occurred before the development of HUS. IgA titres to Stx were found to be higher in children who subsequently developed HUS than in those with UCS. However, logistic regression analysis revealed that titres of Stx antibodies in the serum were not significant risk factors for the development of HUS. Thus, although the levels of Stx antibodies were different in children with HUS, and higher IgA titres to Stx were identifiable in children who subsequently developed HUS compared with those with UCS, the relevance of these findings in the development of HUS remains to be elucidated.

[Functional heterogeneity of polymorphonuclear leukocytes population in peripheral blood of patients with acute dysentery]. / Geterogennost' funktsional'nogo sostoianiia populiatsii polimorfno-iadernykh le'ikotsitov perifericheskoi krovi u bol'nykh ostroi dizenteriei.

AIM: To study chemiluminescence (CL) of polymorphonuclear neutrophils (PMNL) in peripheral blood of patients with acute mild and moderate dysentery caused by Shig. Flexneri in response to Re- and S-chemotype lipopolysaccharide (LPS) Sh. Flexneri and agonists fMLP, PMA and OZ. RESULTS: PMNL population proved functionally heterogeneic, correlating with acute dysentery severity. Subgroups of PMNL varied by CL amplitude, reserve ability to additional stimulation. LPS rather modulated CL of PMNL than stimulated it. CL intensity depended on LPS structure and concentration. Mild acute dysentery was characterized by pool of primed PMNL which had higher functional reserves. CONCLUSION: CL analysis can be used for raising accuracy of dysentery prognosis and severity, assessment of implications of functional state of neutrophil granulocytes for support of antiendotoxin immunity in acute dysentery.