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 ISSinduced toxicity was critically dependent on TNF-α, with IFN-α required for TNF-α induction. In contrast, human PBMCs, human alveolar macrophages, and airway-derived cells from Ascaris suum-allergic cynomolgus monkeys did not produce appreciable TNF-α 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-α induction. These data demonstrate that ISS induce rodent-specific TNF-α-dependent toxicity that is absent in humans and reflective of differential TLR9 expression patterns in rodents versus humans.

The coronavirus disease 2019 (COVID-19) pandemic continues to spread globally, highlighting the urgent need for safe and effective vaccines that could be rapidly mobilized to immunize large populations. We report the preclinical development of a self-amplifying mRNA (SAM) vaccine encoding a prefusion stabilized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein and demonstrate strong cellular and humoral immune responses at low doses in mice and rhesus macaques. The homologous prime-boost vaccination regimen of SAM at 3, 10 and 30 μg induced potent neutralizing antibody (nAb) titers in rhesus macaques following two SAM vaccinations at all dose levels, with the 10 μg dose generating geometric mean titers (GMT) 48-fold greater than the GMT of a panel of SARS-CoV-2 convalescent human sera. Spike-specific T cell responses were observed with all tested vaccine regimens. SAM vaccination provided protective efficacy against SARS-CoV-2 challenge as both a homologous prime-boost and as a single boost following ChAd prime, demonstrating reduction of viral replication in both the upper and lower airways. The SAM vaccine is currently being evaluated in clinical trials as both a homologous prime-boost regimen at low doses and as a boost following heterologous prime.

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 (CpG-ODN) linked to the sucrose polymer Ficoll, forming soluble 50 nm particles (DV230-Ficoll), each containing over 100 molecules of the TLR9 ligand, DV230. DV230-Ficoll was evaluated as an adjuvant for a candidate vaccine for anthrax using a recombinant form of protective antigen (rPA) from Bacillus anthracis. A single immunization with rPA plus DV230-Ficoll induced 10-fold higher titers of toxin-neutralizing antibodies in cynomolgus monkeys at 2 weeks 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 co-localized with rPA in key antigen-presenting cell 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.

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