GC-072: A Novel Therapeutic Candidate for Oral Treatment of Melioidosis and Infections Caused by Select Biothreat Pathogens.

Burkholderia pseudomallei (B. pseudomallei), the etiological agent of melioidosis, is a Gram-negative bacterium with additional concern as a biothreat pathogen. The mortality rate from B. pseudomallei varies depending on the type of infection and extent of available health care, but in the case of septicemia left untreated it can range from 50 - 90%. Current therapy for melioidosis is biphasic, consisting of parenteral acute-phase treatment for two weeks or longer, followed by oral eradication-phase treatment lasting several months. An effective oral therapeutic for outpatient treatment of acute-phase melioidosis is needed. GC-072 is a potent, 4-oxoquinolizine antibiotic with selective inhibitory activity against bacterial topoisomerases. GC-072 has demonstrated in vitro potency against susceptible and drug-resistant strains of B. pseudomallei and is also active against Burkholderia mallei, Bacillus anthracis, Yersinia pestis, and Francisella tularensis GC-072 is bactericidal both extra- and intracellularly, with rapid killing noted within a few hours and reduced development of resistance compared to ceftazidime. GC-072, delivered intragastrically to mimic oral administration, promoted dose-dependent survival in mice using lethal inhalational models of B. pseudomallei infection following exposure to a 24 or 339 LD50 challenge with B. pseudomallei strain 1026b. Overall, GC-072 appears to be a strong candidate for first-line, oral treatment of melioidosis.

Preclinical safety assessment of a recombinant plague vaccine (rF1V).

A recombinant vaccine (rF1V) is being developed to protect adults 18 to 55 years of age from fatal pneumonic plague caused by aerosolized Yersinia pestis. A comprehensive series of studies was conducted to evaluate the general toxicity and local reactogenicity of the rF1V vaccine prior to first use in humans. Toxicity was evaluated in CD-1 mice vaccinated with control material and three dosage concentrations of rF1V with or without Alhydrogel(®) by intramuscular (IM) injection on Study Days 1, 29, 57 and 71 in a volume of 0.1 mL. Total immunizing protein given in each dose was 0, 20 or 60 µg/animal. Local reactogenicity was evaluated in mice at the dosages given and in New Zealand white (NZW) rabbits using the same injection volume and formulations (40, 80, 160 and 320 µg/mL total antigen and 0.3% (w/v) Alhydrogel(®)) intended for human use (0.5 mL). The rF1V vaccine produced no apparent systemic toxicity and only transient edema and erythema at the injection site. Together these results indicated a favorable safety profile for rF1V and supported its use in a Phase 1 clinical trial.

Quantification of botulinum neurotoxin serotypes A and B from serum using mass spectrometry.

Botulinum neurotoxins (BoNT) are the deadliest agents known. Previously, we reported an endopeptidase activity based method (Endopep-MS) that detects and differentiates BoNT serotypes A-G. This method uses serotype specific monoclonal antibodies and the specific enzymatic activity of BoNT against peptide substrates which mimic the toxin's natural target. Cleavage products from the reaction are detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We have now developed a multiple reaction monitoring method to quantify the biological activity of BoNT serotypes A (BoNT/A) and B (BoNT/B) present in 0.5 mL of serum using electrospray mass spectrometry. The limit of quantification for each serotype is 1 mouse intraperitoneal lethal dose (MIPLD(50)) corresponding to 31 pg of BoNT/A and 15 pg of BoNT/B in this study. This method was applied to serum from rhesus macaques with inhalational botulism following exposure to BoNT/B, showing a maximum activity of 6.0 MIPLD(50)/mL in surviving animals and 653.6 MIPLD(50)/mL in animals that died in the study. The method detects BoNT/B in serum 2-5 h after exposure and up to 14 days. This is the first report of a quantitative method with sufficient sensitivity, selectivity, and low sample size requirements to measure circulating BoNT activity at multiple times during the course of botulism.

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.

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.

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.

Portal donor-specific blood transfusion and mycophenolate mofetil allow steroid avoidance and tacrolimus dose reduction with sustained levels of chimerism in a pig model of intestinal transplantation.

BACKGROUND: In a pig model of intestinal transplantation, we previously showed that hepatic conditioning through portal donor-specific blood transfusion (pDSBT), high-dose tacrolimus (TAC), and steroids prevented rejection and increased survival Our current study tests a protocol of pDSBT, short-term mycophenolate mofetil (MMF), and low-dose TAC to eliminate the use of steroids, reduce TAC dosage, and increase the level of chimerism in the peripheral blood. MATERIALS AND METHODS: Four groups of outbred, mixed lymphocyte culture (MLC)-reactive pigs underwent bowel transplants and pDSBT. Immunosuppression (group 1, high-dose TAC and steroids; group 2, low-dose TAC and MMF; group 3, low-dose TAC, MMF, and aminoguanidine; group 4, low-dose TAC, MMF, and arginine) was discontinued after 28 days. RNA was extracted from intestinal graft and native liver biopsies for cytokine measurements. Chimerism levels were determined using a Q-PCR analysis. RESULTS: Pig survival and death rates due to rejection did not significantly differ between the four groups. Chimerism levels determined by Q-PCR analysis were not different until day 28. After discontinuation of immunosuppression, we noted a trend (P = 0.15) toward higher mean chimerism levels on day 60 for groups 2, 3, and 4 (9%) vs. group 1 (0.5%). Tissue cytokine and serum nitrate levels did not significantly differ between the four groups. Attempts to modify nitric oxide synthase activity offered no added benefit. CONCLUSIONS: The combination of pDSBT, MMF, and low-dose TAC (vs. high-dose TAC and steroids) allowed sustained levels of mixed chimerism to develop after discontinuation of immunosuppression.

Preclinical safety assessment of recombinant botulinum vaccine A/B (rBV A/B).

A recombinant botulinum vaccine (rBV A/B) is being developed to protect adults 18-55 years of age from fatal botulism caused by inhalational intoxication with botulinum neurotoxin complex (BoNT) serotype A, subtype A1 (BoNT/A1) and BoNT serotype B, subtype B1 (BoNT/B1). Fundamental to the advanced development process is an initial demonstration of product safety in animals. A comprehensive series of studies was conducted to evaluate the general toxicity, neurobehavioral toxicity and local reactogenicity of the rBV A/B vaccine prior to first use in humans. Toxicity was evaluated in CD-1 mice vaccinated with control material and three dosages of rBV A/B with or without Alhydrogel(®) by intramuscular (IM) injection on Study Days 0, 28, 56 and 70 in a volume of 100µL. Total immunizing protein given in each dose was either 0, 2, 4 or 8 µg/animal. Local reactogenicity was evaluated in mice at the dosages given and in New Zealand white (NZW) rabbits using the same injection volume (0.5 mL) and formulations (10, 20 and 40 g/mL total antigen with 0.2% (w/v) Alhydrogel(®)) intended for human use. The rBV A/B vaccine produced no apparent systemic or neurobehavioral toxicity and only transient mild inflammation at the injection site. Together these results indicated a favorable safety profile for rBV A/B and supported its use in a Phase 1 clinical trial.

Botulinum neurotoxin neutralizing activity of immune globulin (IG) purified from clinical volunteers vaccinated with recombinant botulinum vaccine (rBV A/B).

The basis for efficacy of the recombinant botulinum vaccine, serotypes A and B (rBV A/B) is that neutralizing antibodies induced by vaccination bind to botulinum neurotoxin complex serotype A, subtype A1 (BoNT/A1) and serotype B, subtype B1 (BoNT/B1) and prevent their actions at cholinergic neurons. The protective capacity of BoNT/A1 and BoNT/B1 neutralizing antibodies derived from the serum of clinical volunteers vaccinated with rBV A/B was evaluated in a guinea pig passive transfer model and a mouse bioassay. Guinea pigs passively immunized to achieve circulating neutralizing antibody concentration (NAC) levels representing the lowest measurable concentrations for BoNT/A1 and BoNT/B1 were protected against an intramuscular (IM) challenge more than 10 times the guinea pig IM median lethal dosage for BoNT/A1 and BoNT/B1. The passively immunized guinea pigs were asymptomatic during the 14-day post-challenge observation period. Control guinea pigs died within 48 h after challenge. Calculation of neutralizing efficiency of antibodies using results from a mouse bioassay indicated that a simple linearly proportional relationship does not exist between NAC level and the amount of BoNT neutralized. Based on this finding, estimates of level of protection must consider variability in BoNT neutralizing efficiency at different NAC levels. The protective capacity of human BoNT/A1 and BoNT/B1 neutralizing antibodies induced by rBV A/B vaccination was verified in a guinea pig passive immunization model. Additionally, estimates of the neutralizing efficiency have been established for BoNT/A1 and BoNT/B1 neutralizing antibodies obtained from clinical volunteers vaccinated with the rBV A/B.

Inhalational botulism in rhesus macaques exposed to botulinum neurotoxin complex serotypes A1 and B1.

A recombinant botulinum vaccine (rBV A/B) is being developed for protection against inhalational intoxication with botulinum neurotoxin (BoNT) complex serotype A, subtype A1 (BoNT/A1), and BoNT serotype B, subtype B1 (BoNT/B1). A critical component for evaluating rBV A/B efficacy will be the use of animal models in which the pathophysiology and dose-response relationships following aerosol exposure to well-characterized BoNT are thoroughly understood and documented. This study was designed to estimate inhaled 50% lethal doses (LD(50)) and to estimate 50% lethal exposure concentrations relative to time (LCt(50)) in rhesus macaques exposed to well-characterized BoNT/A1 and BoNT/B1. During the course of this study, clinical observations, body weights, clinical hematology results, clinical chemistry results, circulating neurotoxin levels, and telemetric parameters were documented to aid in the understanding of disease progression. The inhaled LD(50) and LCt(50) for BoNT/A1 and BoNT/B1 in rhesus macaques were determined using well-characterized challenge material. Clinical observations were consistent with the recognized pattern of botulism disease progression. A dose response was demonstrated with regard to the onset of these clinical signs for both BoNT/A1 and BoNT/B1. Dose-related changes in physiologic parameters measured by telemetry were also observed. In contrast, notable changes in body weight, hematology, and clinical chemistry parameters were not observed. Circulating levels of BoNT/B1 were detected in animals exposed to the highest levels of BoNT/B1; however, BoNT/A1 was not detected in the circulation at any aerosol exposure level. The rhesus macaque aerosol challenge model will be used for future evaluations of rBV A/B efficacy against inhalational BoNT/A1 and BoNT/B1 intoxication.

An improved Francisella tularensis live vaccine strain (LVS) is well tolerated and highly immunogenic when administered to rabbits in escalating doses using various immunization routes.

Tularemia is a severe disease for which there is no licensed vaccine. An attenuated F. tularensis live vaccine strain (LVS) was protective when administered to humans but safety concerns precluded its licensure and use in large-scale immunization. An improved F. tularensis LVS preparation was produced under current good manufacturing practice (cGMP) guidelines for evaluation in clinical trials. Preclinical safety, tolerability and immunogenicity were investigated in rabbits that received LVS in escalating doses (1 x 10(5)-1 x 10(9)CFU) by the intradermal, subcutaneous or percutaneous (scarification) route. This improved LVS formulation was well tolerated at all doses; no death or adverse clinical signs were observed and necropsies showed no signs of pathology. No live organisms were detected in liver or spleen. Transient local reactogenicity was observed after scarification injection. Erythema and edema developed after intradermal injection in the highest dose cohorts. High levels of F. tularensis-specific IgM, IgG and IgA developed early after immunization, in a dose-dependent fashion. Scarification elicited higher levels of IgA. Antibodies elicited by LVS also recognized F. tularensis Schu-S4 antigens and there was a significant correlation between antibody titers measured against both LVS and Schu-S4. The ELISA titers also correlated closely with those measured by microagglutination. This is the first report describing comprehensive toxicological and immunological studies of F. tularensis LVS in rabbits. This animal model, which closely resembles human disease, proved adequate to assess safety and immunogenicity of F. tularensis vaccine candidates. This new LVS vaccine preparation is being evaluated in human clinical studies.

Biological activity of an intravenous preparation of human vaccinia immune globulin in mouse models of vaccinia virus infection.

The biological activity of a new intravenous (i.v.) preparation of human vaccinia immune globulin (VIGIV) was evaluated in two mouse models of vaccinia virus (VV) infection. In a mouse tail lesion model, female CD-1 mice were inoculated i.v. with 7 x 10(4) PFU of VV to produce >10 lesions per tail 8 days later. In a mouse lethality model, female severe combined immunodeficient (SCID) mice were inoculated i.v. with 3 x 10(4) PFU of VV to produce 100% mortality within 45 days. The ability of VIGIV to reduce tail lesion formation in CD-1 mice and mortality in SCID mice was determined by (i) pretreatment of a lethal VV dose with VIGIV prior to i.v. inoculation into SCID mice and (ii) i.v. administration of VIGIV to CD-1 and SCID mice the day before and up to 8 days after VV infection. VIGIV reduced the proportion of CD-1 mice with >10 tail lesions in a dose-related manner when VIGIV was given 1 day before and up to 1 day after VV inoculation. The pretreatment of VV with VIGIV prolonged survival and decreased mortality. VIGIV (100 and 400 mg/kg) prolonged survival when given up to 4 days after VV inoculation, and the 400-mg/kg dose reduced the mortality rate by 80% when given the day before or immediately after VV inoculation. The biological activity of VIGIV was demonstrated in both the immunocompetent and immunocompromised murine models. The timing of treatment relative to VV inoculation appeared to be important for the demonstration of VIGIV's biological activity.