Search

rVSV-ΔG-SARS-CoV-2-S is a clinical stage (Phase 2) replication competent recombinant vaccine against SARS-CoV-2. To evaluate the safety profile of the vaccine, a series of non-clinical safety, immunogenicity and efficacy studies were conducted in four animal species, using multiple doses (up to 108 Plaque Forming Units/animal) and dosing regimens. There were no treatment-related mortalities or any noticeable clinical signs in any of the studies. Compared to unvaccinated controls, hematology and biochemistry parameters were unremarkable and no adverse histopathological findings. There was no detectable viral shedding in urine, nor viral RNA detected in whole blood or serum samples seven days post vaccination. The rVSV-ΔG-SARS-CoV-2-S vaccination gave rise to neutralizing antibodies, cellular immune responses, and increased lymphocytic cellularity in the spleen germinal centers and regional lymph nodes. No evidence for neurovirulence was found in C57BL/6 immune competent mice or in highly sensitive type I interferon knock-out mice. Vaccine virus replication and distribution in K18-human Angiotensin-converting enzyme 2-transgenic mice showed a gradual clearance from the vaccination site with no vaccine virus recovered from the lungs. The nonclinical data suggest that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe and immunogenic. These results supported the initiation of clinical trials, currently in Phase 2. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-021-03214-w.

The COVID-19 pandemic caused by SARS-CoV-2 imposes an urgent need for rapid development of an efficient and cost-effective vaccine, suitable for mass immunization. Here, we show the development of a replication competent recombinant VSV-∆G-spike vaccine, in which the glycoprotein of VSV is replaced by the spike protein of SARS-CoV-2. In-vitro characterization of this vaccine indicates the expression and presentation of the spike protein on the viral membrane with antigenic similarity to SARS-CoV-2. A golden Syrian hamster in-vivo model for COVID-19 is implemented. We show that a single-dose vaccination results in a rapid and potent induction of SARS-CoV-2 neutralizing antibodies. Importantly, vaccination protects hamsters against SARS-CoV-2 challenge, as demonstrated by the abrogation of body weight loss, and alleviation of the extensive tissue damage and viral loads in lungs and nasal turbinates. Taken together, we suggest the recombinant VSV-∆G-spike as a safe, efficacious and protective vaccine against SARS-CoV-2., Here, the authors generate a replication-competent VSV based vaccine expressing SARS-CoV-2 spike protein and show protection in the hamster model with one dose. Analysis of the antibody response in mice shows induction of neutralizing antibodies and suggests a desirable Th1-biased response to the vaccine.

The poly- δ- d-glutamic acid capsule of Bacillus anthracis plays a major role in this bacterium pathogenicity. Capsule synthesis relies on a 5 gene operon; capB, C, A, D and E that are regulated by acpA and acpB, that respond to the major virulence regulator - atxA. We took a genetic approach to examine the involvement of acpA and acpB in capsule production in vitro and on B. anthracis virulence in vivo. To complement the effect of the mutations on capsule accumulation in vitro, we applied our toxin independent systemic infection method to study their effects in vivo. We found that though the roles of acpA and axpB are redundant in vitro, deleting acpA had a significant effect on pathogenicity, mainly on the time to death. As expected, deletion of both acpA and acpB resulted in loss of capsule accumulation in vitro and full attenuation in vivo, indicating that capsule production depends exclusively on acpA/B regulation. To identify additional effects of acpA and acpB on pathogenicity via non-capsule related virulence pathways, we bypassed acpA/B regulation by inserting the pagA promotor upstream to the cap operon, diverting regulation directly to atxA. This resulted in restoration of capsule accumulation in vitro and virulence (in intravenous or subcutaneous inoculation) in vivo. To test for additional pXO2-based genes involved in capsule production, we cloned the pagAprom-capA-E into the chromosome of VollumΔpXO2, which restored capsule accumulation. These results indicate that of the pXO2 genes, only capA-E and acpA are required for capsule production.

As Bacillus anthracis spores pose a proven bio-terror risk, the treatment focus has shifted from exposed populations to anthrax patients and the need for effective antibiotic treatment protocols increases. The CDC recommends carbapenems and Linezolid (oxazolidinone), for the treatment of anthrax, particularly for the late, meningeal stages of the disease. Previously we demonstrated that treatment with Meropenem or Linezolid, either as a single treatment or in combination with Ciprofloxacin, fails to protect rabbits from anthrax-meningitis. In addition, we showed that the failure of Meropenem was due to slow BBB penetration rather than low antibacterial activity. Herein, we tested the effect of increasing the dose of the antibiotic on treatment efficacy. We found that for full protection (88% cure rate) the dose should be increased four-fold from 40 mg/kg to 150 mg/kg. In addition, B. anthracis is a genetically stable bacterium and naturally occurring multidrug resistant B. anthracis strains have not been reported. In this manuscript, we report the efficacy of classical β-lactams as a single treatment or in combination with β-lactamase inhibitors in treating anthrax meningitis. We demonstrate that Ampicillin based treatment of anthrax meningitis is largely efficient (66%). The high efficacy (88-100%) of Augmentin (Amoxicillin and Clavulonic acid) and Unasyn (Ampicillin and Sulbactam) makes them a favorable choice due to reports of β-lactam resistant B. anthracis strains. Tazocin (Piperacillin and Tazobactam) proved inefficient compared to the highly efficient Augmentin and Unasyn.

Protective antigen (PA)-based vaccines are effective in preventing the development of fatal anthrax disease both in humans and in relevant animal models. The Bacillus anthracis toxins lethal toxin (lethal factor [LF] plus PA) and edema toxin (edema factor [EF] plus PA) are essential for the establishment of the infection, as inactivation of these toxins results in attenuation of the pathogen. Since the toxins reach high toxemia levels at the bacteremic stages of the disease, the CDC's recommendations include combining antibiotic treatment with antitoxin (anti-PA) immunotherapy. We demonstrate here that while treatment with a highly potent neutralizing monoclonal antibody was highly efficient as postexposure prophylaxis treatment, it failed to protect rabbits with any detectable bacteremia (≥10 CFU/ml). In addition, we show that while PA vaccination was effective against a subcutaneous spore challenge, it failed to protect rabbits against systemic challenges (intravenous injection of vegetative bacteria) with the wild-type Vollum strain or a toxin-deficient mutant. To test the possibility that additional proteins, which are secreted by the bacteria under pathogenicity-stimulating conditions in vitro , may contribute to the vaccine's potency, we immunized rabbits with a secreted protein fraction from a toxin-null mutant. The antiserum raised against the secreted fraction reacts with the bacteria in an immunofluorescence assay. Immunization with the secreted protein fraction did not protect the rabbits against a systemic challenge with the fully pathogenic bacteria. Full protection was obtained only by a combined vaccination with PA and the secreted protein fraction. Therefore, these results indicate that an effective antiserum treatment in advanced stages of anthrax must include toxin-neutralizing antibodies in combination with antibodies against bacterial cell targets.

Nonencapsulated (∆pXO2) Bacillus anthracis strains are commonly used as vaccines and for anthrax research, mainly in the mouse model. Previously, we demonstrated that the infection of rabbits, intranasally or subcutaneously, with the spores of a fully virulent strain results in the systemic dissemination of the bacteria, meningitis, and death, whereas ∆pXO2 strains are fully attenuated in this animal model. We used the intravenous inoculation of rabbits to study the pathogenicity of the ∆pXO2 strain infection. Bacteremia, brain bacterial burden, and pathology were used as criteria to compare the Vollum∆pXO2 disease to the wild type Vollum infection. To test the role of adhesion in the virulence of Vollum∆pXO2, we deleted the major adhesion protein BslA and tested the virulence and immunogenicity of this mutant. We found that 50% of the rabbits succumb to Vollum∆pXO2 strain following i.v. infection, a death that was accompanied with significant neurological symptoms. Pathology revealed severe brain infection coupled with an atypical massive bacterial growth into the parenchyma. Contrary to the Vollum strain, deletion of the bslA gene fully attenuated the ∆pXO2 strain. Though the Vollum∆pXO2 cannot serve as a model for B. anthracis pathogenicity in rabbits, deletion of the bslA gene prevents central nervous system (CNS) infections, possibly leading to the generation of a safer vaccine.

Treatment of anthrax is challenging, especially during the advanced stages of the disease. Recently, the Centers for Disease Control and Prevention (CDC) updated its recommendations for postexposure prophylaxis and treatment of exposed populations (before and after symptom onset). These recommendations distinguished, for the first time, between systemic disease with and without meningitis, a common and serious complication of anthrax. The CDC considers all systemic cases meningeal unless positively proven otherwise. The treatment of patients suffering from systemic anthrax with suspected or confirmed meningitis includes the combination of three antibiotics, i.e., a fluoroquinolone (levofloxacin or ciprofloxacin), a β-lactam (meropenem or imipenem), and a protein synthesis inhibitor (linezolid or clindamycin). In addition, treatment with an antitoxin (anti-protective antigen antibodies) and dexamethasone should be applied. Since the efficacy of most of these treatments has not been demonstrated, especially in animal meningitis models, we developed an anthrax meningitis model in rabbits and tested several of these recommendations. We demonstrated that, in this model, ciprofloxacin, linezolid, and meropenem were ineffective as single treatments, while clindamycin was highly effective. Furthermore, combined treatments of ciprofloxacin and linezolid or ciprofloxacin and dexamethasone failed in treating rabbits with meningitis. We demonstrated that dexamethasone actually hindered blood-brain barrier penetration by antibiotics, reducing the effectiveness of antibiotic treatment of anthrax meningitis in this rabbit model.

Hemorrhagic meningitis is considered a complication of anthrax and was reported in about 50% of deadly cases in humans and non-human primates (NHP). Recently we demonstrated in Guinea pigs and rabbits that 100% of the B. anthracis-infected animals presented histopathology of meningitis at the time of death, some without any sign of hemorrhage. A similar pathology was observed in animals that succumbed following infection with the toxin deficient mutant, thus indicating that anthrax meningitis is a toxin-independent phenomenon. In this manuscript we describe a histopathological study of the B. anthracis infection of the central nervous system (CNS). Though we could find sporadic growth of the bacteria around blood vessels in the cortex, we report that the main infiltration route is the choroid plexus. We found massive destruction of entire sections of the choroid plexus coupled with massive aggregation of bacilli in the ventricles, in close proximity to the parenchyma. The choroid plexus also contained significant amounts of intravascular bacterial aggregates, often enclosed in what appear to be fibrin-like clots. The high concentration of these aggregates in areas of significant tissue destruction combined with the fact that capsular B. anthracis bacteria have a low tendency to adhere to endothelial cells, might suggest that these clots are used as an adherence mechanism by the bacteria. The major histopathological finding is meningitis. We find massive bacterial growth in the meninges without evidence of encephalitis, even when the bacteria emerge from a parenchymal blood vessel. Erythrocytes were present within the meningeal space but no clear vasculitis could be detected. Histology of the brain stem indicates meningitis, edema and hemorrhages that might explain death from suffocation due to direct damage to the respiratory center. All of these processes are toxin-independent, since they were observed following infection with either the wild type strain or the toxin-deficient mutant. Herein, we propose that the first step of anthrax-meningitis is bacterial adhesion to the blood vessels by manipulating coagulation, mainly in the choroid plexus. The trapped bacteria then destroy sections of the choroid plexus, resulting in penetration into the CSF, leading to meningitis and hemorrhage. Death could be the result of increased intracranial pressure and/or damage to the brain stem.

Respiratory anthrax is a fatal disease in the absence of early treatment with antibiotics. Rabbits are highly susceptible to infection with Bacillus anthracis spores by intranasal instillation, succumbing within 2 to 4 days postinfection. This study aims to test the efficiency of antibiotic therapy to treat systemic anthrax in this relevant animal model. Delaying the initiation of antibiotic administration to more than 24 h postinfection resulted in animals with systemic anthrax in various degrees of bacteremia and toxemia. As the onset of symptoms in humans was reported to start on days 1 to 7 postexposure, delaying the initiation of treatment by 24 to 48 h (time frame for mass distribution of antibiotics) may result in sick populations. We evaluated the efficacy of antibiotic administration as a function of bacteremia levels at the time of treatment initiation. Here we compare the efficacy of treatment with clarithromycin, amoxicillin-clavulanic acid (Augmentin), imipenem, vancomycin, rifampin, and linezolid to the previously reported efficacy of doxycycline and ciprofloxacin. We demonstrate that treatment with amoxicillin-clavulanic acid, imipenem, vancomycin, and linezolid were as effective as doxycycline and ciprofloxacin, curing rabbits exhibiting bacteremia levels of up to 10 5 CFU/ml. Clarithromycin and rifampin were shown to be effective only as a postexposure prophylactic treatment but failed to treat the systemic (bacteremic) phase of anthrax. Furthermore, we evaluate the contribution of combined treatment of clindamycin and ciprofloxacin, which demonstrated improvement in efficacy compared to ciprofloxacin alone.

Infection of the central nervous system is considered a complication of Anthrax and was reported in humans and non-human primates. Previously we have reported that Bacillus anthracis possesses a toxin-independent virulent trait that, like the toxins, is regulated by the major virulence regulator, AtxA, in the presence of pXO2. This toxin-independent lethal trait is exhibited in rabbits and Guinea pigs following significant bacteremia and organ dissemination. Various findings, including meningitis seen in humans and primates, suggested that the CNS is a possible target for this AtxA-mediated activity. In order to penetrate into the brain tissue, the bacteria have to overcome the barriers isolating the CNS from the blood stream. Taking a systematic genetic approach, we compared intracranial (IC) inoculation and IV/SC inoculation for the outcome of the infection in rabbits/GP, respectively. The outstanding difference between the two models is exhibited by the encapsulated strain VollumΔpXO1, which is lethal when injected IC, but asymptomatic when inoculated IV/SC. The findings demonstrate that there is an apparent bottleneck in the ability of mutants to penetrate into the brain. Any mutant carrying either pXO1 or pXO2 will kill the host upon IC injection, but only those carrying AtxA either on pXO1 or in the chromosome in the background of pXO2 can penetrate into the brain following peripheral inoculation. The findings were corroborated by histological examination by H&E staining and immunofluorescence of rabbits' brains following IV and IC inoculations. These findings may have major implications on future research both on B. anthracis pathogenicity and on vaccine development.

Since its role in the prevention of osteoporosis in humans was proven some 30 years ago, calcium bioavailability has been the subject of numerous scientific studies. Recent technology allowing the production of a stable amorphous calcium carbonate (ACC) now enables a bioavailability analysis of this unique form of calcium. This study thus compares the solubility and fractional absorption of ACC, ACC with chitosan (ACC-C), and crystalline calcium carbonate (CCC). Solubility was evaluated by dissolving these preparations in dilute phosphoric acid. The results demonstrated that both ACC and ACC-C are more soluble than CCC. Fractional absorption was evaluated by intrinsically labeling calcium carbonate preparations with 45Ca, orally administrated to rats using gelatin capsules. Fractional absorption was determined by evaluating the percentage of the administrated radioactive dose per milliliter that was measured in the serum, calcium absorption in the femur, and whole-body retention over a 34-hour period. Calcium serum analysis revealed that calcium absorption from ACC and ACC-C preparations was up to 40% higher than from CCC, whereas retention of ACC and ACC-C was up to 26.5% higher than CCC. Absorbed calcium in the femurs of ACC-administrated rats was 30% higher than in CCC-treated animals, whereas 15% more calcium was absorbed following ACC-C treatment than following CCC treatment. This study demonstrates the enhanced solubility and bioavailability of ACC over CCC. The use of stable ACC as a highly bioavailable dietary source for calcium is proposed based on the findings of this study. © 2011 American Society for Bone and Mineral Research.