John D. Campbell

CpG-containing immunostimulatory DNA sequences elicit TNF-α–dependent toxicity in rodents but not in humans

CpG-containing immunostimulatory DNA sequences (ISS), which signal through TLR9, are being developed as a therapy for allergic indications and have proven to be safe and well tolerated in humans when administrated via the pulmonary route. In contrast, ISS inhalation has unexplained toxicity in rodents, which express TLR9 in monocyte/macrophage lineage cells as well as in plasmacytoid DCs (pDCs) and B cells, the principal TLR9-expressing cells in humans. We therefore investigated the mechanisms underlying this rodent-specific toxicity and its implications for humans. Mice responded to intranasally administered 1018 ISS, a representative B class ISS, with strictly TLR9-dependent toxicity, including lung inflammation and weight loss, that was fully reversible and pDC and B cell independent. Knockout mouse experiments demonstrated that ISS-induced toxicity was critically dependent on TNF-alpha, with IFN-alpha required for TNF-alpha induction. In contrast, human PBMCs, human alveolar macrophages, and airway-derived cells from Ascaris suum-allergic cynomolgus monkeys did not produce appreciable TNF-alpha in vitro in response to ISS stimulation. Moreover, sputum of allergic humans exposed to inhaled ISS demonstrated induction of IFN-inducible genes but minimal TNF-alpha induction. These data demonstrate that ISS induce rodent-specific TNF-alpha-dependent toxicity that is absent in humans and reflective of differential TLR9 expression patterns in rodents versus humans.

Allergen‐specific T cell responses to immunotherapy monitored by CD154 and intracellular cytokine expression

Background A sensitive measurement of low numbers of intracellular cytokine‐expressing antigen‐specific T cells from peripheral blood mononuclear cells (PBMC) is possible using CD154 as a marker of recently activated T cells. This technique may have potential for monitoring peripheral blood T cell responses to immunotherapy.

A limited CpG-containing oligodeoxynucleotide therapy regimen induces sustained suppression of allergic airway inflammation in mice

Background CpG-containing oligodeoxynucleotides (CpG-ODNs) are potent inhibitors of T helper 2 mediated allergic airway disease in sensitised mice challenged with allergen. A single treatment has transient effects but a limited series of treatments has potential to achieve clinically meaningful sustained inhibition of allergic airway disease. Objective To optimise the treatment regimen for sustained efficacy and to determine the mechanisms of action in mice of an inhaled form of CpG-ODN being developed for human asthma treatment. Methods We set up a chronic allergic-asthma model using ragweed-sensitised mice exposed weekly to intranasal ragweed. Using this model, the effects of a limited series of weekly intranasal 1018 ISS (CpG-ODN; B-class) treatments were evaluated during treatment and for several weeks after treatments had stopped but weekly allergen exposures continued. Treatment efficacy was evaluated by measuring effects on lung T helper 2 cytokines and eosinophilia, and lung dendritic cell function and T-cell responses. Results Twelve intranasal 1018 ISS treatments induced significant suppression of bronchoalveolar lavage eosinophilia and interleukin 4, 5 and 13 levels. This suppression of allergic T helper 2 parameters was maintained through 13 weekly ragweed exposures administered after treatment cessation. Subsequent experiments demonstrated that at least five treatments were required for lasting suppression. Although CpG-ODN induced moderate T helper 1 responses, suppression of allergic airway disease did not require interferon γ but was associated with induction of a regulatory T-cell response. Conclusions A short series of CpG-ODN treatments results in sustained suppression of allergic lung inflammation induced by a clinically relevant allergen.

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.

Optimization, Production, and Characterization of a CpG-Oligonucleotide-Ficoll Conjugate Nanoparticle Adjuvant for Enhanced Immunogenicity of Anthrax Protective Antigen

We have synthesized and characterized a novel phosphorothioate CpG oligodeoxynucleotide (CpG ODN)-Ficoll conjugated nanoparticulate adjuvant, termed DV230-Ficoll. This adjuvant was constructed from an amine-functionalized-Ficoll, a heterobifunctional linker (succinimidyl-[(N-maleimidopropionamido)-hexaethylene glycol] ester) and the CpG-ODN DV230. Herein, we describe the evaluation of the purity and reactivity of linkers of different lengths for CpG-ODN-Ficoll conjugation, optimization of linker coupling, and conjugation of thiol-functionalized CpG to maleimide-functionalized Ficoll and process scale-up. Physicochemical characterization of independently produced lots of DV230-Ficoll reveal a bioconjugate with a particle size of approximately 50 nm and covalent attachment of more than 100 molecules of CpG per Ficoll. Solutions of purified DV230-Ficoll were stable for at least 12 months at frozen and refrigerated temperatures and stability was further enhanced in lyophilized form. Compared to nonconjugated monomeric DV230, the DV230-Ficoll conjugate demonstrated improved in vitro potency for induction of IFN-α from human peripheral blood mononuclear cells and induced higher titer neutralizing antibody responses against coadministered anthrax recombinant protective antigen in mice. The processes described here establish a reproducible and robust process for the synthesis of a novel, size-controlled, and stable CpG-ODN nanoparticle adjuvant suitable for manufacture and use in vaccines.

First‐in‐Human Study With the Inhaled TLR9 Oligonucleotide Agonist AZD1419 Results in Interferon Responses in the Lung, and Is Safe and Well‐Tolerated

Current asthma treatments address symptoms rather than the underlying disease pathophysiology, a better understanding of which has led to the identification of the Th2 high endotype. The activation of Toll‐like receptors to induce Type I interferons directly in the lungs represents a novel therapeutic approach to reset this underlying Th2 pathophysiology with the potential to provide long‐term disease modification. We present the nonclinical data and phase I clinical profile of an inhaled TLR9 agonist, AZD1419, a C‐type CpG designed to induce interferon in the lung. In healthy volunteers, AZD1419 was found to be safe and well‐tolerated. Target engagement in the lung was demonstrated at all dose levels tested. No evidence of tolerization or amplification of responses was evident on repeated dosing and 15.4 mg was defined as the maximum tolerated dose. AZD1419 clinical data supports its continued development as a potentially disease‐modifying therapeutic in asthma.

Self-defense against Bacillus anthracis toxins: Is P-selectin the key?

Anthrax infections result from exposure to spores of the gram positive bacterium Bacillus anthracis. Anthrax spores are hardy and can persist in soil for years, posing an ongoing risk of infection to grazing animals. Natural infections in humans usually result from contact with infected animals or animal-derived products and subsequent spore acquisition through skin abrasions, ingestion, or inhalation. Cutaneous anthrax infections are the most common, but are still relatively rare and usually respond to medical care, while less common inhalational anthrax is the most frequently life-threatening. Prophylactic vaccination is available but is only approved in the US for groups at high risk of exposure, such as veterinarians, laboratory staff working with anthrax, and some members of the military. The US vaccine, anthrax vaccine absorbed (AVA; BioThrax ) consists of anthrax protective antigen (PA) adsorbed to aluminum hydroxide and is administered in 5 doses over 18 months with yearly boosters. Data from animal studies show that induction of toxin neutralizing antibodies to PA is the principal immune correlate of protection against anthrax. The resistance of spores to heat, desiccation, and chemical disinfectants and the potential for widespread infections resulting from deliberate public exposure to spores has led to the employment of anthrax as a biological weapon. Most recently, in 2001, anthrax spores sent through the US postal service to recipients on the eastern seaboard were responsible for a total of 22 confirmed or suspected cases of cutaneous and inhalational anthrax, with 5 deaths due to inhalational infections. This incident and the potential for future bioterror attacks highlight the need for a comprehensive set of post-exposure prophylaxis (PEP) options. In cases of suspected exposure, the AVA vaccine can be used in the US under an Investigational New Drug protocol or Emergency Use Authorization in a 3 dose regimen over 1 month. However, it is likely that sufficient protective antibody responses will take at least 4 weeks to develop and patients will be at risk of disease in the interim given a median incubation period of 4 days between exposure and signs of symptoms. Therefore, administration of antimicrobials (ciprofloxacin or doxycycline) is recommended as complementary PEP to help prevent onset of disease following germination of spores to vegetative bacilli in the body (antimicrobials are inactive against non-germinated spores). Continuation on antimicrobials for 60 days is considered necessary as dormant spores can persist in the lungs for several weeks. While this PEP approach appears to have been effective in the aftermath of the 2001 anthrax exposure incident, a follow up study of the individuals who were prescribed antimicrobials revealed only 44% overall compliance with the complete 60 day antimicrobials regimen, raising the possibility that inadequate compliance may risk development of infections in potential future exposure incidents. Even with 100% patient regimen compliance and significant suppression of bacterial replication, cellular uptake and activity of anthrax toxins is not affected by antimicrobials. Anthrax spores are ingested by macrophages, in which spores germinate into bacilli. The bacilli subsequently multiply and burst macrophages in lymph nodes. In the blood, bacilli secrete exotoxins which largely drive the pathology of anthrax infections. In addition to PA, which mediates binding of toxins to cells, anthrax bacteria produce a couple of enzymatically active proteins; lethal factor (LF) and edema factor (EF) which, respectively associate with PA to form lethal toxin (LT) and edema toxin (ET). The toxins cause irreversible tissue injury with hemorrhagic mediastinitis being the main clinical manifestation of inhalational anthrax. To directly counteract this pathology, the monoclonal antibodies raxibacumab and obiltoxaximab, both of which block