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We report that a toxin neutralization assay (TNA) can detect a decrease in the immunogenicity of anthrax vaccines as a consequence of brief exposure to elevated temperature. This attribute of TNA may help in adopting immunogenicity as a replacement of the current potency test, which involves protection from lethal challenge.

The common perception that pertussis is only a childhood disease is not correct. Vaccination or infection with Bordetella pertussis provides only short-lived protection against pertussis-and the majority of the population is consequently at risk of contracting pertussis. We evaluated the seroprevalence of pertussis antibodies (IgG against pertussis toxin) in serum samples from 265 Danish patients, aged 8 years and older, with coughs of unknown etiology. Depending on the cutoff chosen, we found that 2.6% to 10.9% of these patients were seropositive for pertussis. Of 178 patients with a reported duration of cough between 2 weeks and 3 months, 3.4% to 12.4% were seropositive for pertussis, indicating recent infection. Our study indicates that B. pertussis infection may be underdiagnosed among older children and adults with coughs in Denmark.

Two toxigenic Corynebacterium ulcerans isolates recovered from pharyngeal swabs of two patients from the same hospital in Japan during 2001-2002 were characterized by PFGE and ribotyping. Toxin production in different culture media was examined and serological analysis of patient sera was performed. The two isolates could not be distinguished by PFGE; however, their ribotypes were distinguishable. One of the isolates could represent a novel ribotype. Analysis of toxin production in different culture media demonstrated that the two isolates produced varying amounts of the diphtheria toxin. Serological analysis showed a greater than sevenfold increase in the serum antitoxin titre during the course of infection in one patient.

The aim of the present study was to evaluate and standardize the ToBI test in vitro as a substitute for the serum neutralization test in mice for quality control of clostridial vaccines. The ToBI test in vitro was used to evaluate 40 serum samples of known antibody content, obtained from rabbits immunized against clostridiosis with experimental vaccine. The correlation between epsilon antitoxin titers in rabbit sera, determined by the ToBI test and serum neutralization in mice, ranged from 0.222% to 0.452% in polyvalent vaccines and from 0.154% to 0.387% in monovalent vaccines. Interplate coefficients of variation were not significant, reaching 0.350% in polyvalent vaccines and 0.400% in monovalent vaccines, indicating high homogeneity. In conclusion, the ToBI test in vitro is suitable for assessing the potency of clostridial vaccines and may be used as an alternative method able to replace current in vivo tests., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The technical and diagnostic performances of five commercially available enzyme-linked immunosorbent assays for the measurement of anti-diphtheria toxoid IgG antibodies were evaluated. There was good agreement between the relative sensitivities of the five assays, but the relative specificity of one of the assays differed from that of the other four assays. Three of the five assays possessed recoveries of the international reference material NIBSC 00/496 within the range of 90% to 110% at antibody levels >0.1 IU/ml. The data suggest that there are manufacture-dependent differences in relative sensitivity, specificity, and accuracy for the determination of anti-diphtheria toxoid IgG antibodies that could result in different diagnostic interpretations.

The licensed anthrax vaccine and many of the new anthrax vaccines being developed are based on protective antigen (PA), a nontoxic component of anthrax toxin. For this reason, an understanding of the immune response to PA vaccination is important. In this study, we examined the antibody response elicited by PA-based vaccines and identified the domains of PA that contribute to that response in humans as well as nonhuman primates (NHPs) and rabbits, animal species that will be used to generate efficacy data to support approval of new anthrax vaccines. To this end, we developed a competitive enzyme-linked immunosorbent assay (ELISA), using purified recombinant forms of intact PA and its individual domains. We found that PA-based vaccines elicited IgG antibodies to each of the four PA domains in all three species. We also developed a competitive toxin neutralization assay, which showed that rabbits, NHPs, and humans all have functional antibody populations that bind to domains 1, 3, and 4. While the domain specificities of the antibody responses elicited by PA-based vaccines were similar in humans, NHPs, and rabbits, competitive assays suggested that humans may have a more significant secondary population of IgG antibodies that bind to partially unfolded or incorrectly folded PA. These findings provide information that will be useful when linking animal protection data to humans via an antibody bridge to establish efficacy of new anthrax vaccines.

Background: Menstrual Toxic Shock Syndrome (mTSS) is thought to be associated with the vaginal colonization with specific strains of Staphylococcus aureus TSST-1 in women who lack sufficient antibody titers to this toxin. There are no published studies that examine the seroconversion in women with various colonization patterns of this organism. Thus, the aim of this study was to evaluate the persistence of Staphylococcus aureus colonization at three body sites (vagina, nares, and anus) and serum antibody to toxic shock syndrome toxin-producing Staphylococcus aureus among a small group of healthy, menstruating women evaluated previously in a larger study., Methods: One year after the completion of that study, 311 subjects were recalled into 5 groups. Four samples were obtained from each participant at several visits over an additional 6-11 month period: 1) an anterior nares swab; 2) an anal swab; 3) a vagina swab; and 4) a blood sample. Gram stain, a catalase test, and a rapid S. aureus-specific latex agglutination test were performed to phenotypically identify S. aureus from sample swabs. A competitive ELISA was used to quantify TSST-1 production. Human TSST-1 IgG antibodies were determined from the blood samples using a sandwich ELISA method., Results: We found only 41% of toxigenic S. aureus and 35.5% of non-toxigenic nasal carriage could be classified as persistent. None of the toxigenic S. aureus vaginal or anal carriage could be classified as persistent. Despite the low persistence of S. aureus colonization, subjects colonized with a toxigenic strain were found to display distributions of antibody titers skewed toward higher titers than other subjects. Seven percent (5/75) of subjects became seropositive during recall, but none experienced toxic shock syndrome-like symptoms., Conclusions: Nasal carriage of S. aureus appears to be persistent and the best predicator of subsequent colonization, whereas vaginal and anal carriage appear to be more transient. From these findings, it appears that antibody titers in women found to be colonized with toxigenic S. aureus remained skewed toward higher titers whether or not the colonies were found to be persistent or transient in nature. This suggests that colonization at some point in time is sufficient to elevate antibody titer levels and those levels appear to be persistent. Results also indicate that women can become seropositive without experiencing signs or symptoms of toxic shock syndrome.

Aim: Induction of protective immunity against pathogenic microbes, including Bacillus anthracis, requires efficient vaccines that potentiate antibody avidity and increase T-cell longevity. We recently reported that the delivery of targeted B. anthracis protective antigen (PA) genetically fused to a DC-binding peptide (DCpep) by Lactobacillus acidophilus induced mucosal and systemic immunity against B. anthracis challenge in mice., Materials & Methods: Improvement of this oral vaccine strategy was attempted by use of the high copy and genetically stable q-replicating vector, pTRKH2, for expression of the targeted PA fusion protein in Lactobacillus gasseri, a common human commensal microbe, to vaccinate animals against anthrax Sterne infection., Results: Oral application of L. gasseri expressing the PA-DCpep fusion proteins elicited robust PA-neutralizing antibody and T-cell mediated immune responses against anthrax Sterne challenge, resulting in complete animal survival. Collectively, this improved expression vaccine strategy reduced the number of inoculations and length of the boosting period, leading to animal protection via efficacious bacterial adjuvanticity and safe oral delivery of this vaccine to mucosal immune cells, including dendritic cells., Conclusion: Lactobacillus-based delivery offers tremendous practical advantages. Recombinant antigens such as PA would not require chemical coupling agents, and the recombinant bacteria can be administered orally where upon both mucosal and systemic immune responses are elicited.

Enterotoxigenic Escherichia coli (ETEC)-associated diarrhea causes a substantial economic loss to swine producers worldwide. The majority of ETEC strains causing porcine diarrhea, especially postweaning diarrhea (PWD), produce heat-labile toxin (LT) and heat-stable toxin b (STb). LT is commonly used in vaccine development, but STb has not been included because of its poor immunogenicity. As a virulence factor in porcine diarrhea, STb needs to be included as an antigen for development of broad-spectrum vaccines. In this study, we used an LT toxoid (LT(R192G) [hereafter, LT(192)]) derived from porcine ETEC to carry a mature STb peptide for LT(192)-STb fusions to enhance STb immunogenicity for potential vaccine application. Anti-LT and anti-STb antibodies were detected in immunized rabbits and pigs. In addition, when challenged with an STb-positive ETEC strain, all 10 suckling piglets borne by immunized gilts remained healthy, whereas 7 out 9 piglets borne by unimmunized gilts developed moderate diarrhea. This study indicates that the LT(192)-STb fusion enhanced anti-STb immunogenicity and suggests the LT(192)-STb fusion antigen can be used in future vaccine development against porcine ETEC diarrhea.

Despite the potential for its use as an agent of biowarfare or bioterrorism, no approved vaccine against staphylococcal enterotoxin B (SEB) exists. Nontoxic, mutant forms of SEB have been developed; however, it has been difficult to determine the efficacy of such subunit vaccine candidates due to the lack of superantigen activity of native SEB in rodents and due to the limitations of primate models. Since pigs respond to SEB in a manner similar to that of human subjects, we utilized this relevant animal model to investigate the safety and immunogenicity of a triple mutant of SEB carrying the amino acid changes L45R, Y89A, and Y94A. This recombinant mutant SEB (rmSEB) did not possess superantigen activity in pig lymphocyte cultures. Furthermore, rmSEB was unable to compete with native SEB for binding to pig leukocytes. These in vitro studies suggested that rmSEB could be a safe subunit vaccine. To test this possibility, piglets immunized orally with rmSEB formulations experienced no significant decrease in food consumption and no weight loss during the vaccination regimen. Oral vaccination with 1-mg doses of rmSEB on days 0, 7, 14, and 24 resulted in serum IgG and fecal IgA levels by day 36 that cross-reacted with native SEB. Surprisingly, the inclusion of cholera toxin adjuvant in vaccine formulations containing rmSEB did not result in increased antibody responses compared to formulations using the immunogen alone. Taken together, these studies provide additional evidence for the potential use of nontoxic forms of SEB as vaccines.

Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni(+) affinity chromatography. Mice immunized with three injections containing 5 microg of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 10(5) against the native toxin complex, which enabled protection against a high-dose toxin challenge (10(3) to 10(6) mouse 50% lethal dose [MsLD(50)]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 10(5) MsLD(50) toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.

This study was undertaken to estimate the magnitude of Bordetella pertussis infections in a highly vaccinated population in Israel in order to evaluate the relationship between clinical notification data and serology-based evidence of infection. A cross-sectional survey was conducted on a total of 1982 serum samples from the National Serum Bank, collected from January 2000 through December 2001, in order to monitor high levels of pertussis toxin (PT) IgG antibody indicative of recent B. pertussis infection, by standardized methods. The estimation yielded an infection incidence rate of 2448 per 100,000 population (> or =3 years of age) for the year 2000 compared to an annual incidence of reported pertussis of 5.6 per 100,000 for the same period. The peaks of estimated incidence of infection were found in the groups of 15-19-year olds (5245 per 100,000) and older than 60 years (6469 per 100,000), whereas the majority of clinical pertussis cases were reported for the 10-14-year olds (20.5 per 100,000). The findings clearly show that despite a high vaccination coverage rate (>93%), there is still a considerable circulation of B. pertussis, particularly in adolescents and elderly. Population-based serosurveillance for pertussis offers the potential to assist interpretation of trends independent of notification and diagnostic bias., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Shiga-like toxin 2 (Stx2)-producing enterohemorrhagic Escherichia coli (referred to as EHEC or STEC) strains are the primary etiologic agents of hemolytic-uremic syndrome (HUS), which leads to renal failure and high mortality rates. Expression of Stx2 is the most relevant virulence-associated factor of EHEC strains, and toxin neutralization by antigen-specific serum antibodies represents the main target for both preventive and therapeutic anti-HUS approaches. In the present report, we describe two Salmonella enterica serovar Typhimurium aroA vaccine strains expressing a nontoxic plasmid-encoded derivative of Stx2 (Stx2DeltaAB) containing the complete nontoxic A2 subunit and the receptor binding B subunit. The two S. Typhimurium strains differ in the expression of flagellin, the structural subunit of the flagellar shaft, which exerts strong adjuvant effects. The vaccine strains expressed Stx2DeltaAB, either cell bound or secreted into the extracellular environment, and showed enhanced mouse gut colonization and high plasmid stability under both in vitro and in vivo conditions. Oral immunization of mice with three doses of the S. Typhimurium vaccine strains elicited serum anti-Stx2B (IgG) antibodies that neutralized the toxic effects of the native toxin under in vitro conditions (Vero cells) and conferred partial protection under in vivo conditions. No significant differences with respect to gut colonization or the induction of antigen-specific antibody responses were detected in mice vaccinated with flagellated versus nonflagellated bacterial strains. The present results indicate that expression of Stx2DeltaAB by attenuated S. Typhimurium strains is an alternative vaccine approach for HUS control, but additional improvements in the immunogenicity of Stx2 toxoids are still required.

Ricin is a CDC level B biothreat. Our recombinant ricin A chain vaccine (RiVax) contains two mutations, rendering it non-toxic at high doses. Frozen or alum formulations of RiVax protected mice against ricin administered by injection, gavage or aerosol. Without alum, RiVax was safe and immunogenic in rabbits and human volunteers. For military use, the predominant target group, it would be optimal not to require a cold chain for transport and storage. We have now developed a lyophilized formulation and demonstrated stability and efficacy for at least 1 year stored refrigerated or at room temperature administered with or without alum.

It has recently been reported that the worldwide increase in the number of pertussis cases is a result of the waning of whole-cell vaccine-induced immunity. Thus, in this study, we aimed to investigate the pertussis immunity status of primary and secondary school students in a district of Ankara, Turkey. A total of 997 healthy students, aged 9-17 years, who had been immunized with four doses of whole-cell pertussis vaccine were included in the study. The subjects were divided into two age groups: 9-14 and 15-17 years. To determine the immune status, serum levels of IgG anti-pertussis toxin (aPT) antibody were tested by in-house ELISA and arbitrarily evaluated as non-immune [< 10 ELISA units (EU)/ml], immune (10-100 EU/ml), and recent infection (> 100 EU/ml). Serum samples of 997 students (559 females, 438 males) aged between 9 and 17 years (mean 13.02 +/- 2.25, median 13 years) were tested. Non-immune, immune and recent infection levels of aPT were found in 27.3%, 59.3% and 13.4% of individuals, respectively. The immune group did not have statistically significant differences between males and females (P = 0.68). In the 9-14 and 15-17 years age groups, serum aPT antibody levels 10 EU/ml were 73.1% and 72.2%, respectively, which did not represent any statistical difference (P = 0.81). Students aged 15-17 years had a higher immunity rate than the 9-14 years group, and the percentage of students with recent infection in the 9-14 years group was higher than the 15-17 years group (P < 0.001). The peak age of non-immunized subjects was 9 years (47.0%), and decreased to a minimum at age 12-13 years, and began to increase again from age 13-14 years. In contrast, the ratio of recent infection was least at age 9-10 years, began to increase, and reached a peak at 12 years, and then decreased. On the other hand, it was observed that household size and monthly income were not associated with the immunity status (P = 0.65, P = 0.37, respectively). The results of the present study show that levels of antibody against pertussis decreased in the younger age groups and, as a result, there is an increase in the number of pertussis cases. Thus, in order to decrease the incidence of pertussis and protect infants, we recommend the application of booster doses at regular intervals.

Background: Previous studies have demonstrated a correlation between Clostridium difficile anti-toxin A serum antibodies and protection against symptomatic disease and recurrence., Methods: A neutralizing monoclonal antibody to C. difficile toxin A (CDA1) developed by MBL and Medarex, Inc. was studied in a phase II, randomized, double-blind, placebo-controlled trial in patients receiving standard of care treatment for C. difficile infection (CDI). Twenty-nine subjects received a single intravenous infusion of 10mg/kg CDA1 and 17 subjects received placebo and were evaluated for recurrence of CDI during the 56-day study period. Serum antibodies against C. difficile toxin A and B were measured by ELISA and cytotoxicity assay at various time points before and after infusion., Findings: CDI recurrence occurred in 5 of 29 (17%) in the CDA1 group and 3 of 17 (18%) (p=NS) in the placebo group with a trend toward delay in time to recurrence in the group treated with CDA1. The geometric mean concentration of antibody to an epitope of the receptor-binding domain of toxin B (0.300 and 1.20microg/ml, respectively; p=0.02) and geometric mean titer of neutralizing B antibody (8.00 and 100, respectively; p=0.02) at study day 28 were lower for those subjects with recurrence compared to those who did not recur. In addition, a significantly greater proportion of subjects who recurred were infected with the epidemic BI/NAP1/027 strain compared with those that did not recur (88% vs. 22%; p=0.002). Finally, in a multiple logistic regression analysis neutralizing anti-toxin B at day 14 (p<0.001), anti-toxin A at day 28 (p<0.001) and infection with the BI/NAP1/027 strain at enrollment (p=0.002) were all predictive of CDI recurrence., Interpretation: In this prospective study, lower concentrations of neutralizing anti-toxin B and anti-toxin A antibody and infection with the BI/NAP1/027 strain of C. difficile were significantly associated with recurrence of CDI.

DNA vaccination is a promising immunization strategy that could be applied in the development of vaccines for a variety of prophylactic and therapeutic indications. Utilizing anthrax protective antigen as a model antigen, we demonstrate that electroporation mediated delivery enhanced the immunogenicity of DNA vaccines in nonhuman primates over 100-fold as compared to conventional intramuscular injection. Two administrations of a DNA vaccine with electroporation elicited anthrax toxin neutralizing antibody responses in 100% of rhesus macaques. Toxin neutralizing antibodies were sustained for the nearly 1-year study duration and were correlated with protection against subsequent lethal Bacillus anthracis spore challenge. Collectively, electroporation mediated DNA vaccination conferred protection comparable to that observed following vaccination with an FDA approved anthrax vaccine.

We previously showed that a multiple antigenic peptide (MAP) displaying amino acids (aa) 305 to 319 from the 2beta2-2beta3 loop of protective antigen (PA) can elicit high-titered antibody that neutralizes lethal toxin (LeTx) in vitro and that this loop-neutralizing determinant (LND) specificity is absent in PA-immune rabbits. Some immune rabbits were, however, nonresponders to the MAP. We hypothesized that the immunogen elicited suboptimal major histocompatibility complex (MHC) class II-restricted T-cell help and that introduction of a functional helper T-cell epitope would increase MHC-restricted responsiveness and the magnitude and affinity of the antibody responses. In the current study, we characterized the T- and B-cell responses to LND peptides in mice, then designed second-generation MAP immunogens for eliciting LND-specific immunity, and tested them in rabbits. The 305-319 sequence was devoid of helper T-cell epitopes in three strains of mice; however, a T-B peptide comprising aa 305 to 319, colinearly synthesized with the P30 helper epitope of tetanus toxin, elicited robust LeTx-neutralizing immunity in mice. T-B MAPs displaying B-cell epitopes 304 to 319 (MAP304) or 305 to 319 (MAP305) elicited high-titer, durable antibody responses in rabbits which exhibited potent neutralization of LeTx in vitro. All MAP304-immune rabbits demonstrated neutralization titers exceeding that of hyperimmune sera of rabbits immunized with PA in Freund's adjuvant, with peak neutralization titers 23-, 6-, and 3-fold higher than that of the PA antiserum. Overall, immunization with MAPs containing the P30 epitope elicited higher antibody and toxin neutralization titers and peptide-specific affinity than immunization with an LND MAP lacking a helper epitope. P30-containing MAP304 represents a promising LND-specific vaccine for anthrax.

Annual vaccination with trivalent influenza vaccine (TIV), and a single dose of tetanus toxoid-reduced diphtheria toxoid-acellular pertussis (Tdap) vaccine, are both recommended for adults in the US. This study was conducted to obtain information on the safety and immunogenicity of co-administered TIV and a Tdap vaccine (Boostrix) in US adults. The immunogenicity and safety of Tdap and TIV was evaluated in 1,497 adult subjects 19-64 years of age, who were randomized to receive Tdap and TIV either concomitantly or one month apart (TIV followed by Tdap). Seroprotection rates for diphtheria, tetanus and influenza antigens were high (>or=94.1%) for both vaccine regimens, and immune responses to these antigens in the concomitant group were non-inferior to those observed in the sequential group. Although antibody concentrations for pertussis antigens were lower in the concomitant group than in the sequential group, concomitant administration was shown to be non-inferior to sequential administration with respect to anti-pertussis toxoid concentrations one month after Tdap vaccination. For filamentous haemagglutinin and pertactin, the between-group differences in antibody concentrations marginally exceeded pre-specified limits for defining non-inferiority. In both groups, anti-pertussis antibody concentrations were greater than those observed in infants following primary DTaP vaccination, in whom vaccine efficacy against pertussis was demonstrated. Reporting of adverse events appeared to be similar between groups. The data support the conclusion that Tdap and TIV vaccines may be co-administered without compromising either the effectiveness or tolerability of either vaccine.

Bordetella pertussis infection may cause severe illness in newborns. Mothers with B. pertussis infection during delivery can infect newborns. The seroprevalence of B. pertussis infection in pregnancy was measured in pregnant women by detection of immunoglobulin G against pertussis toxin; 6.3% had serological evidence of infection. Maternal vaccination should be considered to prevent pertussis in newborns.

Botulinum neurotoxins, predominantly serotypes C and D, cause equine botulism through forage poisoning. The C-terminal part of the heavy chain of botulinum neurotoxin types C and D (HcBoNT/C and D) was expressed in Escherichia coli and evaluated as a recombinant mono- and bivalent vaccine in twelve horses in comparison to a commercially available toxoid vaccine. A three-dose subcutaneous immunization of adult horses elicited robust serum antibody response in an ELISA using the immunogen as a capture antigen. Immune sera showed dose-dependent high potency in neutralizing specifically the active BoNT/C and D in the mouse protection assay. The aluminium hydroxide based mono- and bivalent recombinant HcBoNT/C and D vaccines were characterized by good compatibility and the ability to elicit protective antibody titers similar or superior to the commercially available toxoid vaccine.

There has been an increased demand for the development of novel vaccine adjuvants that lead to enhanced induction of protection from infectious challenges and development of immunological memory. A novel vaccine adjuvant was developed comprising a complex containing CpG oligonucleotide and the synthetic cationic innate defence regulator peptide HH2 that has enhanced immune modulating activities. The complex of HH2 and the CpG oligonucleotide 10101 was a potent inducer of cytokine/chemokine expression ex vivo, retained activity following extended storage, had low associated cytotoxicity, and upregulated surface marker expression in dendritic cells, a critical activity for a vaccine adjuvant. Immunization of mice with a coformulation of the HH2-CpG complex and pertussis toxoid significantly enhanced the induction of toxoid-specific antibody titres when compared to toxoid alone, inducing high titres of IgG1 and IgG2a, typical of a balanced Th1/Th2 response, and also led to high IgA titres. This study demonstrates the potential application of the HH2-CpG complex as a vaccine adjuvant.

The aim of this study was to evaluate the seroprevalence and correlates of diphtheria and tetanus in Edirne, Turkey. Tetanus and diphtheria antitoxin levels were determined by enzyme-linked immunosorbent assay. Among 99 participants, a diphtheria antitoxin level of >or=0.1 IU/mL was found in 97 (98%), while 2 (2%) had antitoxin levels of 0.011-0.099 IU/mL. The geometric mean titres (GMTs) in men were statistically higher. Among 295 participants, a tetanus antitoxin level of >or=0.1 IU/mL was found in 291 (98.6%), while 4 (1.4%) had antitoxin levels of 0.011-0.099 IU/mL. Participants who had completed secondary school or higher education showed higher GMT values. Additionally, participants vaccinated within the previous 5 years had higher GMT values and the percentage of participants who had completed secondary school or higher education was higher among them. GMTs decrease with increasing age and increase as the poverty index increases. The average socioeconomic status index of the participants was high for both diphtheria and tetanus seroepidemiology. In this community-based study, antitoxin levels of diphtheria and tetanus were high. However, revaccination of adults with tetanus-diphtheria toxoids at every opportunity (military service, pregnancy, post-injury prophylaxis, etc.) together with a single booster every 10 years should be considered as an immunization policy.

Botulinum neurotoxins are known to be among the most toxic known substances. They produce severe paralysis by preventing the release of acetylcholine at the neuromuscular junction. Thus, new strategies for efficient production of safe and effective anti-botulinum neurotoxin antisera have been a high priority. Here we describe the use of DNA electrotransfer into the skeletal muscle to enhance antiserum titers against botulinum toxin serotypes A, B, and E in mice. We treated animals with codon-optimized plasmid DNA encoding the nontoxic but highly immunogenic C-terminal heavy chain fragment of the toxin. By employing both codon optimization and the electrotransfer procedure, the immune response and corresponding neutralizing antiserum titers were markedly increased. The cellular localization of the antigen and the immunization regimens were also shown to increase neutralizing titers to >100 IU/ml. This study demonstrates that DNA electrotransfer is an effective procedure for raising neutralizing antiserum titers to remarkably high levels.

Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A(1) peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A(2) peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2DeltaAB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A(2) sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2DeltaAB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.

The lack of a clear correlation between the levels of antibody to pertussis antigens and protection against disease lends credence to the possibility that cell-mediated immunity provides primary protection against disease. This phase I comparative trial had the aim of comparing the in vitro cellular immune response and anti-pertussis toxin (anti-PT) immunoglobulin G (IgG) titers induced by a cellular pertussis vaccine with low lipopolysaccharide (LPS) content (wP(low) vaccine) with those induced by the conventional whole-cell pertussis (wP) vaccine. A total of 234 infants were vaccinated at 2, 4, and 6 months with the conventional wP vaccine or the wP(low) vaccine. Proliferation of CD3(+) T cells was evaluated by flow cytometry after 6 days of peripheral blood mononuclear cell culture with stimulation with heat-killed Bordetella pertussis or phytohemagglutinin (PHA). CD3(+), CD4(+), CD8(+), and T-cell receptor gammadelta-positive (gammadelta(+)) cells were identified in the gate of blast lymphocytes. Gamma interferon, tumor necrosis factor alpha, interleukin-4 (IL-4), and IL-10 levels in supernatants and serum anti-PT IgG levels were determined using enzyme-linked immunosorbent assay (ELISA). The net percentage of CD3(+) blasts in cultures with B. pertussis in the group vaccinated with wP was higher than that in the group vaccinated with the wP(low) vaccine (medians of 6.2% for the wP vaccine and 3.9% for the wP(low) vaccine; P = 0.029). The frequencies of proliferating CD4(+), CD8(+), and gammadelta(+) cells, cytokine concentrations in supernatants, and the geometric mean titers of anti-PT IgG were similar for the two vaccination groups. There was a significant difference between the T-cell subpopulations for B. pertussis and PHA cultures, with a higher percentage of gammadelta(+) cells in the B. pertussis cultures (P < 0.001). The overall data did suggest that wP vaccination resulted in modestly better specific CD3(+) cell proliferation, and gammadelta(+) cell expansions were similar with the two vaccines.

Enzyme-linked immunosorbent assay (ELISA) for measurement of antibodies to pertussis toxin (PT) is a widely used method for diagnosis of whooping cough and is also frequently used for seroprevalence studies and for assessment of antibodies after vaccinations against whooping cough. The recommended ELISA procedure for assessment of PT antibodies in human serum does, however, have a serious problem with false-positive results when heat-treated sera are analyzed. Historic sera might have been exposed to routine heat treatment, and diagnostic sera from warm geographic regions are at risk of unintentional exposure to heat. A modified version of the PT ELISA, incorporating a blocking step with 1% milk and addition of 0.1% milk to the dilution buffer, eliminates the false-positive phenomenon occurring with heat-treated sera. Results for serum antibody concentrations correlate well with the currently recommended method. This ELISA modification is straightforward and cheap, and it should be recommended at all analyses incorporating sera with unknown history of heat exposure.

Transcutaneous immunization is a promising vaccination delivery strategy which targets potent immune cells residing in the outer layer of the skin. In this study, the immunogenicity and neutralizing potency of the non-toxic Hc fragment of tetanus toxin (HcWT) and a mutant of Hc lacking ganglioside binding activity were compared with that of tetanus toxoid (TTxd) following transcutaneous immunization (TCI) of mice. Mice immunized with HcWT in the absence of an adjuvant induced highest anti-toxoid and anti-Hc antibody titres, with a significant increase in the toxin neutralizing antibody response compared with TTxd. These results are in contrast to previous studies employing subcutaneous delivery, where TTxd was found to be a more potent immunogen than the Hc fragment of the toxin. We conclude that the HcWT protein is more immunogenic than TTxd when given via the transcutaneous route. Our results suggest that TCI may provide an opportunity for effective delivery of toxin-like antigens which harbor protective epitopes and that traditional toxoid proteins may not be optimal antigens for skin immunization.

The original license for production of the anthrax vaccine, Anthrax Vaccine Adsorbed (AVA), was issued in 1970. Since that time, over 8 million AVA immunizations have been administered to 2+ million individuals. In 2002, the National Academy of Sciences, Institute of Medicine, reviewed the safety and efficacy of AVA. They concluded that the vaccine is acceptably safe and effective in protecting humans against anthrax. The vaccine should protect people against all known strains of anthrax bacteria, as well as against any strains that might be created by potential terrorists or others. Although the Institute of Medicine concluded that AVA was reasonably safe, they noted that it is fairly common for people to experience local reactions (e.g., redness and swelling at the injection site) and for a smaller number to experience systemic reactions such as fever and malaise, within hours or days of vaccination. Results of animal studies done previously and subsequent to this report are generally in agreement. For instance, AVA vaccination increases the level of anthrax anti-protective antigen IgG (anti-PA IgG), which is thought to be one possible correlate of protection (although absolute protective concentrations have not been identified in humans). Anthrax lethal factor neutralization has also been identified as possibly being an important additional correlate of immunity. Future vaccine research efforts include developing a recombinant anthrax vaccine and anthrax monoclonal antibodies to block the anthrax toxin(s). It is projected that the next-generation vaccine will elicit a markedly increased anti-anthrax immune response within a shorter time period and consequently, will enable the easier inoculations of individuals working within high-risk areas.

This study investigated the use of two doses of three different meningococcal group C conjugate (MCC) vaccines when given for primary immunization with a seven-valent pneumococcal conjugate vaccine (PCV7) and Pediacel, a combination product containing five acellular pertussis components, diphtheria and tetanus toxoids, Haemophilus influenzae type b (Hib) conjugate, and inactivated-poliovirus vaccine. The immune response after a single dose of MCC is also presented. Infants were randomized to receive two doses of one of the MCC vaccines and PCV7 at 2 and 3 months or at 2 and 4 months of age. Meningococcal group C serum bactericidal antibody (SBA) geometric mean titers, Hib-polyribosylribitol phosphate (PRP) immunoglobulin G (IgG) geometric mean concentrations (GMCs), and diphtheria and tetanus antitoxin GMCs, together with the proportions of infants achieving putative protective levels, were determined. A total of 393 infants were recruited. Following the first dose of NeisVac-C (MCC conjugated to tetanus toxoid), 97% of infants achieved protective levels (SBA titer of >or=8), compared with 80% and 53%, respectively, for Menjugate and Meningitec (both of which are conjugated to CRM(197)). SBA responses to MCC vaccines were not significantly different when administered at 2 and 3 or 2 and 4 months of age. Following two doses of each MCC, 98 to 100% of infants achieved protective levels. Both PRP IgG and tetanus responses were significantly enhanced when Pediacel was coadministered with NeisVac-C. This study demonstrates that NeisVac-C and Menjugate generate good immunogenicity after the first dose at 2 months of age when coadministered with PCV7 and Pediacel and merit further investigation in single-dose priming strategies.

Anthrax toxins significantly contribute to anthrax disease pathogenesis, and mechanisms by which the toxins affect host cellular responses have been identified with purified toxins. However, the contribution of anthrax toxin proteins to dissemination, disease progression, and subsequent immunity after aerosol infection with spores has not been clearly elucidated. To better understand the role of anthrax toxins in pathogenesis in vivo and to investigate the contribution of antibody to toxin proteins in protection, we completed a series of in vivo experiments using a murine aerosol challenge model and a collection of in-frame deletion mutants lacking toxin components. Our data show that after aerosol exposure to Bacillus anthracis spores, anthrax lethal toxin was required for outgrowth of bacilli in the draining lymph nodes and subsequent progression of infection beyond the lymph nodes to establish disseminated disease. After pulmonary exposure to anthrax spores, toxin expression was required for the development of protective immunity to a subsequent lethal challenge. However, immunoglobulin (immunoglobulin G) titers to toxin proteins, prior to secondary challenge, did not correlate with the protection observed upon secondary challenge with wild-type spores. A correlation was observed between survival after secondary challenge and rapid anamnestic responses directed against toxin proteins. Taken together, these studies indicate that anthrax toxins are required for dissemination of bacteria beyond the draining lymphoid tissue, leading to full virulence in the mouse aerosol challenge model, and that primary and anamnestic immune responses to toxin proteins provide protection against subsequent lethal challenge. These results provide support for the utility of the mouse aerosol challenge model for the study of inhalational anthrax.

Humoral and cell-mediated immunity (CMI) against B. pertussis was assessed in a sample of adolescent, adult and senior subjects distributed in five different geographical areas in Italy. Most (99.1%) subjects had IgG anti-pertussis toxin (PT) antibodies exceeding the minimum detection level [> or = 2 ELISA units (EU)/ml]. There were no significant differences between the genders; 6.2% samples recorded titres > or = 100 EU/ml. CMI was positive [stimulation index (SI) > or = 5] against PT in 39.0% of all samples. This study suggests that B. pertussis continues to circulate in age groups that have been previously considered to be uninvolved in the circulation of this pathogen and that adolescent and adult pertussis boosters may be of value in these populations. Nevertheless, over the last 10 years, large increases in vaccination coverage rates have contributed to reduce the spread of the aetiological agent, especially in the immunized population.

Background: Recent data suggest that Clostridium difficile-associated diarrhea is becoming more severe and difficult to treat. Antibody responses to C. difficile toxin A are protective against symptomatic disease and recurrence. We examined the safety and pharmacokinetics (pk) of a novel neutralizing human monoclonal antibody against C. difficile toxin A (CDA1) in healthy adults., Methods: Five cohorts with 6 subjects each received a single intravenous infusion of CDA1 at escalating doses of 0.3, 1, 5, 10, and 20 mg/kg. Safety evaluations took place on days 1, 2, 3, 7, 14, 28, and 56 post-infusion. Samples for pk analysis were obtained before and after infusion, and at each safety evaluation. Serum CDA1 antibody concentrations and human anti-human antibody (HAHA) titers were measured with enzyme-linked immunosorbent assays. A noncompartmental model was used for pk analysis., Results: Thirty subjects were enrolled. The median age was 27.5 yrs. There were no serious adverse events (AE) related to CDA1. Twenty-one of the 48 reported non-serious adverse events were possibly related to CDA1, and included transient blood pressure changes requiring no treatment, nasal congestion, headache, abdominal cramps, nausea, and self-limited diarrhea. Serum CDA1 concentrations increased with escalating doses: mean C(max) ranged from 6.82 microg/ml for the 0.3 mg/kg cohort to 511 microg/ml for the 20 mg/kg cohort. The geometric mean values of the half-life of CDA1 ranged between 25.3 and 31.8 days, and the volume of distribution approximated serum. No subject formed detectable HAHA titers., Conclusion: Administration of CDA1 as a single intravenous infusion was safe and well tolerated. C(max) increased proportionally with increasing doses. A randomized study of CDA1 in patients with C. difficile associated diarrhea is underway.

Objective: Monitor the long-term immunogenicity of a single dose of acellular pertussis vaccine in health-care workers., Design: German health-care workers and child-care workers received a single dose of a monovalent acellular pertussis vaccine (PAC-Mérieux) in an open-label study. Blood samples were taken before (n=261), 4 weeks after (n=246), 1 year (n=187), 2 years (n=53), 3 years (n=134) and 4 years (n=37) after vaccination. IgG- and IgA-anti-pertussis-toxin (PT), IgG- and IgA-anti-filamentous hemagglutinin (FHA), and IgG-anti-pertactin (PRN) were measured by ELISA., Results: Of all subjects, 97.1%, 99.2% and 97.2% had an antibody response to PT, FHA and PRN, respectively. Four weeks after vaccination the median titres of IgG antibodies to PT, FHA and PRN were 314, 785 and 84 EU/l, respectively, and all vaccinees had an immune response to at least one pertussis antigen. IgA-anti-PT and IgA-anti-FHA responses were found in 63.4% and 96.3% of subjects with a median titre of 30 and 196 EU/ml, respectively. The titre of IgG-anti-PT decreased slowly with a median concentration of 76, 71, 71 and 63 EU/ml after 1, 2, 3 and 4 years, respectively. Secondary titre increases were observed in 0.5%, 3.3%, 5.6% and 12.5% of the vaccinees 1, 2, 3, and 4 years after vaccination., Conclusion: In German health paediatric care workers long-lasting immune responses with high antibody levels could be induced by a single dose of acellular pertussis vaccine. A renewed contact with B. pertussis antigens resulted in a measurable immune response to PT between 0.5% (1 year p.v.) and 12.5% (4 years p.v.).

In USA, the potency of commercial vaccines containing Clostridium botulinum type C toxoid is determined by a mink vaccination-challenge assay outlined in the Code of Federal Regulations, Title 9, Part 113.110. A more humane potency test is desired, and this study provides preliminary data in support of a serological assay that correlates post-vaccination antitoxin titers of guinea pigs to vaccine efficacy in mink. Mink and guinea pigs were injected with varying dilutions of a vaccine containing C. botulinum type C toxoid. Blood samples were collected from each animal prior to challenging the mink with type C toxin. Serum antitoxin titers of mink and guinea pigs were measured by a mouse protection test, and the results were compared to the outcome of the toxin challenge in mink. A dose-dependent antitoxin response was observed in guinea pigs vaccinated with the critical dilutions of vaccine bracketing the minimum protective dose in mink. These preliminary data suggest that it may be possible to correlate post-vaccination antitoxin titers in guinea pigs to vaccine efficacy in mink. This correlation could be used as the basis for a more humane potency test for C. botulinum type C toxoids.

Botulinum neurotoxins (BoNTs) are the most toxic proteins for humans and are classified as category A toxins. There are seven serotypes of BoNTs defined by the lack of cross-serotype toxin neutralization. Thus, an effective vaccine must neutralize each BoNT serotype. BoNTs are organized as dichain A-B toxins, where the N-terminal domain (light chain) is a zinc metalloprotease targeting soluble NSF attachment receptor proteins that is linked to the C-terminal domain (heavy chain [HC]) by a disulfide bond. The HC comprises a translocation domain and a C-terminal receptor binding domain (HCR). HCRs of the seven serotypes of BoNTs (hepta-HCR) were engineered for expression in Escherichia coli, and each HCR was purified from E. coli lysates. Immunization of mice with the E. coli-derived hepta-serotype HCR vaccine elicited an antibody response to each of the seven BoNT HCRs and neutralized challenge by 10,000 50% lethal doses of each of the seven BoNT serotypes. A solid-phase assay showed that the anti-hepta-serotype HCR sera inhibited the binding of HCR serotypes A and B to the ganglioside GT1b, the first step in BoNT intoxication of neurons. This is the first E. coli-derived vaccine that effectively neutralizes each of the seven BoNT serotypes.

Enterohemorrhagic Escherichia coli (EHEC) strains are important human food-borne pathogens. EHEC strains elaborate potent Shiga toxins (Stx1, and/or Stx2) implicated in the development of hemorrhagic colitis (HC) or hemolytic-uremic syndrome (HUS). In this report, we evaluated the immunogenicity and protective efficacy of Stx1 subunit B (StxB1) administered by transcutaneous immunization (TCI). Three groups of Dutch Belted rabbits received patches containing StxB1, StxB1 in combination with Escherichia coli heat-labile enterotoxin (LT), or LT alone. An additional group of naïve rabbits served as controls. The protective efficacy following TCI with StxB1 was assessed by challenging rabbits with a virulent Stx1-producing strain, RDEC-H19A, capable of inducing HC and HUS in rabbits. Antibodies specific to StxB1 from serum and bile samples were determined by enzyme-linked immunosorbent assay and toxin neutralization test. Rabbits immunized with StxB1 demonstrated improved weight gain and reduced Stx-induced histopathology. Rabbits receiving StxB or StxB1/LT showed a significant increase in serum immunoglobulin G titers specific to StxB1 as well as toxin neutralization titers. These data demonstrated that the StxB delivered by TCI could induce significant systemic immune responses. Thus, Stx subunit B vaccine delivered by a patch for a high-risk population may be a practical approach to prevent (and/or reduce) Stx-induced pathology.

Anthrolysin O (ALO) is a toxin produced by Bacillus anthracis, the causative agent of anthrax. It is a member of the cholesterol-dependent cytolysin (CDC) group of toxins, many of which are potential vaccine candidates that protect against their producing organisms. Pore formation by ALO was studied by transmission electron microscopy and pores were found to be consistent with those formed by other members of this toxin family. We constructed and characterised a novel genetic toxoid of anthrolysin O, Delta6mALO, which was able to bind to cells but was incapable of pore-formation or haemolysis. The capacity of the haemolytic and non-haemolytic forms of ALO to protect against challenge with the toxin or B. anthracis was determined. Immunisation with both active and non-haemolytic forms of ALO elicited protection against lethal i.v. challenge with ALO but neither was protective against B. anthracis in a murine i.p. challenge model. Immunisation with another CDC, pneumolysin, did not confer cross-protection against challenge with ALO. Histopathological investigation following lethal i.v. challenge with ALO revealed acute pathology in the lungs with occlusion of alveolar vessels by fibrin deposits.

In this study, the authors report the purification and characterization of antitoxic proteins from the serum of Agkistrodon halys Pallas. Two antitoxic proteins have been successfully isolated by the methods of (NH4)(2)SO(4) fractional precipitation, chromatography and preparative discontinuous polyacrylamide gel electrophoresis (PAGE). We have measured their molecular weights by Sephadex G-150 chromatography and 0.1% SDS-Tris-HCl discontinue PAGE respectively. Antitoxin I was about 138,000+/-40 Da and antitoxin II was about 76,000+/-40 Da, they are all single-chain peptides. We have measured their capacity to neutralize the toxicity of agkistrodotoxin (ATX), and their capacity to inhibit the PLA(2) activity of ATX. The results showed that antitoxin I could increase LD(50) of ATX from 0.25+/-0.05 to 0.445+/-0.13 mg/kg, decrease its PLA(2) activity from 2.36 to 1.72 microm/mg min, and antitoxin II could increase LD(50) of ATX from 0.25+/-0.05 to 0.56+/-0.12 mg/kg, decrease Phospholipase A(2) (PLA(2)) activity from 2.36 to 1.2 microm/mg min. When the natural antitoxins were mixed with different amounts of ATX and inoculated intraperitonially into eight mice, it was found that 0.5 mg antitoxin I could neutralize the toxicity of 0.4 mg ATX and 0.5 mg antitoxin II could neutralize the toxicity of 0.5 mg ATX completely. These antitoxic proteins could neutralize the toxicity of ATX completely and inhibit ATX's PLA(2) activity partially.

The currently available commercial human anthrax vaccine requires multiple injections for efficacy and has side effects due to its alum adjuvant. These factors limit its utility when immunizing exposed populations in emergent situations. We evaluated a novel mucosal adjuvant that consists of a nontoxic, water-in-oil nanoemulsion (NE). This material does not contain a proinflammatory component but penetrates mucosal surfaces to load antigens into dendritic cells. Mice and guinea pigs were intranasally immunized with recombinant Bacillus anthracis protective antigen (rPA) mixed in NE as an adjuvant. rPA-NE immunization was effective in inducing both serum anti-PA immunoglobulin G (IgG) and bronchial anti-PA IgA and IgG antibodies after either one or two mucosal administrations. Serum anti-PA IgG2a and IgG2b antibodies and PA-specific cytokine induction after immunization indicate a Th1-polarized immune response. rPA-NE immunization also produced high titers of lethal-toxin-neutralizing serum antibodies in both mice and guinea pigs. Guinea pigs nasally immunized with rPA-NE vaccine were protected against an intradermal challenge with approximately 1,000 times the 50% lethal dose ( approximately 1,000x LD(50)) of B. anthracis Ames strain spores (1.38 x 10(3) spores), which killed control animals within 96 h. Nasal immunization also resulted in 70% and 40% survival rates against intranasal challenge with 10x LD(50) and 100x LD(50) (1.2 x 10(6) and 1.2 x 10(7)) Ames strain spores. Our results indicate that NE can effectively adjuvant rPA for intranasal immunization. This potentially could lead to a needle-free anthrax vaccine requiring fewer doses and having fewer side effects than the currently available human vaccine.

Recombinant Bacillus subtilis strains, either in the form of spores or vegetative cells, may be employed as safe and low-cost vaccine vehicles. In this study, we studied the role of promoter sequences and antigen-sorting signals on the immunogenicity based on previously constructed B. subtilis episomal expression systems. Mice orally immunized with spores or cells encoding the B subunit of the heat labile toxin (LTB), originally expressed by some enterotoxigenic Escherichia coli (ETEC) strains, under control of the stress-inducible gsiB promoter developed higher anti-LTB serum IgG and fecal IgA responses with regard to vaccine strains transformed with plasmids encoding the antigen under control of IPTG-inducible (Pspac) or constitutive (PlepA) promoters. Moreover, surface expression of the vaccine antigen under the control of the PgsiB promoter enhanced the immunogenicity of vegetative cells, while intracellular accumulation of LTB led to higher antibody responses in mice orally immunized with recombinant B. subtilis spores. Specific anti-LTB antibodies raised in vaccinated mice recognized and neutralized in vitro the native toxin produced by ETEC strains. Nonetheless, only mice orally immunized with recombinant B. subtilis strains, either as vegetative cells or spores, expressing intracellular LTB under the control of the gsiB promoter conferred partial protection to lethal challenges with purified LT. The present report further demonstrates that B. subtilis plasmid-based heterologous protein expression systems are adequate for antigen delivery via the oral route.

We have evaluated an oral vaccine based on an Salmonella enteric serovar typhi (S. typhi) Ty2 derivative TSB7 harboring deletion mutations in ssaV (SPI-2) and aroC together with a chromosomally integrated copy of eltB encoding the B subunit of enterotoxigenic Escherichia coli heat labile toxin (LT-B) in volunteers. Two oral doses of 10(8) or 10(9)CFU were administered to two groups of volunteers and both doses were well tolerated, with no vaccinemia, and only transient stool shedding. Immune responses to LT-B and S. typhi lipopolysaccharide were demonstrated in 67 and 97% of subjects, respectively, without evidence of anti-carrier immunity preventing boosting of LT-B responses in many cases. Further development of this salmonella-based (spi-VEC) system for oral delivery of heterologous antigens appears warranted.

Ricin is a potent toxin associated with bioterrorism for which no vaccine or specific countermeasures are currently available. A stable, non-toxic and immunogenic recombinant ricin A-chain vaccine (RTA 1-33/44-198) has been developed by protein engineering. We identified optimal formulation conditions for this vaccine under which it remained stable and potent in storage for up to 18 months, and resisted multiple rounds of freeze-thawing without stabilizing co-solvents. Reformulation from phosphate buffer to succinate buffer increased adherence of the protein to aluminum hydroxide adjuvant from 15 to 91%, with a concomitant increase of nearly threefold in effective antigenicity in a mouse model. Using Fourier-transform infrared spectroscopy, we examined the secondary structure of the protein while it was adhered to aluminum hydroxide. Adjuvant adsorption produced only a small apparent change in secondary structure, while significantly stabilizing the protein to thermal denaturation. The vaccine therefore may be safely stored in the presence of adjuvant. Our results suggest that optimization of adherence of a protein antigen to aluminum adjuvant can be a useful route to increasing both stability and effectiveness, and support a role for a "depot effect" of adjuvant.

The development of small-animal models is necessary to understand host responses and immunity to emerging infectious diseases and potential bioterrorism agents. In this report we have characterized a murine model of intestinal ricin intoxication. Ricin administered intragastrically (i.g.) to BALB/c mice at doses ranging from 1 to 10 mg/kg of body weight induced dose-dependent morphological changes in the proximal small intestine (i.e., duodenum), including widespread villus atrophy and epithelial damage. Coincident with epithelial damage was a localized increase in monocyte chemotactic protein 1, a chemokine known to be associated with inflammation of the intestinal mucosa. Immunity to intestinal ricin intoxication was achieved by immunizing mice i.g. with ricin toxoid and correlated with elevated levels of antitoxin mucosal immunoglobulin A (IgA) and serum IgG antibodies. We expect that this model will serve as a valuable tool in identifying the inflammatory pathways and protective immune responses that are elicited in the intestinal mucosa following ricin exposure and will prove useful in the evaluation of antitoxin vaccines and therapeutics.

The anti-PA IgG1, IgG2, IgG3, and IgG4 subclass responses to clinical anthrax and to different numbers of anthrax vaccine adsorbed (AVA, BioThrax) injections were determined in a cross-sectional study of sera from 63 vaccinees and 13 clinical anthrax patients. The data show that both vaccination with three AVA injections and clinical anthrax elicit anti-PA IgG1, IgG2, and IgG3 subclass responses. An anti-PA IgG4 response was detected in AVA recipients after the fourth injection. The anthrax lethal toxin (LTx) neutralization efficacy of sera from recipients who received 4 to > or =10 AVA injections did not vary significantly in relation to changes in distribution of anti-PA IgG1 and IgG4 subclasses.

Seroepidemiological studies need sensitive, practicle and cost-effective methods. For pertussis serosurveillance, an in-house ELISA for antipertussis-toxin (PT) and anti-filamentous-hemagglutinin (FHA) were established in our laboratory and compared by a Ball-ELISA which had been reported to be reliable previously. Sixty sera with various antibody titers were tested by both of the methods. The correlation coefficients between two methods were 0.729 and 0.776 for anti-PT and anti-FHA, respectively, and regression coefficients were 0.623 and 0.693, respectively. The in-house ELISA was applied to a serosurvey including 373 healthy subjects (6 months-91 years old) in Turkey to observe the results. The moving averages of both antibodies were increased until 10 years old, reaching to 31 EU/ml for anti-PT and 65 EU/ml for anti-FHA and kept around this level in the older ages. The in-house ELISA was found to be reliable and the serosurvey results obtained by ELISA showed a characteristic distribution of antibody titers in each age group.

The shore crab Hemigrapsus sanguineus hemolymph contains soluble proteins that bind tetrodotoxin (TTX) and are responsible for high resistance of the crab to TTX. The TTX-binding protein was purified from the hemolymph by ultrafiltration, lectin affinity chromatography and gel filtration HPLC. The purified protein gave only one band in native-polyacrylamide gel electrophoresis (PAGE), confirming its homogeneity. Its molecular weight was estimated to be about 400k by gel filtration HPLC, while it was estimated to be about 82k under non-reducing conditions and about 72 and 82k under reducing conditions by SDS-PAGE, indicating that the TTX-binding protein was composed of at least two distinct subunits. The TTX-binding protein was an acidic glycoprotein with pI 3.5, abundant in Asp and Glu but absent in Trp, and contained 6% reducing sugar and 12% amino sugar. The protein selectively bound to TTX, with a neutralizing ability of 6.7 mouse unit TTX/mg protein, but not to paralytic shellfish poisoning toxins. However, its neutralizing activity was almost lost by treatments with enzymes (protease XIV, thermolysin, trypsin, amyloglucosidase and alpha-amylase) and denaturing agents (1% SDS, 1% dithiothreitol, 8 M urea and 6 M guanidine hydrochloride), suggesting the involvement of both proteinaceous and sugar moieties in the binding to TTX and the importance of the steric conformation of the TTX-binding protein., (Copright 2002 Elsevier Science Ltd.)

The toxin binding inhibition test (ToBI) were developed for potency testing of C. novyi type B alpha toxoid containing veterinary vaccines to replace the currently used toxin neutralisation test in mice (TNT). The antitoxin titres of rabbit sera (AN-, HV- and SP sera) were determined with ToBI using the international reference serum with known antitoxin titre. In order to show the validity of the methods, the results were compared with those of the manufacturers/regulatory authorities and correlation coefficients were calculated. The correlation coefficients were r = 0.93 (AN sera), r = 0.73 (HV sera) and r = 0.85 (SP sera). All correlations were statistically significant. The specificity of the methods could be proved using heterologous antisera. The results of the ToBI were reproducible. Thus, the ToBI offers a suitable in vitro method for the determination of the antitoxin titre of rabbit antisera as an alternative to the toxin neutralisation in mice for potency testing of vaccines containing C. novyi type B alpha toxoid.

Endogenous proteins isolated from the serum of snakes have been found to be natural inhibitors displaying anti-hemorrhagic, anti-neurotoxic or anti-myotoxic activity. Some of these proteins inhibit phospholipase A(2) (PLA(2)) activity. We review in brief here the properties, structure and classification of these PLA(2) inhibitors (PLIs), focusing in particular on the mechanism of neutralization of the toxic PLA(2)s by anti-neurotoxic PLIs. We also discuss: 1) the protection provided by these molecules against endogenous snake venom PLA(2)s; 2) their specificity for neurotoxic snake venom PLA(2)s (beta-neurotoxins) and non-toxic mammalian secreted sPLA(2)s; and 3) the domains of the inhibitor and PLA(2) potentially involved in the binding of these two molecules. Purified and characterized natural inhibitors of PLA(2)s may be used to develop more effective therapeutic strategies for dealing with snake envenomation. Moreover, the structural and, in some cases, functional similarity of natural inhibitors to various mammalian proteins suggests that these mammalian proteins may themselves behave as PLA(2) inhibitors. Thus, these proteins may have important physiological functions in regulating the activities of neurotoxic PLA(2) and non-toxic sPLA(2).

Data from two Swedish pertussis vaccine trials with various combination vaccines were used to compare anti-diphtheria antitoxin concentrations over time between different vaccines, vaccine lots and vaccine schedules. The immune responses were measured with a validated ELISA method.Results are given for 1326 children, born 1992, that were recruited to the placebo (DT)-controlled Trial I which used a 2, 4, 6 month schedule. Two DTP acellular and one DTP whole cell vaccine were used. No DT boosters were given until 5 years of age. Trial II recruited children born 1993-94 and compared three DTP acellular vaccines with one DTP whole cell vaccine. Results are given for 306 children in a 2, 4, 6 month schedule and for 531 children in a 3, 5, 12 month schedule. The latter schedule gave significantly higher diphtheria antitoxin concentrations post third dose. The various DTP acellular vaccines and an inefficacious DTP whole cell vaccine gave lower antitoxin concentrations than both an efficacious DTP whole cell vaccine and the DT vaccine. The larger differences in antigen response between vaccines was reduced in the course of time. Generally, an initial rapid decline of antitoxin concentration was followed by a slower decline; the change typically occurring when the antitoxin concentration reached 0.13-0.16 EU/ml. The time needed to reach this level was between 6 and 10 months based on the initial vaccine response.A "best-fit" combined exponential regression model was used to predict the optimal timing for booster vaccinations against diphtheria.Our data support a 3, 5, 12 month schedule followed by a fourth dose 4-5 years after the third dose, depending upon the vaccine used.