United States' regulatory approved pharmacotherapies for nuclear reactor explosions and anthrax-associated bioterrorism.

INTRODUCTION: Nuclear reactor incidents and bioterrorism outbreaks are concerning public health disasters. Little is known about US Food and Drug Administration (FDA)-approved agents that can mitigate consequences of these events. We review FDA data supporting regulatory approvals of these agents. AREAS COVERED: We reviewed pharmaceutical products approved to treat Hematopoietic Acute Radiation Syndrome (H-ARS) and to treat or prevent pulmonary infections following Bacillus anthracis (anthrax) exposure. Four drugs were approved for H-ARS: granulocyte-colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor, pegylated G-CSF, and romiplostim. For bioterrorism-associated anthrax, the FDA approved five antibiotics (doxycycline, penicillin-G, levofloxacin, moxifloxacin, and ciprofloxacin), two monoclonal antibodies (obiltoxaximab and raxibacumab), one polyclonal antitoxin (Anthrax Immune Globulin Intravenous) and two vaccines (Anthrax Vaccine Adsorbed and Anthrax Vaccine Adsorbed with an adjuvant). A national stockpile system ensures that communities have ready access to these agents. Our literature search was based on data included in drugs@FDA (2001-2023). EXPERT OPINION: Two potential mass public health disasters are aerosolized anthrax dissemination and radiological incidents. Five agents authorized for anthrax emergencies only have FDA approval for this indication, five antibiotics have FDA approvals as antibiotics for common infections and for bacillus anthrax, and four agents have regulatory approvals for supportive care for cancer and for radiological incidents.

A putative exosporium lipoprotein GBAA0190 of Bacillus anthracis as a potential anthrax vaccine candidate.

BACKGROUND: Bacillus ancthracis causes cutaneous, pulmonary, or gastrointestinal forms of anthrax. B. anthracis is a pathogenic bacterium that is potentially to be used in bioterrorism because it can be produced in the form of spores. Currently, protective antigen (PA)-based vaccines are being used for the prevention of anthrax, but it is necessary to develop more safe and effective vaccines due to their prolonged immunization schedules and adverse reactions. METHODS: We selected the lipoprotein GBAA0190, a potent inducer of host immune response, present in anthrax spores as a novel potential vaccine candidate. Then, we evaluated its immune-stimulating activity in the bone marrow-derived macrophages (BMDMs) using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Protective efficacy of GBAA0190 was evaluated in the guinea pig (GP) model. RESULTS: The recombinant GBAA0190 (r0190) protein induced the expression of various inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) in the BMDMs. These immune responses were mediated through toll-like receptor 1/2 via activation of mitogen-activated protein (MAP) kinase and Nuclear factor-κB (NF-κB) pathways. We demonstrated that not only immunization of r0190 alone, but also combined immunization with r0190 and recombinant PA showed significant protective efficacy against B. anthracis spore challenges in the GP model. CONCLUSIONS: Our results suggest that r0190 may be a potential target for anthrax vaccine.

Two approaches for the stabilization of Bacillus anthracis recombinant protective antigen.

Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacteria Bacillus anthracis. There is a need for safe, highly effective, long-term storage vaccine formulations for mass vaccination. However, the development of new subunit vaccines based on recombinant protective antigen (rPA) faces the problem of vaccine antigen instability. Here, the potential of simultaneous application of two different approaches to stabilize rPA was demonstrated. Firstly, we employed spherical particles (SPs) obtained from the tobacco mosaic virus (TMV). Previously, we had reported that SPs can serve as an adjuvant and platform for antigen presentation. In the current work, SPs were shown to increase the stability of the full-size rPA without loss of its antigenic properties. The second direction was site-specific mutagenesis of asparagine residues to avoid deamidation that causes partial protein degradation. The modified recombinant protein comprising the PA immunogenic domains 3 and 4 (rPA3 + 4) was stable during storage at 4 and 25°C. rPA3 + 4 interacts with antibodies to rPA83 both individually and as a part of a complex with SPs. The results obtained can underpin the development of a recombinant vaccine with a full-size modified rPA (with similar amino acid substitutions that stabilize the protein) and SPs.

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens.

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is requisite to phagocytosis and killing, the assay is an alternative method to opsono-phagocytic killing assays. An advantage of the opsono-adherence assay is the option of using inactivated pathogens and mammalian cell lines, which allows standardization across multiple experiments. The use of an inactivated pathogen in the assay also facilitates work with biosafety level 3 infectious agents and other virulent pathogens. In our work, the opsono-adherence assay was used to assess the functional ability of antibodies, from sera of animals immunized with an anthrax capsule-based vaccine, to induce adherence of fixed Bacillus anthracis to a mouse macrophage cell line, RAW 264.7. Automated fluorescence microscopy was used to capture images of bacilli adhering to macrophages. Increased adherence was correlated with the presence of anti-capsule antibodies in the serum. Non-human primates that exhibited high serum anti-capsule antibody concentrations were protected from anthrax challenge. Thus, the opsono-adherence assay can be used to elucidate the biological functions of antigen specific antibodies in sera, to evaluate the efficacy of vaccine candidates and other therapeutics, and to serve as a possible correlate of immunity.

Crystalline and Amorphous Preparation of Aluminum Hydroxide Nanoparticles Enhances Protective Antigen Domain 4 Specific Immunogenicity and Provides Protection Against Anthrax.

INTRODUCTION: Aluminum salts, although they have been used as adjuvants in many vaccine formulations since 1926, exclusively induce a Th2-biased immune response, thereby limiting their use against intracellular pathogens like Mycobacterium tuberculosis. METHODS AND RESULTS: Herein, we synthesized amorphous and crystalline forms of aluminum hydroxide nanoparticles (AH nps) of 150-200 nm size range. Using Bacillus anthracis protective antigen domain 4 (D4) as a model antigen, we demonstrated that both amorphous and crystalline forms of AH nps displayed enhanced antigen D4 uptake by THP1 cells as compared to commercial adjuvant aluminum hydroxide gel (AH gel). In a mouse model, both amorphous and crystalline AH nps triggered an enhanced D4-specific Th2- and Th1-type immune response and conferred superior protection against anthrax spore challenge as compared to AH gel. Physicochemical characterization of crystalline and amorphous AH nps revealed stronger antigen D4 binding and release than AH gel. CONCLUSION: These results demonstrate that size and crystallinity of AH nps play important roles in mediating enhanced antigen presenting cells (APCs) activation and potentiating a strong antigen-specific immune response, and are critical parameters for the rational design of alum-based Th1-type adjuvant to induce a more balanced antigen-specific immune response.

Functional characterization and evaluation of protective efficacy of EA752-862 monoclonal antibody against B. anthracis vegetative cell and spores.

The most promising means of controlling anthrax, a lethal zoonotic disease during the early infection stages, entail restricting the resilient infectious form, i.e., the spores from proliferating to replicating bacilli in the host. The extractible antigen (EA1), a major S-layer protein present on the vegetative cells and spores of Bacillus anthracis, is highly immunogenic and protects mice against lethal challenge upon immunization. In the present study, mice were immunized with r-EA1C, the C terminal crystallization domain of EA1, to generate a neutralizing monoclonal antibody EA752-862, that was evaluated for its anti-spore and anti-bacterial properties. The monoclonal antibody EA752-862 had a minimum inhibitory concentration of 0.08 mg/ml, was bactericidal at a concentration of 0.1 mg/ml and resulted in 100% survival of mice against challenge with B. anthracis vegetative cells. Bacterial cell lysis as observed by scanning electron microscopy and nucleic acid leakage assay could be attributed as a possible mechanism for the bactericidal property. The association of mAb EA752-862 with spores inhibits their subsequent germination to vegetative cells in vitro, enhances phagocytosis of the spores and killing of the vegetative cells within the macrophage, and subsequently resulted in 90% survival of mice upon B. anthracis Ames spore challenge. Therefore, owing to its anti-spore and bactericidal properties, the present study demonstrates mAb EA752-862 as an efficient neutralizing antibody that hinders the establishment of early infection before massive multiplication and toxin release takes place.

Detection of Bacillus anthracis in animal tissues using InBios active anthrax detect rapid test lateral flow immunoassay.

The Active Anthrax Detect (AAD) Rapid Test lateral flow immunoassay is a point-of-care assay that was under investigational use for detecting Bacillus anthracis capsular polypeptide (polyglutamic acid) in human blood, serum and plasma. Small sample volumes, rapid results and no refrigeration required allow for easy use in either the field or laboratory. Although the test was developed for use in suspect cases of human inhalation anthrax, its features also make it a potentially powerful tool for testing suspect animal cases. We tested animal tissue samples that were confirmed or ruled out for B. anthracis. The AAD Rapid Tests were also deployed in the field, testing animal carcasses during an anthrax outbreak in hippopotami (Hippopotamus amphibius) and Cape buffalo (Syncerus caffer) in Namibia. Evaluation of all samples showed a specificity of 82% and sensitivity of 98%. However, when the assay was used on specimens from only fresh carcasses (dead for <24 h), the specificity increased to 96%. The AAD Rapid Test is a rapid and simple screening assay, but confirmatory testing needs to be done, especially when the age of the sample (days animal has been deceased) is unknown. SIGNIFICANCE AND IMPACT OF THE STUDY: In countries where anthrax is endemic, many human outbreaks are often caused by epizootics. Earlier detection of infected animals may allow for identification of exposed people, early implementation of prevention and control methods, and ultimately lessen the number of people and animals affected. Detection of Bacillus anthracis in animal tissues using a simple, rapid and field-deployable method would allow for faster outbreak response. We evaluated a simple sample collection and processing method for use with the Active Anthrax Detect Rapid Test lateral flow immunoassay to screen dead animals for anthrax.

A lanthanide-based magnetic nanosensor as an erasable and visible platform for multi-color point-of-care detection of multiple targets and the potential application by smartphone.

The sensitive, selective and point-of-care detection of dipicolinic acid (DPA) is of great significance for the prevention of the anthrax virus and the containment of bioterrorism. In this work, a multi-color fluorescent nanoprobe composed of lanthanides and magnetic nanoparticles (Fe3O4@CePO4:Tb-EDTA-Eu) has been designed, in which the portion of Fe3O4@CePO4:Tb can be used as the internal stable signal of green fluorescence, while the EDTA-Eu part can be used as the sensitive reaction signal for monitoring DPA. Upon the addition of DPA, the red fluorescence of Eu3+ ions is significantly enhanced, while the fluorescent color of the nanoprobes can change from green to red (such as yellow-green, orange-yellow and orange-red), achieving visual multi-color fluorescent detection even by the naked eye. By using the magnetic separation method, the composites can be easily purified for point-of-care testing. More importantly, the nanoprobe fixed test pieces enable real-time analysis of DPA by using an easy-to-access color-scanning application on a smartphone. Furthermore, the fluorescence intensity can be quenched by the addition of Cu2+, which leads to a rewritable nanosensor and can be used in the detection of cysteine (Cys) with high sensitivity. With the addition of Cys, this erasable nano detection platform can also display the original multi-color visual point-of-care detection. With further optimization, this new type of multi-color fluorescent assay is promising in point-of-care clinics for multi-target diagnostics.

A niosome formulation modulates the Th1/Th2 bias immune response in mice and also provides protection against anthrax spore challenge.

INTRODUCTION: In this study, we have investigated the immunogenicity and protective efficacy of a niosomal formulation encapsulating protective antigen (PA) and PA domain 4 (D4) of Bacillus anthracis. METHODS: Nonionic surfactant-based vesicles (NISV) + PA and NISV + D4 were prepared from span-60 and cholesterol by reverse-phase evaporation method and were evaluated for in vitro characteristics and immunological studies. RESULTS: Particle characterization using transmission electron microscopy and atomic force microscopy analysis showed that the niosomal formulation was spherical in shape. The entrapment efficiency values were calculated to be 58.5% and 44.75% for PA and D4, respectively. Confocal microscopy and flow cytometry studies showed an enhanced uptake of antigen in THP1 macrophages by niosome as compared to antigen only. An in vitro release assay showed a burst release of antigen from niosome within 24 hours followed by a gradual release for 144 hours. Immunological studies showed that both PA- and D4-encapsulated niosome elicited a robust IgG titer. Antibody isotyping and cytokine profile showed that NISV + PA and NISV + D4 enhanced both Th1 and Th2 responses in mice, suggesting a mixed Th1/Th2 response. Both NISV + PA and NISV + D4 elicited high levels of anti-inflammatory cytokine interleukin-10 with low levels of pro-inflammatory cytokine tumor necrosis factor-α, suggesting the anti-inflammatory property of niosome. Both the niosomal formulations were also able to confer protection against BA infection as compared to only PA and D4. CONCLUSION: PA and D4 encapsulated NISV formulation could modulate both the Th1 and Th2 adaptive immune system and was found to be a better prophylactic against anthrax.

Tetrazole-Based trans-Translation Inhibitors Kill Bacillus anthracis Spores To Protect Host Cells.

Bacillus anthracis, the causative agent of anthrax, remains a significant threat to humans, including potential use in bioterrorism and biowarfare. The capacity to engineer strains with increased pathogenicity coupled with the ease of disseminating lethal doses of B. anthracis spores makes it necessary to identify chemical agents that target and kill spores. Here, we demonstrate that a tetrazole-based trans-translation inhibitor, KKL-55, is bactericidal against vegetative cells of B. anthracis in culture. Using a fluorescent analog, we show that this class of compounds colocalizes with developing endospores and bind purified spores in vitro KKL-55 was effective against spores at concentrations close to its MIC for vegetative cells. Spore germination was inhibited at 1.2× MIC, and spores were killed at 2× MIC. In contrast, ciprofloxacin killed germinants at concentrations close to its MIC but did not prevent germination even at 32× MIC. Because toxins are released by germinants, macrophages infected by B. anthracis spores are killed early in the germination process. At ≥2× MIC, KKL-55 protected macrophages from death after infection with B. anthracis spores. Ciprofloxacin required concentrations of ≥8× MIC to exhibit a similar effect. Taken together, these data indicate that KKL-55 and related tetrazoles are good lead candidates for therapeutics targeting B. anthracis spores and suggest that there is an early requirement for trans-translation in germinating spores.

Protection of farm goats by combinations of recombinant peptides and formalin inactivated spores from a lethal Bacillus anthracis challenge under field conditions.

BACKGROUND: Bacillus (B.) anthracis, the causal agent of anthrax, is effectively controlled by the Sterne live spore vaccine (34F2) in animals. However, live spore vaccines are not suitable for simultaneous vaccination and antibiotic treatment of animals being at risk of infection in an outbreak situation. Non-living vaccines could close this gap. RESULTS: In this study a combination of recombinant protective antigen and recombinant Bacillus collagen-like antigen (rBclA) with or without formalin inactivated spores (FIS), targeted at raising an immune response against both the toxins and the spore of B. anthracis, was tested for immunogenicity and protectiveness in goats. Two groups of goats received from local farmers of the Kars region of Turkey were immunized thrice in three weeks intervals and challenged together with non-vaccinated controls with virulent B. anthracis, four weeks after last immunization. In spite of low or none measurable toxin neutralizing antibodies and a surprisingly low immune response to the rBclA, 80% of the goats receiving the complete vaccine were protected against a lethal challenge. Moreover, the course of antibody responses indicates that a two-step vaccination schedule could be sufficient for protection. CONCLUSION: The combination of recombinant protein antigens and FIS induces a protective immune response in goats. The non-living nature of this vaccine would allow for a concomitant antibiotic treatment and vaccination procedure. Further studies should clarify how this vaccine candidate performs in a post infection scenario controlled by antibiotics.

Combined virtual screening, MMPBSA, molecular docking and dynamics studies against deadly anthrax: An in silico effort to inhibit Bacillus anthracis nucleoside hydrolase.

Anthrax is a deadly disease caused by Bacillus anthracis, a dangerous biological warfare agent employed for both military and terrorist purposes. A critical selective target for chemotherapy against this disease is nucleoside hydrolase (NH), an enzyme still not found in mammals. In the current study, we have performed molecular docking and dynamics studies, aiming to propose the new potent inhibitors of B. anthracis NH among National Cancer Institute (NCI) Diversity Set. We also analyzed the principal interactions of proposed compounds with the active site residues of NH and the relevant factors to biological activity. Additionally, the physic-chemical properties of free and inhibitor bound NH were evaluated and discussed. Our data showed that compound NSC79887 is a good candidate to inhibit NH and also for biological tests and further development. Also, ADMET prediction revealed that all physic-chemical parameters are within the acceptable range defined for human use.

Comparisons of the humoral and cellular immunity induced by live A16R attenuated spore and AVA-like anthrax vaccine in mice.

The live attenuated anthrax vaccine and anthrax vaccine adsorbed (AVA) are two main types of anthrax vaccines currently used in human. However, the immunoprotective mechanisms are not fully understood. In this study, we compared humoral and cellular immunity induced by live A16R spore vaccine and A16R strain derived AVA-like vaccine in mice peripheral blood, spleen and bone marrow. Both A16R spores and AVA-like vaccines induced a sustained IgG antibody response with IgG1/IgG2b subtype dominance. However, A16R spores vaccine induced higher titer of IgG2a compared with AVA-like vaccine, indicating a stronger Th1 response to A16R spores. Using antigen-specific ELISpot assay, we observed a significant response of ASCs (antibody secreting cells) and IL4-CSCs (cytokine secreting cells) in mice. Specially, there was a positive correlation between the frequencies of antigen specific ASCs and IL4-CSCs in bone marrow derived cells, either by A16R spore or AVA-like vaccine vaccination. Moreover, we also found A16R spore vaccine, not AVA-like vaccine, could induce sustained frequency of IFN-γ-CSCs in bone marrow derived cells. Collectively, both the vaccines induced a mixed Th1/Th2 response with Th2 dominance in mice and A16R spore vaccine might provide a more comprehensive protection because of humoral and cellular immunity induced in bone marrow.

Generation of a novel chimeric PALFn antigen of Bacillus anthracis and its immunological characterization in mouse model.

Bacillus anthracis chimeric molecule PALFn, comprising the immunodominant domains of protective antigen (PA) and lethal factor (LF), has been developed in the past and has been shown to confer enhanced protection against anthrax in mouse model when challenged with anthrax lethal toxin (LeTx). However, the immunological correlates for this chimeric antigen, both in terms of humoral as well as cell-mediated immune responses, have not been described in detail. To address this gap, we have determined the immunological responses both at humoral as well as cellular levels for the protection conferred by the novel chimeric antigen PALFn constructed in our laboratory in comparison to PA antigen. The biological functionality of the chimeric antigen was ascertained by the trypsin digestion assay. The trypsin cleavage activated the functionality of PALFn and rendered it to interact and bind with the LF molecule. Similarly, the LFn component in the chimera could independently interact and bind to the trypsin-activated wild-type PA. Further, it was observed that the PALFn-immunized mice sera could readily react to both PA and LF antigens while PA-immunized mice sera showed reaction to PA and PALFn alone and not to the individual LF antigen. The in vitro toxin neutralizing ability of PALFn antisera on macrophage cell line J774.1 was robust but with 1.3-fold lesser titer than PA-immunized antisera. PALFn-immunized mouse splenocytes showed a significant lymphocyte proliferation when stimulated with PALFn. There was a remarkable increase in the level of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin 10 (IL-10), interferon-γ (IFN- γ), and tumor necrosis factor α (TNFα) from PALFn- and PA-stimulated splenocytes. In addition, there was a significant increase in antigen-specific CD4+ and CD8+ T-cell counts from both PALFn- and PA-immunized mouse splenocytes. The results clearly demonstrate the ability of chimeric molecule PALFn in eliciting robust humoral and cell-mediated immune responses in mouse model that is parallel to the wild-type PA but has additional anti-LF antibody response. Considering the enhanced protection offered by the chimera PALFn, we can conclude that it can be a better alternative to the wild-type PA-based recombinant vaccine against anthrax.

[PERSPECTIVES OF DEVELOPMENT OF LIVE RECOMBINANT ANTHRAX VACCINES BASED ON OPPORTUNISTIC AND APATHOGENIC MICROORGANISMS].

Live genetic engineering anthrax vaccines on the platform of avirulent and probiotic micro-organisms are a safe and adequate alternative to preparations based on attenuated Bacillus anthracis strains. Mucosal application results in a direct contact of the vaccine preparations with mucous membranes in those organs arid tissues of the macro-organisms, that are exposed to the pathogen in the first place, resulting in a development of local and systemic immune response. Live recombinant anthrax vaccines could be used both separately as well as in a prime-boost immunization scheme. The review focuses on immunogenic and protective properties of experimental live genetic engineering prearations, created based on members of geni of Salmonella, Lactobacillus and adenoviruses.

Production of Functionally Active and Immunogenic Non-Glycosylated Protective Antigen from Bacillus anthracis in Nicotiana benthamiana by Co-Expression with Peptide-N-Glycosidase F (PNGase F) of Flavobacterium meningosepticum.

Bacillus anthracis has long been considered a potential biological warfare agent, and therefore, there is a need for a safe, low-cost and highly efficient anthrax vaccine with demonstrated long-term stability for mass vaccination in case of an emergency. Many efforts have been made towards developing an anthrax vaccine based on recombinant protective antigen (rPA) of B. anthracis, a key component of the anthrax toxin, produced using different expression systems. Plants represent a promising recombinant protein production platform due to their relatively low cost, rapid scalability and favorable safety profile. Previous studies have shown that full-length rPA produced in Nicotiana benthamiana (pp-PA83) is immunogenic and can provide full protection against lethal spore challenge; however, further improvement in the potency and stability of the vaccine candidate is necessary. PA of B. anthracis is not a glycoprotein in its native host; however, this protein contains potential N-linked glycosylation sites, which can be aberrantly glycosylated during expression in eukaryotic systems including plants. This glycosylation could affect the availability of certain key epitopes either due to masking or misfolding of the protein. Therefore, a non-glycosylated form of pp-PA83 was engineered and produced in N. benthamiana using an in vivo deglycosylation approach based on co-expression of peptide-N-glycosidase F (PNGase F) from Flavobacterium meningosepticum. For comparison, versions of pp-PA83 containing point mutations in six potential N-glycosylation sites were also engineered and expressed in N. benthamiana. The in vivo deglycosylated pp-PA83 (pp-dPA83) was shown to have in vitro activity, in contrast to glycosylated pp-PA83, and to induce significantly higher levels of toxin-neutralizing antibody responses in mice compared with glycosylated pp-PA83, in vitro deglycosylated pp-PA83 or the mutated versions of pp-PA83. These results suggest that pp-dPA83 may offer advantages in terms of dose sparing and enhanced immunogenicity as a promising candidate for a safe, effective and low-cost subunit vaccine against anthrax.

Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice.

Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 µg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 µg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 µg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors.

A CpG-Ficoll Nanoparticle Adjuvant for Anthrax Protective Antigen Enhances Immunogenicity and Provides Single-Immunization Protection against Inhaled Anthrax in Monkeys.

Nanoparticulate delivery systems for vaccine adjuvants, designed to enhance targeting of secondary lymphoid organs and activation of APCs, have shown substantial promise for enhanced immunopotentiation. We investigated the adjuvant activity of synthetic oligonucleotides containing CpG-rich motifs linked to the sucrose polymer Ficoll, forming soluble 50-nm particles (DV230-Ficoll), each containing >100 molecules of the TLR9 ligand, DV230. DV230-Ficoll was evaluated as an adjuvant for a candidate vaccine for anthrax using recombinant protective Ag (rPA) from Bacillus anthracis. A single immunization with rPA plus DV230-Ficoll induced 10-fold higher titers of toxin-neutralizing Abs in cynomolgus monkeys at 2 wk compared with animals immunized with equivalent amounts of monomeric DV230. Monkeys immunized either once or twice with rPA plus DV230-Ficoll were completely protected from challenge with 200 LD50 aerosolized anthrax spores. In mice, DV230-Ficoll was more potent than DV230 for the induction of innate immune responses at the injection site and draining lymph nodes. DV230-Ficoll was preferentially colocalized with rPA in key APC populations and induced greater maturation marker expression (CD69 and CD86) on these cells and stronger germinal center B and T cell responses, relative to DV230. DV230-Ficoll was also preferentially retained at the injection site and draining lymph nodes and produced fewer systemic inflammatory responses. These findings support the development of DV230-Ficoll as an adjuvant platform, particularly for vaccines such as for anthrax, for which rapid induction of protective immunity and memory with a single injection is very important.