GRT-R910: a self-amplifying mRNA SARS-CoV-2 vaccine boosts immunity for ≥6 months in previously-vaccinated older adults.

SARS-CoV-2 has resulted in high levels of morbidity and mortality world-wide, and severe complications can occur in older populations. Humoral immunity induced by authorized vaccines wanes within 6 months, and frequent boosts may only offer transient protection. GRT-R910 is an investigational self-amplifying mRNA (samRNA)-based SARS-CoV-2 vaccine delivering full-length Spike and selected conserved non-Spike T cell epitopes. This study reports interim analyses for a phase I open-label dose-escalation trial evaluating GRT-R910 in previously vaccinated healthy older adults (NCT05148962). Primary endpoints of safety and tolerability were assessed. Most solicited local and systemic adverse events (AEs) following GRT-R910 dosing were mild to moderate and transient, and no treatment-related serious AEs were observed. The secondary endpoint of immunogenicity was assessed via IgG binding assays, neutralization assays, interferon-gamma ELISpot, and intracellular cytokine staining. Neutralizing antibody titers against ancestral Spike and variants of concern were boosted or induced by GRT-R910 and, contrasting to authorized vaccines, persisted through at least 6 months after the booster dose. GRT-R910 increased and/or broadened functional Spike-specific T cell responses and primed functional T cell responses to conserved non-Spike epitopes. This study is limited due to small sample size, and additional data from ongoing studies will be required to corroborate these interim findings.

Low-dose self-amplifying mRNA COVID-19 vaccine drives strong protective immunity in non-human primates against SARS-CoV-2 infection.

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.

Preclinical development of the TLR9 agonist DV281 as an inhaled aerosolized immunotherapeutic for lung cancer: Pharmacological profile in mice, non-human primates, and human primary cells.

CpG-motif-containing oligodeoxynucleotides (CpG-ODN) activate innate immunity through Toll-Like Receptor (TLR) 9 signaling and generate local immune responses when delivered directly to the lung. Herein we describe pharmacological studies in mice, cynomolgus monkeys, and in human primary cells which support the development of DV281, a C-class CpG-ODN, as an inhaled aerosolized immunotherapeutic for lung cancer to be combined with an inhibitor of the anti-programmed cell death protein 1 (PD­1) immune checkpoint. In vitro, DV281 potently induced Interferon (IFN)­α from monkey and human peripheral blood mononuclear cells (PBMCs), stimulated interleukin­6 production and proliferation in human B cells, and induced TLR9-dependent cytokine responses from mouse splenocytes. Intranasal delivery of DV281 to mice led to substantial but transient cytokine and chemokine responses in the lung. Lung responses to repeated intranasal DV281 were partially to fully reversible 2 weeks after the final dose and were absent in TLR9-deficient mice. Single escalating doses of aerosolized DV281 in monkeys induced dose-dependent induction of IFN-regulated genes in bronchoalveolar lavage cells and blood. In a repeat-dose safety study in monkeys, inhaled DV281 was well-tolerated, and findings were mechanism of action-related and non-adverse. Co-culture of human PBMC with DV281 and anti-PD­1 antibody did not augment cytokine or cellular proliferation responses compared to DV281 alone, indicating that the combination did not lead to dysregulated cytokine responses. These studies support clinical development of inhaled aerosolized DV281 as a combination therapy with anti-PD­1 antibody for lung cancer immunotherapy.

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.

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.

CpG-containing immunostimulatory DNA sequences elicit TNF-alpha-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.