Toxin Antigen Research Articles

Antibody levels to Bordetella pertussis and Neisseria meningitidis in immunodeficient patients receiving immunoglobulin replacement therapy.

Patients with antibody deficiency rely on immunoglobulin products for protection against many vaccine-preventable diseases. We measured antibody titers against Bordetella pertussis and Neisseria meningitidis in patients receiving immunoglobulin (IG) therapy to determine if they have any protection against infections from these organisms. In an unblinded, prospective assessment we measured antibody titers against B. pertussis filamentous hemagglutinin (FHA) and pertussis toxin (PT) antigens and N. meningitidis serogroups A, C, W-135, and Y in patients receiving immunoglobulin therapy for primary immune deficiency (PI). We measured steady state levels in patients receiving subcutaneous immunoglobulin therapy while in patients receiving intravenous immunoglobulin therapy we measured titers immediately before and after infusion to more clearly define the contribution of the infused product. Thirty subjects, 17 females and 13 males, participated in the study, 22 were receiving intravenous IG products and 8 were receiving subcutaneous IG products. Diagnoses included common variable immunodeficiency in 12, combined immunodeficiency in 6, specific antibody deficiency in 6, X-linked agammaglobulinemia in 4 and ataxia telangiectasia and hyper-IgE syndrome in one each. All subjects had detectable IgG antibodies against the pertussis antigens measured and most had antibody to the meningococcus serotypes measured. However, only 26.6 % had protective levels (>= 2 mcg/mL) against serogroup C at trough or steady state. Patients receiving immunoglobulin therapy have antibodies against B. pertussis and most of them have antibodies against the four measured serogroups of N. meningitidis. There is significant variability in the levels of antibody between patients and the low titers against group C may suggest a role for active immunization in those who may respond to conjugated polysaccharide vaccine administration.

Characterization and immunological activity of different forms of recombinant secreted Hc of botulinum neurotoxin serotype B products expressed in yeast

The recombinant Hc proteins of botulinum neurotoxins and tetanus toxin are exclusively produced by intracellular heterologous expression in Pichia pastoris for use in subunit vaccines; the same Hc proteins produced by secreted heterologous expression are hyper-glycosylated and immunologically inert. Here, several different recombinant secreted Hc proteins of botulinum neurotoxin serotype B (BHc) were expressed in yeast and we characterized and assessed their immunological activity in detail. Recombinant low-glycosylated secreted BHc products (BSK) were also immunologically inert, similar to hyper-glycosylated BHc products (BSG), although deglycosylation restored their immunological activities. Unexpectedly, deglycosylated proBHc contained an unexpected pro-peptide of an α-factor signal and fortuitous N-linked glycosylation sites in the non-cleaved pro-peptide sequences, but not in the BHc sequences. Notably, a non-glycosylated secreted homogeneous BHc isoform (mBHc), which we successfully prepared after deleting the pro-peptide and removing its single potential glycosylation site, was immunologically active and could confer effective protective immunity, similarly to non-glycosylated rBHc. In summary, we conclude that a non-glycosylated secreted BHc isoform can be prepared in yeast by deleting the pro-peptide of the α-factor signal and mutating its single potential glycosylation site. This approach provides a rational and feasible strategy for the secretory expression of botulism or other toxin antigens.

Doubly Phosphorylated Peptide Vaccines to Protect Transgenic P301S Mice against Alzheimer's Disease Like Tau Aggregation.

Intracellular neurofibrillary tangles and extracellular senile plaques are potential targets for active and passive immunotherapies. In this study we used the transgenic mouse model P301S for active immunizations with peptide vaccines composed of a double phosphorylated tau neoepitope (pSer202/pThr205, pThr212/pSer214, pThr231/pSer235) and an immunomodulatory T cell epitope from the tetanus toxin or tuberculosis antigen Ag85B. Importantly, the designed vaccine combining Alzheimer’s disease (AD) specific B cell epitopes with foreign (bacterial) T cell epitopes induced fast immune responses with high IgG1 titers after prophylactic immunization that subsequently decreased over the observation period. The effectiveness of the immunization was surveyed by evaluating the animal behavior, as well as the pathology in the brain by biochemical and histochemical techniques. Immunized mice clearly lived longer with reduced paralysis than placebo-treated mice. Additionally, they performed significantly better in rotarod and beam walk tests at the age of 20 weeks, indicating that the disease development was slowed down. Forty-eight weeks old vaccinated mice passed the beam walk test significantly better than control animals, which together with the increased survival rates undoubtedly prove the treatment effect. In conclusion, the data provide strong evidence that active immune therapies can reduce toxic effects of deposits formed in AD.

A case of toxic epidermal necrolysis with extensive intestinal involvement

Toxic epidermal necrolysis (TEN) is life-threatening cutaneous adverse drug reaction characterized by extensive keratinocyte death. Gastrointestinal (GI) involvement in TEN is rare, however it contributes to poor prognosis. Here, we report a case of TEN with extensive intestinal involvement. A 56-year-old female was treated by zonisamide for fibromyalgia. Ten days later, generalized erythema appeared with blister, superficial punctate keratitis, and severe diarrhea. Skin biopsy specimen revealed that marked keratinocyte death. She was diagnosed with Stevens-Johnson syndrome (SJS). In spite of methylprednisolone pulse therapy for three days and plasma exchange three times, the bullous lesions spread to cover 60% of the body surface area. She was finally diagnosed with TEN, and treated with high-dose intravenous immunoglobulins for three days and another methylprednisolone pulse therapy. After these therapies, skin lesions were gradually improved. Despite skin manifestation improvement, the patient's diarrhea persisted (2000ml/day), resulting in hypoalbuminemia (0.8g/dL). Upper endoscopy revealed extensive erosions in esophagus and duodenum. Colonoscopy revealed multiple erosions and edematous structures in distal ileum and colon. Biopsy specimens of ileum and colon showed extensive mucosal detachment and granulation tissues. Examinations of Clostridium difficile toxin and cytomegalovirus antigen were negative. According to these findings, we considered her diarrhea to be consistent with GI lesions related in TEN. We treated her with mucoprotective agent, total parenteral nutrition management, albumin supplementation, and tapered systemic corticosteroid slowly. GI symptoms were gradually improved. Approximately 15 cases of SJS/TEN with GI manifestations have been reported in English literature. The mortality rate of SJS/TEN with GI manifestations is about 45%. There is no specific treatment, and treatment has largely been supportive. GI symptoms are often delayed, as in our case. GI mucosal damage may result in enteral nutrition difficulty, hypoalbuminemia, poor wound healing and bacterial translocation. The prolonged intestinal lesions may be attributable to difficulties with re-epithelization of intestinal mucosa by digestive fluid and formation of granulation tissue. Intestinal involvement of SJS/TEN is refractory and life-threatening complication. Regardless of the severity of skin manifestations, we should carefully monitor the GI symptoms.

Generation of an attenuated Salmonella-delivery strains expressing adhesin and toxin antigens for progressive atrophic rhinitis, and evaluation of its immune responses in a murine model

An expression/secretion plasmid containing genes encoding the FimA, CP39, PtfA, ToxA and F1P2 antigens associated with porcine pneumonic pasteurellosis and progressive atrophic rhinitis (PAR) was constructed and harbored in an attenuated Salmonella Typhimurium, which was used as the vaccine candidate. The immune responses induced by this delivery strain were investigated in a murine model. Each antigen secreted from the delivery strain was confirmed by Western blot analysis. Thirty BALB/c mice were divided equally into two groups; group A were intranasally inoculated with the mixture of the five delivery strains, and group B were inoculated with sterile PBS. In group A, all antigen-specific serum IgG were significantly increased compared to those of group B from the 2nd week post-inoculation (WPI) till the 8th WPI. All antigen-specific mucosal IgA in group A were also significantly greater than those of group B. In addition, the significant splenic lymphocyte proliferative responses, the elevations of CD3+CD4+, CD3+CD8+ and B-cell populations, and the induction of IFN-γ expression in group A were observed. In conclusion, the mixture of five delivery strains expressing specific antigen for these diseases was found to be capable of inducing significant humoral and cellular immune responses.

Mo1949 Rapamycin Protects Intestinal Ischemia/Reperfusion-Induced Remote Lung Injury in Mice

Background: Enterotoxigenic Escherichia coli (ETEC), defined by their production of heat labile (LT) and/or heat stable (ST) toxins, are major causes of infantile diarrhea in developing countries and of travelers' diarrhea in visitors to the developing world. Despite our understanding of ETEC pathogenesis, no broadly protective ETEC vaccine is available. This is largely a result of the difficulty in inducing immunity to the small ST molecule. Our goal is to develop a broadly protective live attenuated vaccine against ETEC by taking advantage of the recent demonstration (Zhang et al.,2013,PLoSONe 8 (10):e77386) that genetically produced fusions of mutant ST with LT components can induce effective immunity against both toxins. Methods: To achieve this aim, we engineered an attenuated strain of E. coli, which we have developed as a vaccine vector for mucosal antigen delivery, to express immunogenic fusion constructs of LT and mutant ST. Our vaccine vector strain ZCR533, is a derivative of O157:H7 enterohemorrhagic E. coli (EHEC) strain 86-24 attenuated by deleting its genes for Shiga toxin production and truncating its major adhesin, intimin, such that it can no longer adhere intimately, but can still induce anti-intimin antibody. To express LT-ST fusions in immunogenic form in our vector strain, we used restriction-free cloning technology to incorporate them into the passenger domain of an autotransporter protein (EspP) expressed on a medium copy number plasmid. This system is designed to express the toxin antigens in either surface-bound or secreted forms, and to provide effective delivery to the mucosal immune system as we have done for other toxin antigens. Results: The incorporation of the LT-ST fusions into the passenger domain of the EspP autotransporter plasmids was confirmed by PCR and DNA sequencing. Protein expression by ZCR533 containing the autotransporter plasmids was confirmed by SDS-PAGE, and Western blot of whole cell lysates and culture supernates, using poly-clonal antisera to the LT component. Moreover, appropriate expression of the LT-ST fusions on the bacterial surface of ZCR533 was confirmed by immuno-fluorescent staining with the specific antisera. Conclusions: These studies show that immunogenic fusions of LT and mutant ST can be expressed on the surface, and in the culture supernates, of our attenuated E. coli vaccine vector ZCR533 via the EspP autotransporter. Since our recent studies using the similarly-sized Shiga toxin B subunit antigen have indicated the validity of this approach to mucosal immunization against toxin antigens, these studies could lead to the development of broadly protective vaccines directed against both LTand ST-producing ETEC, as well as against attaching/ effacing E. coli strains expressing intimin.

Mo1948 Toward a Broadly Protective Vaccine Against Enterotoxigenic E.coli (ETEC) Infections

Background: Enterotoxigenic Escherichia coli (ETEC), defined by their production of heat labile (LT) and/or heat stable (ST) toxins, are major causes of infantile diarrhea in developing countries and of travelers' diarrhea in visitors to the developing world. Despite our understanding of ETEC pathogenesis, no broadly protective ETEC vaccine is available. This is largely a result of the difficulty in inducing immunity to the small ST molecule. Our goal is to develop a broadly protective live attenuated vaccine against ETEC by taking advantage of the recent demonstration (Zhang et al.,2013,PLoSONe 8 (10):e77386) that genetically produced fusions of mutant ST with LT components can induce effective immunity against both toxins. Methods: To achieve this aim, we engineered an attenuated strain of E. coli, which we have developed as a vaccine vector for mucosal antigen delivery, to express immunogenic fusion constructs of LT and mutant ST. Our vaccine vector strain ZCR533, is a derivative of O157:H7 enterohemorrhagic E. coli (EHEC) strain 86-24 attenuated by deleting its genes for Shiga toxin production and truncating its major adhesin, intimin, such that it can no longer adhere intimately, but can still induce anti-intimin antibody. To express LT-ST fusions in immunogenic form in our vector strain, we used restriction-free cloning technology to incorporate them into the passenger domain of an autotransporter protein (EspP) expressed on a medium copy number plasmid. This system is designed to express the toxin antigens in either surface-bound or secreted forms, and to provide effective delivery to the mucosal immune system as we have done for other toxin antigens. Results: The incorporation of the LT-ST fusions into the passenger domain of the EspP autotransporter plasmids was confirmed by PCR and DNA sequencing. Protein expression by ZCR533 containing the autotransporter plasmids was confirmed by SDS-PAGE, and Western blot of whole cell lysates and culture supernates, using poly-clonal antisera to the LT component. Moreover, appropriate expression of the LT-ST fusions on the bacterial surface of ZCR533 was confirmed by immuno-fluorescent staining with the specific antisera. Conclusions: These studies show that immunogenic fusions of LT and mutant ST can be expressed on the surface, and in the culture supernates, of our attenuated E. coli vaccine vector ZCR533 via the EspP autotransporter. Since our recent studies using the similarly-sized Shiga toxin B subunit antigen have indicated the validity of this approach to mucosal immunization against toxin antigens, these studies could lead to the development of broadly protective vaccines directed against both LTand ST-producing ETEC, as well as against attaching/ effacing E. coli strains expressing intimin.

Seroepidemiology of Bordetella pertussis Infection in Fresh College Students in Iran: A Multicenter Study

Background: Pertussis is a highly communicable, vaccine-preventable respiratory disease; which may circulate even in populations with high vaccination coverage. Although frequent, but it is often underestimated as a cause of prolonged cough illness in both children and adults. Without boosting, the protection of the childhood vaccination would be minimal after 10 years. The need for adult booster vaccination depends on the national epidemiology. Objectives: The aim of this study was to evaluate the seroepidemiological incidence of Pertussis in fresh college students in four majors. Patients and Methods: In a cross sectional multicenter study, blood samples were obtained from 1617 junior students of four universities. None had received Pertussis booster vaccine in the preceding 10 years. Serum immunoglobulin G (IgG) antibody for Pertussis toxin antigen was measured. Some social and demographic determinants including age, sex and number of family members were recorded. Results: The mean age of participants was 19.64 ± 2.1 years; positive anti Pertussis toxin IgG levels (by cut point of 94 U/mL) was detected in 31.6%. Positivity rate was associated with sex but not with age or residential area. Conclusions: Pertussis continues to challenge medical and public health professionals. Recent reports of increases in the prevalence and incidence may be because of the limited time of protection of childhood vaccination. Our study can serve as one of the scarce population-based reports from developing countries. A universal cut point should be determined for diagnosis of seropositivity, and a booster of a cellular vaccine is recommended in adolescence.

Equivalent T Cell Epitope Promiscuity in Ecologically Diverse Human Pathogens

BackgroundThe HLA (human leukocyte antigen) molecules that present pathogen-derived epitopes to T cells are highly diverse. Correspondingly, many pathogens such as HIV evolve epitope variants in order to evade immune recognition. In contrast, another persistent human pathogen, Mycobacterium tuberculosis, has highly conserved epitope sequences. This raises the question whether there is also a difference in the ability of these pathogens’ epitopes to bind diverse HLA alleles, referred to as an epitope’s binding promiscuity. To address this question, we compared the in silico HLA binding promiscuity of T cell epitopes from pathogens with distinct infection strategies and outcomes of human exposure.MethodsWe used computer algorithms to predict the binding affinity of experimentally-verified microbial epitope peptides to diverse HLA-DR, HLA-A and HLA-B alleles. We then analyzed binding promiscuity of epitopes derived from HIV and M. tuberculosis. We also analyzed promiscuity of epitopes from Streptococcus pyogenes, which is known to exhibit epitope diversity, and epitopes of Bacillus anthracis and Clostridium tetani toxins, as these bacteria do not depend on human hosts for their survival or replication, and their toxin antigens are highly immunogenic human vaccines.ResultsWe found that B. anthracis and C. tetani epitopes were the most promiscuous of the group that we analyzed. However, there was no consistent difference or trend in promiscuity in epitopes contained in HIV, M. tuberculosis, and S. pyogenes.ConclusionsOur results show that human pathogens with distinct immune evasion strategies and epitope diversities exhibit equivalent levels of T cell epitope promiscuity. These results indicate that differences in epitope promiscuity do not account for the observed differences in epitope variation and conservation.

Index of Suspicion

* ALT: : alanine aminotransferase AST: : aspartate aminotransferase BUN: : blood urea nitrogen CBC: : complete blood count CNS: : central nervous system CSF: : cerebrospinal fluid CT: : computed tomography ECG: : electrocardiography ED: : emergency department EEG: : electroencephalography ESR: : erythrocyte sedimentation rate GI: : gastrointestinal GU: : genitourinary Hct: : hematocrit Hgb: : hemoglobin MRI: : magnetic resonance imaging WBC: : white blood cell An 11-year-old boy presents to the emergency department with a 1-week history of frequent episodes of nonbloody, nonbilious emesis associated with diarrhea and abdominal cramping. He has urgency and fecal incontinence. He has been afebrile and denies any rashes, joint pain, sick contacts, headaches, or other symptoms. On further questioning, his mother reveals that he has had 2 similar episodes of abdominal distension and diarrhea during the past 6 to 7 years. Between these episodes, she states that he normally has nonbloody, hard stools accompanied by straining and chronic abdominal pain and pain with defecation. When asked specifically, his mother does not remember when he passed his first stool after he was born. On physical examination, his temperature is 37.6°C, pulse is 100 beats per minute, respiratory rate is 16 breaths per minute, blood pressure is 113/62 mm Hg, oxygen saturation is 100% on room air, height is 63 inches (95%), and weight is 36.7 kg (25%). He has abdominal distention and diffuse abdominal tenderness with voluntary guarding. There is palpable stool in the left quadrant. There is no perianal erythema or skin tag, and rectal examination reveals a low anal tone. A CT scan of the abdomen (Figure 1) reveals a markedly distended rectosigmoid colon with circumferential, confluent, and contiguous mural thickening. Basic metabolic profile, CBC, and urinalysis results are unremarkable, but the C-reactive protein level is elevated at 131 mg/L (1248 nmol/L). Because of the concern for enterocolitis, he is empirically given broad-spectrum intravenous antibiotics and transferred to a children’s hospital for further evaluation. Figure 1. A CT scan of the abdomen shows a markedly distended rectosigmoid colon with circumferential mural thickening. A 4-year-old boy presents for evaluation of intermittent fevers of 4 weeks’ duration after 2 separate antibiotic courses prescribed for acute otitis media. He has experienced clear rhinorrhea and …

THU0034 CD19 Distinguishes Memory and Pre-Memory Antibody Secreting Cells

BackgroundPlasma cells (PC), are differentiated B cells that play a major role in humoral immunity as they actively secrete antibodies which identify and neutralize foreign antigens. Normally, plasma cells develop...

Mycoplasma pneumoniae in children with acute and refractory asthma

BackgroundThe presence of Mycoplasma pneumoniae has been associated with worsening asthma in children. Sensitive assays have been developed to detect M pneumoniae–derived community-acquired respiratory distress syndrome (CARDS) toxin. ObjectivesTo identify the frequency and persistence of M pneumoniae detection in respiratory secretions of children with and without asthma and to evaluate antibody responses to M pneumoniae and the impact of M pneumoniae on biological markers, asthma control, and quality of life. MethodsWe enrolled 143 pediatric patients (53 patients with acute asthma, 26 patients with refractory asthma, and 64 healthy controls; age range, 5-17 years) during a 20-month period with 2 to 5 follow-up visits. We detected M pneumoniae using CARDS toxin antigen capture and polymerase chain reaction and P1 adhesin polymerase chain reaction. Immune responses to M pneumoniae were determined by IgG and IgM levels directed against CARDS toxin and P1 adhesin. pH was measured in exhaled breath condensates, and asthma control and quality of life were assessed using the Asthma Control Test and Pediatric Asthma Quality of Life Questionnaire. ResultsM pneumoniae was detected in 64% of patients with acute asthma, 65% with refractory asthma, and 56% of healthy controls. Children with asthma had lower antibody levels to M pneumoniae compared with healthy controls. Exhaled breath condensate pHs and asthma control and quality of life scores were lower in M pneumoniae–positive patients with asthma. ConclusionThe results suggest that M pneumoniae detection is common in children, M pneumoniae detection is associated with worsening asthma, and children with asthma may have poor humoral immune responses to M pneumoniae.

Toxin–antigen conjugates as selection tools for antibody producing cells

The generation of antibodies with designated specificity requires cost-intensive and time-consuming screening procedures. Here we present a new method by which hybridoma cells can be selected based on the specificity of the produced antibody by the use of antigen–toxin-conjugates thus eliminating the need of a screening procedure. Initial experiments were done with methotrexate as low molecular weight toxin and fluorescein as model antigen. Methotrexate and a methotrexate–fluorescein conjugate were characterized regarding their toxicity. Afterwards the effect of the fluorescein-specific antibody B13-DE1 on the toxicity of the methotrexate–fluorescein conjugate was determined. Finally, first results showed that hybridoma cells that produce fluorescein specific antibodies are able to grow in the presence of fluorescein–toxin-conjugates.

A non-invasive in vivo imaging system to study dissemination of bioluminescent Yersinia pestis CO92 in a mouse model of pneumonic plague

The gold standard in microbiology for monitoring bacterial dissemination in infected animals has always been viable plate counts. This method, despite being quantitative, requires sacrificing the infected animals. Recently, however, an alternative method of in vivo imaging of bioluminescent bacteria (IVIBB) for monitoring microbial dissemination within the host has been employed. Yersinia pestis is a Gram-negative bacterium capable of causing bubonic, septicemic, and pneumonic plague. In this study, we compared the conventional counting of bacterial colony forming units (cfu) in the various infected tissues to IVIBB in monitoring Y. pestis dissemination in a mouse model of pneumonic plague. By using a transposon mutagenesis system harboring the luciferase (luc) gene, we screened approximately 4000 clones and obtained a fully virulent, luc-positive Y. pestis CO92 (Y. pestis-luc2) reporter strain in which transposition occurred within the largest pMT1 plasmid which possesses murine toxin and capsular antigen encoding genes. The aforementioned reporter strain and the wild-type CO92 exhibited similar growth curves, formed capsule based on immunofluorescence microscopy and flow cytometry, and had a similar LD50. Intranasal infection of mice with 15 LD50 of CO92-luc2 resulted in animal mortality by 72 h, and an increasing number of bioluminescent bacteria were observed in various mouse organs over a 24–72 h period when whole animals were imaged. However, following levofloxacin treatment (10 mg/kg/day) for 6 days 24 h post infection, no luminescence was observed after 72 h of infection, indicating that the tested antimicrobial killed bacteria preventing their detection in host peripheral tissues. Overall, we demonstrated that IVIBB is an effective and non-invasive way of monitoring bacterial dissemination in animals following pneumonic plague having strong correlation with cfu, and our reporter CO92-luc2 strain can be employed as a useful tool to monitor the efficacy of antimicrobial countermeasures in real time.

The Oral, Live Attenuated Enterotoxigenic Escherichia coli Vaccine ACE527 Reduces the Incidence and Severity of Diarrhea in a Human Challenge Model of Diarrheal Disease

An oral, live attenuated, three-strain recombinant bacterial vaccine, ACE527, was demonstrated to generate strong immune responses to colonization factor and toxin antigens of enterotoxigenic Escherichia coli (ETEC) in human volunteers. The vaccine was safe and well tolerated at doses of up to 10(11) CFU, administered in each of two doses given 21 days apart. These observations have now been extended in a phase 2b study with a total of 70 subjects. Fifty-six of these subjects were challenged 28 days after the second dose of vaccine with the highly virulent ETEC strain H10407 to obtain preliminary indicators of efficacy against disease and to support further development of the vaccine for both travelers and infants in countries where ETEC is endemic. The vaccine had a significant impact on intestinal colonization by the challenge strain, as measured by quantitative fecal culture 2 days after challenge, demonstrating the induction of a functional immune response to the CFA/I antigen. The incidence and severity of diarrhea were also reduced in vaccinees as measured by a number of secondary and ad hoc endpoints, although the 27% reduction seen in the primary endpoint, moderate to severe diarrhea, was not statistically significant. Together, these observations support the hypothesis that the ACE527 vaccine has a dual mode of action, targeting both colonization factors and the heat-labile enterotoxin (LT), and suggest that it should be further developed for more advanced trials to evaluate its impact on the burden of ETEC disease in field settings.

High selective and sensitive capillary electrophoresis-based electrochemical immunoassay enhanced by gold nanoparticles

A novel capillary electrophoresis (CE)-based electrochemical (EC) immunoassay (IA) enhanced by gold nanoparticles (AuNPs) was developed for the determination of shellfish toxins. After competitive immunoreaction between shellfish toxin antigen (Ag), horseradish peroxidase (HRP)-labeled antigen (Ag⁎), and limited antibody (Ab), the immune sample was introduced into capillary and conjugated with AuNPs. The presence of AuNPs modifies the mobilities of analytes and improves separation resolution between analytes. Furthermore, the AuNPs were used as multianalyte carriers of the excess signaling Ag* and the bound enzyme-labeled complex (Ag*–Ab) to achieve an amplification of EC signal. Enhanced resolution and sensitivity were obtained by using bioconjugates featuring HRP labels linked to AuNPs. The four shellfish toxins were baseline separated by using AuNPs. The relative standard deviation (RSD) values of the migration time were 1.3–3.5% for intraday and 3.1–4.6% for interday, respectively. The RSDs of the peak area were 2.8–4.6% for intraday and 4.1–6.9% for interday, respectively. The limits of detection (S/N=3) were in the range of 3.1–36.7ng/L. The enhanced CE based EC-IA with AuNPs labels was successfully applied for the simultaneous determination of four shellfish toxins in shellfish samples. With high resolving power and sensitivity, the proposed method has shown great potential to become an attractively alternative method for simultaneous determination of diverse red tide poisons in shellfish samples.

A fully liquid DTaP-IPV-Hep B-PRP-T hexavalent vaccine for primary and booster vaccination of healthy Mexican children

ObjectivesTo evaluate an investigational, fully liquid hexavalent diphtheria-tetanus-acellular pertussis-inactivated poliovirus-hepatitis B-Haemophilus influenzae type b (DTaP-IPV-Hep B-PRP-T: Hexaxim™) vaccine for primary and booster vaccination of healthy children in Mexico. MethodsInfants (N=1189) were randomized to receive one of three lots of the DTaP-IPV-Hep B-PRP-T vaccine or a licensed hexavalent control vaccine (Infanrix™ hexa) for primary vaccination at 2, 4 and 6 months. All participants who completed the primary series and agreed to participate in the booster part of the study received a dose of the investigational vaccine at 15–18 months of age. Validated serological assays and parental reports were used to assess immunogenicity and safety, respectively. ResultsPost-primary vaccination, ≥95.8% of participants in both the DTaP-IPV-Hep B-PRP-T and control groups were seroprotected (SP) against diphtheria, tetanus, poliovirus, hepatitis B and PRP, or had seroconverted (SC) to the pertussis toxin (PT) and filamentous hemagglutinin (FHA) pertussis antigens. The SP/SC rates induced by the three DTaP-IPV-Hep B-PRP-T lots were equivalent. No differences in SP/SC rates were observed between the pooled lots of investigational vaccine and the control vaccine. Antibody persistence at 15–18 months was comparable between groups, with strong increases in all antibody concentrations post-DTaP-IPV-Hep B-PRP-T booster. Both vaccines were well tolerated for primary vaccination, as was the booster dose of DTaP-IPV-Hep B-PRP-T. ConclusionThese study findings confirm the suitability of the combined, fully liquid DTaP-IPV-Hep B-PRP-T vaccine for inclusion in routine childhood vaccination schedules.

Study on immunopathogenic effect of bacterial protein antigen and the cytolytic toxin antigen of vibrio vulnificus in BALB / c Mice

Objective To investigate whether Vibrio vulnificus bacterial protein antigen and the cytolytic toxin antigen can induce the effective immune protection against Vibrio vulnificus infection.Methods BALB/c mice were immunized with bacterial cytolytic toxin antigen protein antigen of Vibrio vulnificus to evaluate its ability to stimulate immune response.The protective efficacy of immunized mice was evaluated by active immunization and intraperitoneal challenge with V.vulnificus in mice.Results The immunized mice produced higher percentage of CD19+ B lymphocytes and high level specific antibodies (titers up to 1∶25600).All immunized mice survived from lethal challenge with V.vulnificus,while only 13.33％ of mice in control group survived at the end of challenged experiment.Conclusions The bacterial protein antigen and cytolytic toxin antigen of Vibrio vulnificus are capable of inducing specific antibody response in mice to confer effective protection against lethal challenge with V.vulnificus. Key words: Vibrio vulnificus; Cytolytic toxin; Bacterial protein antigen; Active immune

Recent progress toward an enterotoxigenic Escherichia coli vaccine

Enterotoxigenic Escherichia coli(ETEC) is the most common cause of bacterial diarrhea in children in Africa, Asia and Latin America and in travelers to these regions. Despite this, no effective vaccine for ETEC is available. ETEC causes disease by colonizing the small intestine with colonization factors, most of which are fimbriae, and production of heat-labile and/or heat-stable enterotoxins. Antibodies against heat-labile enterotoxin and the colonization factors have been shown to be protective, and local immunity in the gut seems to be of prime importance for protection. Hence, several inactivated and live candidate ETEC vaccines consisting of toxin antigens, alone or together with colonization factors, have been evaluated in clinical trials. In this review, the authors describe ETEC vaccine development in progress and the rationale for constructing different types of vaccines. They also discuss possibilities of enhancing immune responses to candidate ETEC vaccines, particularly in children.

Impact of treatment reduction for childhood acute lymphoblastic leukemia on serum immunoglobulins and antibodies against vaccine‐preventable diseases

The consequences of current intensive chemotherapy for childhood acute lymphoblastic leukemia (ALL) for immune defense are a matter of concern. The purpose of this study was to examine the effect of reduced compared with intensive (conventional) ALL chemotherapy on serum immunoglobulin levels and specific antibody concentrations against vaccine-preventable diseases. Patients treated according to Dutch Childhood Oncology Group ALL 10 protocol were stratified by minimal residual disease to receive reduced (standard risk; SR) or intensive (medium risk; MR) intensification/maintenance treatment. Between November 2004 and July 2009 we compared serum immunoglobulins of 110 patients and specific antibodies against diphtheria toxin, tetanus toxin, and Bordetella pertussis antigens of 41 patients of SR and MR groups during chemotherapy. Immunoglobulin levels showed significantly different patterns between the SR and MR groups. In the MR group IgG, IgA, and IgM levels decreased towards the end of intensive treatment; in the SR group IgG levels increased while IgA and IgM stabilized. In both groups IgM and IgG levels were most affected. Specific antibody levels against vaccine-preventable diseases decreased in both groups, but more profound in MR group. Although reduced chemotherapy is beneficial for immunoglobulin level recovery and might prevent susceptibility for infections, specific antibodies remain decreased.