Evaluation of the AV7909 Anthrax Vaccine Toxicity in Sprague Dawley Rats Following Three Intramuscular Administrations.

AV7909 is a next-generation anthrax vaccine under development for post-exposure prophylaxis following suspected or confirmed Bacillus anthracis exposure, when administered in conjunction with the recommended antibacterial regimen. AV7909 consists of the FDA-approved BioThrax® vaccine (anthrax vaccine adsorbed) and an immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide adjuvant, CPG 7909. The purpose of this study was to evaluate the potential systemic and local toxicity of AV7909 when administered via repeat intramuscular injection to the right thigh muscle (biceps femoris) to male and female Sprague Dawley rats. The vaccine was administered on Days 1, 15, and 29 and the animals were assessed for treatment-related effects followed by a 2-week recovery period to evaluate the persistence or reversibility of any toxic effects. The AV7909 vaccine produced no apparent systemic toxicity based on evaluation of clinical observations, body weights, body temperature, clinical pathology, and anatomic pathology. Necrosis and inflammation were observed at the injection sites as well as in regional lymph nodes and adjacent tissues and were consistent with immune stimulation. Antibodies against B. anthracis protective antigen (PA) were detected in rats treated with the AV7909 vaccine, confirming relevance of this animal model for the assessment of systemic toxicity of AV7909. In contrast, sera of rats that received saline or soluble CPG 7909 alone were negative for anti-PA antibodies. Overall, 3 intramuscular immunizations of Sprague Dawley rats with AV7909 were well tolerated, did not induce mortality or any systemic adverse effects, and did not result in any delayed toxicity.

Repeat Dose Toxicity Study of the AV7909 Anthrax Vaccine Candidate in Juvenile Rats.

AV7909 is a next-generation anthrax vaccine candidate indicated for post-exposure prophylaxis of exposure to Bacillus anthracis. AV7909 consists of the Anthrax Vaccine Adsorbed (AVA) bulk drug substance and the immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide adjuvant, CPG 7909. Safety testing for pediatric population is warranted to support the potential emergency use of AV7909 in children. This study was conducted to investigate the local tolerance and potential systemic toxicity and their reversibility in juvenile rats by repeat intramuscular injections of the AV7909 vaccine candidate. Animals were dosed on postnatal day (PND) 21 (at weaning), PND 28, and PND 35, with the test article (AV7909), the adjuvant alone (Alhydrogel + CPG 7909), or sterile water for injection. Core group animals were necropsied on PND 37 and recovery group on PND 49. Study end points included survival, clinical observations, injection site observations, body weights, clinical pathology (hematology, coagulation, and clinical chemistry), pro-inflammatory biomarker analysis (alpha-2 macroglobulin [A2M] and alpha-1 acid glycoprotein [AGP]), and anatomic pathology. Immune response to vaccination was measured using the high-throughput anthrax lethal toxin neutralization assay (htpTNA). The AV7909 vaccine candidate produced no apparent systemic or local toxicity. The AGP and A2M levels were elevated in both the adjuvant-alone and AV7909 groups at the end of treatment but were comparable to control levels by the end of the recovery period. All animals in the AV7909 group demonstrated a robust neutralizing antibody response. The results indicate that AV7909 has a favorable safety profile in juvenile rats.

Repeat-Dose Toxicity Study of a Lyophilized Recombinant Protective Antigen-Based Anthrax Vaccine Adjuvanted With CpG 7909.

A recombinant protective antigen (rPA) anthrax vaccine candidate (rPA7909) was developed as a next-generation vaccine indicated for postexposure prophylaxis of disease resulting from suspected or confirmed Bacillus anthracis exposure. The lyophilized form of rPA7909-vaccinated candidate contains 75 µg purified rPA, 750 µg aluminum (as Alhydrogel adjuvant), and 250 µg of an immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide CpG 7909 in a 0.5 mL phosphate-buffered suspension. General toxicity and local reactogenicity were evaluated in Sprague Dawley rats vaccinated with the full human dose of rPA7909 by intramuscular injection. Animals were immunized on study days 1, 15, and 29. Control groups were administered diluent only or adjuvant control (excipients, CpG 7909, and Alhydrogel adjuvant in diluent) intramuscularly at the same dose volume and according to the same schedule used for rPA7909. Toxicity was assessed based on the results of clinical observations, physical examinations, body weights, injection site reactogenicity, ophthalmology, clinical pathology (hematology, coagulation, and serum chemistry), organ weights, and macroscopic and microscopic pathology evaluation. The immune response to rPA7909 vaccination was confirmed by measuring serum anti-PA immunoglobulin G levels. The rPA7909 vaccine produced no apparent systemic toxicity and only transient reactogenicity at the injection site. The injection site reaction from animals receiving the adjuvant control was very similar to those receiving rPA7909 with respect to the inflammation. The inflammatory response observed in the injection site and the draining lymph nodes was consistent with expected immune stimulation. The overall results indicated a favorable safety profile for rPA7909.

Efficacy and safety of AVP-21D9, an anthrax monoclonal antibody, in animal models and humans.

Anthrax is an acute infectious disease caused by the spore-forming bacterium Bacillus anthracis. Timely administration of antibiotics approved for the treatment of anthrax disease may prevent associated morbidity and mortality. However, any delay in initiating antimicrobial therapy may result in increased mortality, as inhalational anthrax progresses rapidly to the toxemic phase of disease. An anthrax antitoxin, AVP-21D9, also known as Thravixa (fully human anthrax monoclonal antibody), is being developed as a therapeutic agent against anthrax toxemia. The efficacy of AVP-21D9 in B. anthracis-infected New Zealand White rabbits and in cynomolgus macaques was evaluated, and its safety and pharmacokinetics were assessed in healthy human volunteers. The estimated mean elimination half-life values of AVP-21D9 in surviving anthrax-challenged rabbits and nonhuman primates (NHPs) ranged from approximately 2 to 4 days and 6 to 11 days, respectively. In healthy humans, the mean elimination half-life was in the range of 20 to 27 days. Dose proportionality was observed for the maximum serum concentration (Cmax) of AVP-21D9 and the area under the concentration-time curve (AUC). In therapeutic efficacy animal models, treatment with AVP-21D9 resulted in survival of up to 92% of the rabbits and up to 67% of the macaques. Single infusions of AVP-21D9 were well tolerated in healthy adult volunteers across all doses evaluated, and no serious adverse events were reported. (This study has been registered at ClinicalTrials.gov under registration no. NCT01202695.).

Pathology and pathophysiology of inhalational anthrax in a guinea pig model.

Nonhuman primates (NHPs) and rabbits are the animal models most commonly used to evaluate the efficacy of medical countermeasures against anthrax in support of licensure under the FDA's "Animal Rule." However, a need for an alternative animal model may arise in certain cases. The development of such an alternative model requires a thorough understanding of the course and manifestation of experimental anthrax disease induced under controlled conditions in the proposed animal species. The guinea pig, which has been used extensively for anthrax pathogenesis studies and anthrax vaccine potency testing, is a good candidate for such an alternative model. This study was aimed at determining the median lethal dose (LD50) of the Bacillus anthracis Ames strain in guinea pigs and investigating the natural history, pathophysiology, and pathology of inhalational anthrax in this animal model following nose-only aerosol exposure. The inhaled LD50 of aerosolized Ames strain spores in guinea pigs was determined to be 5.0 × 10(4) spores. Aerosol challenge of guinea pigs resulted in inhalational anthrax with death occurring between 46 and 71 h postchallenge. The first clinical signs appeared as early as 36 h postchallenge. Cardiovascular function declined starting at 20 h postexposure. Hematogenous dissemination of bacteria was observed microscopically in multiple organs and tissues as early as 24 h postchallenge. Other histopathologic findings typical of disseminated anthrax included suppurative (heterophilic) inflammation, edema, fibrin, necrosis, and/or hemorrhage in the spleen, lungs, and regional lymph nodes and lymphocyte depletion and/or lymphocytolysis in the spleen and lymph nodes. This study demonstrated that the course of inhalational anthrax disease and the resulting pathology in guinea pigs are similar to those seen in rabbits and NHPs, as well as in humans.

Effect of anthrax immune globulin on response to BioThrax (anthrax vaccine adsorbed) in New Zealand white rabbits.

Development of anthrax countermeasures that may be used concomitantly in a postexposure setting requires an understanding of the interaction between these products. Anthrax immune globulin intravenous (AIGIV) is a candidate immunotherapeutic that contains neutralizing antibodies against protective antigen (PA), a component of anthrax toxins. We evaluated the interaction between AIGIV and BioThrax (anthrax vaccine adsorbed) in rabbits. While pharmacokinetics of AIGIV were not altered by vaccination, the vaccine-induced immune response was abrogated in AIGIV-treated animals.

Evaluation of intravenous anthrax immune globulin for treatment of inhalation anthrax.

Bacillus anthracis toxins can be neutralized by antibodies against protective antigen (PA), a component of anthrax toxins. Anthrivig (human anthrax immunoglobulin), also known as AIGIV, derived from plasma of humans immunized with BioThrax (anthrax vaccine adsorbed), is under development for the treatment of toxemia following exposure to anthrax spores. The pharmacokinetics (PK) of AIGIV was assessed in naive animals and healthy human volunteers, and the efficacy of AIGIV was assessed in animals exposed via inhalation to aerosolized B. anthracis spores. In the clinical study, safety, tolerability, and PK were evaluated in three dose cohorts (3.5, 7.1, and 14.2 mg/kg of body weight of anti-PA IgG) with 30 volunteers per cohort. The elimination half-life of AIGIV in rabbits, nonhuman primates (NHPs), and humans following intravenous infusion was estimated to be approximately 4, 12, and 24 days, respectively, and dose proportionality was observed. In a time-based treatment study, AIGIV protected 89 to 100% of animals when administered 12 h postexposure; however, a lower survival rate of 39% was observed when animals were treated 24 h postexposure, underscoring the need for early intervention. In a separate set of studies, animals were treated on an individual basis upon detection of a clinical sign or biomarker of disease, namely, a significant increase in body temperature (SIBT) in rabbits and presence of PA in the serum of NHPs. In these trigger-based intervention studies, AIGIV induced up to 75% survival in rabbits depending on the dose and severity of toxemia at the time of treatment. In NHPs, up to 33% survival was observed in AIGIV-treated animals. (The clinical study has been registered at ClinicalTrials.gov under registration no. NCT00845650.).

Developmental and reproductive safety evaluation of AV7909 anthrax vaccine candidate in rats.

The AV7909 vaccine, consists of the Anthrax Vaccine Adsorbed (AVA) bulk drug substance and the immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide adjuvant CPG 7909. The purpose of this research was to evaluate the potential maternal, reproductive, and developmental toxicity of AV7909 in rats to support licensure for use in women of childbearing potential. Groups of first generation (F0 ) female Sprague Dawley rats were dosed by intramuscular injection with water for injection, adjuvant or AV7909 at a volume of 0.5 ml/dose. Each rat received three vaccinations: 14 days prior to start of the mating period, on the first day of the mating period and on gestation day (GD) 7. There was no maternal mortality. Body weights, weight gain, and food consumption were comparable between groups. Findings in F0 females were limited to transient injection site edema and nodules consistent with immunostimulatory effects of the vaccine and adjuvant. Administration of AV7909 did not affect mating, fertility, pregnancy, embryo-fetal viability, growth, or morphologic development, parturition, maternal care of offspring or postnatal survival, growth, or development. There was no evidence of systemic inflammation in pregnant rats, based on evaluation of serum concentrations of the acute phase proteins alpha-2-macroglobulin and alpha-1-acid glycoprotein on GD 21. Anthrax lethal toxin-neutralizing antibodies were detected in AV7909-vaccinated F0 females. The antibodies were also detected in the sera of fetuses and F1 pups. Exposure of the fetuses and pups to maternally derived anthrax lethal toxin-neutralizing antibodies was not associated with developmental toxicity.

Efficacy of the AV7909 anthrax vaccine candidate in guinea pigs and nonhuman primates following two immunizations two weeks apart.

The anthrax vaccine candidate AV7909 is being developed as a next-generation vaccine for post-exposure prophylaxis (PEP) against inhalational anthrax. In clinical studies, two vaccinations with AV7909 administered either two or four weeks apart induced an enhanced immune response compared to BioThrax® (Anthrax Vaccine Adsorbed) (AVA). Anthrax toxin-neutralizing antibody (TNA) levels on Day 70 following initial vaccination that were associated with protection of animals exposed to inhalational anthrax were previously reported for the 0, 4-week AV7909 vaccination regimen. The current study shows that a 0, 2-week AV7909 vaccination regimen protected guinea pigs (GPs) and nonhuman primates (NHPs) against a lethal inhalational anthrax challenge on Days 28 and 70 after the first immunization. An earlier induction of protective TNA levels using a 0, 2-week AV7909 vaccination regimen may provide benefit over the currently approved AVA PEP 0, 2, and 4-week vaccination regimen.

Current Status and Trends in Prophylaxis and Management of Anthrax Disease.

Bacillus anthracis has been identified as a potential military and bioterror agent as it is relatively simple to produce, with spores that are highly resilient to degradation in the environment and easily dispersed. These characteristics are important in describing how anthrax could be used as a weapon, but they are also important in understanding and determining appropriate prevention and treatment of anthrax disease. Today, anthrax disease is primarily enzootic and found mostly in the developing world, where it is still associated with considerable mortality and morbidity in humans and livestock. This review article describes the spectrum of disease caused by anthrax and the various prevention and treatment options. Specifically we discuss the following; (1) clinical manifestations of anthrax disease (cutaneous, gastrointestinal, inhalational and intravenous-associated); (2) immunology of the disease; (3) an overview of animal models used in research; (4) the current World Health Organization and U.S. Government guidelines for investigation, management, and prophylaxis; (5) unique regulatory approaches to licensure and approval of anthrax medical countermeasures; (6) the history of vaccination and pre-exposure prophylaxis; (7) post-exposure prophylaxis and disease management; (8) treatment of symptomatic disease through the use of antibiotics and hyperimmune or monoclonal antibody-based antitoxin therapies; and (9) the current landscape of next-generation product candidates under development.

Development of a guinea pig inhalational anthrax model for evaluation of post-exposure prophylaxis efficacy of anthrax vaccines.

A next-generation anthrax vaccine candidate, AV7909, is being developed for post-exposure prophylaxis (PEP) of inhalational anthrax in combination with the recommended course of antimicrobial therapy. Clinical efficacy studies of anthrax countermeasures in humans are not ethical or feasible, therefore, licensure of AV7909 for PEP is being pursued under the US Food and Drug Administration (FDA) Animal Rule, which requires that evidence of effectiveness be demonstrated in an animal model of anthrax, where results of studies in such a model can establish reasonable likelihood of AV7909 to produce clinical benefit in humans. Initial development of a PEP model for inhalational anthrax included evaluation of post-exposure ciprofloxacin pharmacokinetics (PK), tolerability and survival in guinea pigs treated with various ciprofloxacin dosing regimens. Three times per day (TID) intraperitoneal (IP) dosing with 7.5 mg/kg of ciprofloxacin initiated 1 day following inhalational anthrax challenge and continued for 14 days was identified as a well tolerated partially curative ciprofloxacin treatment regimen. The added benefit of AV7909 vaccination was evaluated in guinea pigs given the partially curative ciprofloxacin treatment regimen. Groups of ciprofloxacin-treated guinea pigs were vaccinated. 1 and 8 days post-challenge with serial dilutions of AV7909, a 1:16 dilution of AVA, or normal saline. A group of untreated guinea pigs was included as a positive control to confirm lethal B. anthracis exposure. Post-exposure vaccination with the AV7909 anthrax vaccine candidate administered in combination with the partially curative ciprofloxacin treatment significantly increased survival of guinea pigs compared to ciprofloxacin treatment alone. These results suggest that the developed model can be useful in demonstrating added value of the vaccine for PEP.

Comparative immunogenicity and efficacy of thermostable (lyophilized) and liquid formulation of anthrax vaccine candidate AV7909.

The anthrax vaccine candidate AV7909 is being developed as a next-generation vaccine for a post-exposure prophylaxis (PEP) indication against anthrax. AV7909 consists of the anthrax vaccine adsorbed (AVA) (Emergent BioSolutions Inc., Lansing, MI) bulk drug substance adjuvanted with the immunostimulatory oligodeoxynucleotide (ODN) compound, CPG 7909. The addition of CPG 7909 to AVA enhances both the magnitude and the kinetics of antibody responses in animals and human subjects, making AV7909 a suitable next-generation vaccine for use in a PEP setting. Emergent has produced a thermostable (lyophilized) formulation of AV7909 vaccine utilizing drying technology. The purpose of the study described here was to assess the immunogenicity and efficacy of the lyophilized formulation of the AV7909 vaccine candidate as compared with the liquid formulation in the guinea pig general-use prophylaxis (GUP) model. The study also provides initial information on the relationship between the immune response induced by the thermostable formulation of the vaccine, as measured by the toxin neutralization assay (TNA), and animal survival following lethal anthrax aerosol challenge. Results demonstrated that there were no significant differences in the immunogenicity or efficacy of lyophilized AV7909 against lethal anthrax spore aerosol challenge in the guinea pig model as compared to liquid AV7909. For both vaccine formulations, logistic regression modeling showed that the probability of survival increased as the pre-challenge antibody levels increased.

Identification of staphylococcal enterotoxin B domains involved in binding to cultured human kidney proximal tubular cells: imparting proliferation and death.

Studies suggest that staphylococcal enterotoxin B (SEB) is initially harbored in the kidney by binding to digalactosylceramide molecules in the proximal tubular cells. However, little is known in regard to the peptide motif within SEB that binds to these cells and imparts toxic effects. Herein, using human kidney proximal tubular cells (PTs) we have performed a systematic study on the binding of various peptides and peptide analogs of SEB and demonstrate a structure-functional relationship. Using [(125)I]labeled SEB peptides, we show a high affinity and displaceable binding of SEB 191-220 to human PT cells. Binding was mitigated by the use of antibody against SEB, by digalactosylceramide (the putative receptor), and by the use of endoglycoceramidase, which selectively removes the oligosaccharide backbones from glycosphingolipids. Our structure/ functional studies revealed that peptide 130-160 induces a concentration-dependent increase in programmed cell death/ apoptosis in human proximal tubular cells. Mechanistic studies further suggest that SEB/SEB peptide (130-160) impart apoptosis via the activation of neutral sphingomyelinase, which hydrolizes sphingomyelin to ceramide and phosphocholine. SEB 130-160 mediated apoptosis was mitigated by preincubation of cells with antibody against SEB and an SEB 130-160 antibody.

Correlation between anthrax lethal toxin neutralizing antibody levels and survival in guinea pigs and nonhuman primates vaccinated with the AV7909 anthrax vaccine candidate.

The anthrax vaccine candidate AV7909 is being developed as a next generation vaccine for a post-exposure prophylaxis (PEP) indication against anthrax. AV7909 consists of the Anthrax Vaccine Adsorbed (AVA, BioThrax®) bulk drug substance adjuvanted with the immunostimulatory oligodeoxynucleotide (ODN) compound, CPG 7909. The addition of CPG 7909 to AVA enhances both the magnitude and the kinetics of antibody responses in animals and human subjects, making AV7909 a suitable next-generation vaccine for use in a PEP setting. The studies described here provide initial information on AV7909-induced toxin-neutralizing antibody (TNA) levels associated with the protection of animals from lethal Bacillus anthracis challenge. Guinea pigs or nonhuman primates (NHPs) were immunized on Days 0 and 28 with various dilutions of AV7909, AVA or a saline or Alhydrogel+CPG 7909 control. Animals were challenged via the inhalational route with a lethal dose of aerosolized B. anthracis (Ames strain) spores and observed for clinical signs of disease and mortality. The relationship between pre-challenge serum TNA levels and survival following challenge was determined in order to calculate a threshold TNA level associated with protection. Immunisation with AV7909 induced a rapid, highly protective TNA response in guinea pigs and NHPs. Surprisingly, the TNA threshold associated with a 70% probability of survival for AV7909 immunized animals was substantially lower than the threshold which has been established for the licensed AVA vaccine. The results of this study suggest that the TNA threshold of protection against anthrax could be modified by the addition of an immune stimulant such as CPG 7909 and that the TNA levels associated with protection may be vaccine-specific.

Immunogenicity of the histidine-to-tyrosine staphylococcal enterotoxin B mutant protein in C3H/HeJ mice.

Staphylococcal enterotoxin B (SEB) is a common cause of food poisoning and toxic shock. A safe and effective vaccine is needed to protect against the superantigenic effects of this toxin. We previously constructed and produced an apparently nontoxic SEB mutant having four histidine-to-tyrosine substitutions in positions 12, 32, 105, and 121. In the present study, we found that this H1.2.3.4 SEB mutant had low toxicity, was able to induce high levels of specific IgG antibodies, and protected mice in both the actinomycin D-primed and intranasal SEB intoxication model systems, despite the absence of detectable specific IgM and IgA antibodies. We propose further development of the H1.2.3.4 recombinant protein as a potential anti-SEB vaccine candidate.

Cancer information resources: digital and online sources.

The Internet is becoming an increasingly important source of information on cancer. This article highlights major sites of credible information on cancer and describes the types of information each of these sites contains. Large directories that help point the visitor to additional cancer-related sites are described, as are searchable databases of information on cancer as a disease, its diagnosis, and treatment. Sources of information on chemical carcinogens, mechanistic studies, and applied cancer toxicology are also described. These Internet sources of cancer information address the needs of toxicologists, environmental and occupational health scientists, cancer researchers and clinicians, government regulators, the public, and cancer survivors and their caregivers.

Cancer information resources: digital and online sources.

The Internet is becoming an increasingly important source of information on cancer. This article highlights major sites of credible information on cancer and describes the types of information each of these sites contains. Large directories that help point the visitor to additional cancer-related sites are described, as are searchable databases of information on cancer as a disease, its diagnosis, and treatment. Sources of information on chemical carcinogens, mechanistic studies, and applied cancer toxicology are also described. These Internet sources of cancer information address the needs of toxicologists, environmental and occupational health scientists, cancer researchers and clinicians, government regulators, the public, and cancer survivors and their caregivers.

Evaluation of immunogenicity and efficacy of anthrax vaccine adsorbed for postexposure prophylaxis.

Antimicrobials administered postexposure can reduce the incidence or progression of anthrax disease, but they do not protect against the disease resulting from the germination of spores that may remain in the body after cessation of the antimicrobial regimen. Such additional protection may be achieved by postexposure vaccination; however, no anthrax vaccine is licensed for postexposure prophylaxis (PEP). In a rabbit PEP study, animals were subjected to lethal challenge with aerosolized Bacillus anthracis spores and then were treated with levofloxacin with or without concomitant intramuscular (i.m.) vaccination with anthrax vaccine adsorbed (AVA) (BioThrax; Emergent BioDefense Operations Lansing LLC, Lansing, MI), administered twice, 1 week apart. A significant increase in survival rates was observed among vaccinated animals compared to those treated with antibiotic alone. In preexposure prophylaxis studies in rabbits and nonhuman primates (NHPs), animals received two i.m. vaccinations 1 month apart and were challenged with aerosolized anthrax spores at day 70. Prechallenge toxin-neutralizing antibody (TNA) titers correlated with animal survival postchallenge and provided the means for deriving an antibody titer associated with a specific probability of survival in animals. In a clinical immunogenicity study, 82% of the subjects met or exceeded the prechallenge TNA value that was associated with a 70% probability of survival in rabbits and 88% probability of survival in NHPs, which was estimated based on the results of animal preexposure prophylaxis studies. The animal data provide initial information on protective antibody levels for anthrax, as well as support previous findings regarding the ability of AVA to provide added protection to B. anthracis-infected animals compared to antimicrobial treatment alone.

Design and evaluation in mice of a broadly protective meningococcal group B native outer membrane vesicle vaccine.

A vaccine based on native outer membrane vesicles (NOMV) that has potential to provide safe, broad based protection against group B strains of Neisseria meningitidis has been developed. Three antigenically diverse group B strains of N. meningitidis were chosen and genetically modified to improve safety and expression of desirable antigens. Safety was enhanced by disabling three genes: synX, lpxL1, and lgtA. The vaccine strains were genetically configured to have three sets of antigens each with potential to induce protective antibodies against a wide range of group B strains. Preliminary immunogenicity studies with combined NOMV from the three strains confirmed the capacity of the vaccine to induce a broad based bactericidal antibody response. Analysis of the bactericidal activity indicated that antibodies to the LOS were responsible for a major portion of the bactericidal activity and that these antibodies may enhance the bactericidal activity of anti-protein antibodies.

Staphylococcal enterotoxin B causes differential expression of Rnd3 and RhoA in renal proximal tubule epithelial cells while inducing actin stress fiber assembly and apoptosis.

Staphylococcal enterotoxin B (SEB) is a toxic shock-inducing agent produced by Staphylococcus aureus. The hallmark of SEB-induced lethal shock is acute vasodilation leading to severe hypotension. Animal studies reveal that approximately 70% of intravenously administered toxin localizes to renal proximal tubule epithelial cells (RPTEC). This evidence, together with the well-documented role of the kidney in regulation of vascular tone, suggests that molecular events induced in RPTEC by SEB may contribute to the blood pressure dysregulation seen in enterotoxic shock. In an attempt to elucidate these molecular mechanisms, differential display was performed on SEB-treated and untreated RPTEC, and 32 differentially expressed transcripts (DETs) were identified. One of the down-regulated DETs matched the sequence for Rnd3, which normally inhibits Rho protein function. Consistent with Rnd3 down-regulation, message for RhoA was shown to increase upon SEB exposure, and actin stress fiber formation was dramatically increased. Further, SEB-exposed cells showed both increased enzymatic activity of caspase-3 and an increase in the percentage of apoptotic cells. Taken together, these results support the hypothesis that RPTEC undergo apoptosis upon exposure to SEB. Furthermore, these data implicate the involvement of the Rho family proteins in the molecular signaling pathway induced by SEB in RPTEC.