Immunogenicity phase II study evaluating booster capacity of nonadjuvanted AKS-452 SARS-Cov-2 RBD Fc vaccine.

AKS-452, a subunit vaccine comprising an Fc fusion of the ancestral wild-type (WT) SARS-CoV-2 virus spike protein receptor binding domain (SP/RBD), was evaluated without adjuvant in a single cohort, non-randomized, open-labelled phase II study (NCT05124483) at a single site in The Netherlands for safety and immunogenicity. A single 90 µg subcutaneous booster dose of AKS-452 was administered to 71 adults previously primed with a registered mRNA- or adenovirus-based vaccine and evaluated for 273 days. All AEs were mild and no SAEs were attributable to AKS-452. While all subjects showed pre-existing SP/RBD binding and ACE2-inhibitory IgG titers, 60-68% responded to AKS-452 via ≥2-fold increase from days 28 to 90 and progressively decreased back to baseline by day 180 (days 28 and 90 mean fold-increases, 14.7 ± 6.3 and 8.0 ± 2.2). Similar response kinetics against RBD mutant proteins (including omicrons) were observed but with slightly reduced titers relative to WT. There was an expected strong inverse correlation between day-0 titers and the fold-increase in titers at day 28. AKS-452 enhanced neutralization potency against live virus, consistent with IgG titers. Nucleocapsid protein (Np) titers suggested infection occurred in 66% (46 of 70) of subjects, in which only 20 reported mild symptomatic COVID-19. These favorable safety and immunogenicity profiles support booster evaluation in a planned phase III universal booster study of this room-temperature stable vaccine that can be rapidly and inexpensively manufactured to serve vaccination at a global scale without the need of a complex distribution or cold chain.

A randomized phase I/II safety and immunogenicity study of the Montanide-adjuvanted SARS-CoV-2 spike protein-RBD-Fc vaccine, AKS-452.

BACKGROUND: Previous interim data from a phase I study of AKS-452, a subunit vaccine comprising an Fc fusion of the respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor binding domain (SP/RBD) emulsified in the water-in-oil adjuvant, Montanide™ ISA 720, suggested a good safety and immunogenicity profile in healthy adults. This phase I study was completed and two dosing regimens were further evaluated in this phase II study. METHODS: This phase II randomized, open-labelled, parallel group study was conducted at a single site in The Netherlands with 52 healthy adults (18 - 72 years) receiving AKS-452 subcutaneously at one 90 µg dose (cohort 1, 26 subjects) or two 45 µg doses 28 days apart (cohort 2, 26 subjects). Serum samples were collected at the first dose (day 0) and at days 28, 56, 90, and 180. Safety and immunogenicity endpoints were assessed, along with induction of IgG isotypes, cross-reactive immunity against viral variants, and IFN-γ T cell responses. RESULTS: All AEs were mild/moderate (grades 1 or 2), and no SAEs were attributable to AKS-452. Seroconversion rates reached 100% in both cohorts, although cohort 2 showed greater geometric mean IgG titers that were stable through day 180 and associated with enhanced potencies of SP/RBD-ACE2 binding inhibition and live virus neutralization. AKS-452-induced IgG titers strongly bound mutant SP/RBD from several SARS-CoV-2 variants (including Omicrons) that were predominantly of the favorable IgG1/3 isotype and IFN-γ-producing T cell phenotype. CONCLUSION: These favorable safety and immunogenicity profiles of the candidate vaccine as demonstrated in this phase II study are consistent with those of the phase I study (ClinicalTrials.gov: NCT04681092) and suggest that a total of 90 µg received in 2 doses may offer a greater duration of cross-reactive neutralizing titers than when given in a single dose.

Phase I interim results of a phase I/II study of the IgG-Fc fusion COVID-19 subunit vaccine, AKS-452.

To address the coronavirus disease 2019 (COVID-19) pandemic caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a recombinant subunit vaccine, AKS-452, is being developed comprising an Fc fusion protein of the SARS-CoV-2 viral spike protein receptor binding domain (SP/RBD) antigen and human IgG1 Fc emulsified in the water-in-oil adjuvant, Montanide™ ISA 720. A single-center, open-label, phase I dose-finding and safety study was conducted with 60 healthy adults (18-65 years) receiving one or two doses 28 days apart of 22.5 µg, 45 µg, or 90 µg of AKS-452 (i.e., six cohorts, N = 10 subjects per cohort). Primary endpoints were safety and reactogenicity and secondary endpoints were immunogenicity assessments. No AEs ≥ 3, no SAEs attributable to AKS-452, and no SARS-CoV-2 viral infections occurred during the study. Seroconversion rates of anti-SARS-CoV-2 SP/RBD IgG titers in the 22.5, 45, and 90 µg cohorts at day 28 were 70%, 90%, and 100%, respectively, which all increased to 100% at day 56 (except 89% for the single-dose 22.5 µg cohort). All IgG titers were Th1-isotype skewed and efficiently bound mutant SP/RBD from several SARS-CoV-2 variants with strong neutralization potencies of live virus infection of cells (including alpha and delta variants). The favorable safety and immunogenicity profiles of this phase I study (ClinicalTrials.gov: NCT04681092) support phase II initiation of this room-temperature stable vaccine that can be rapidly and inexpensively manufactured to serve vaccination at a global scale without the need of a complex distribution or cold chain.

Development of an IgG-Fc fusion COVID-19 subunit vaccine, AKS-452.

AKS-452 is a biologically-engineered vaccine comprising an Fc fusion protein of the SARS-CoV-2 viral spike protein receptor binding domain antigen (Ag) and human IgG1 Fc (SP/RBD-Fc) in clinical development for the induction and augmentation of neutralizing IgG titers against SARS-CoV-2 viral infection to address the COVID-19 pandemic. The Fc moiety is designed to enhance immunogenicity by increasing uptake via Fc-receptors (FcγR) on Ag-presenting cells (APCs) and prolonging exposure due to neonatal Fc receptor (FcRn) recycling. AKS-452 induced approximately 20-fold greater neutralizing IgG titers in mice relative to those induced by SP/RBD without the Fc moiety and induced comparable long-term neutralizing titers with a single dose vs. two doses. To further enhance immunogenicity, AKS-452 was evaluated in formulations containing a panel of adjuvants in which the water-in-oil adjuvant, Montanide™ ISA 720, enhanced neutralizing IgG titers by approximately 7-fold after one and two doses in mice, including the neutralization of live SARS-CoV-2 virus infection of VERO-E6 cells. Furthermore, ISA 720-adjuvanted AKS-452 was immunogenic in rabbits and non-human primates (NHPs) and protected from infection and clinical symptoms with live SARS-CoV-2 virus in NHPs (USA-WA1/2020 viral strain) and the K18 human ACE2-trangenic (K18-huACE2-Tg) mouse (South African B.1.351 viral variant). These preclinical studies support the initiation of Phase I clinical studies with adjuvanted AKS-452 with the expectation that this room-temperature stable, Fc-fusion subunit vaccine can be rapidly and inexpensively manufactured to provide billions of doses per year especially in regions where the cold-chain is difficult to maintain.

Rapid estrogen signaling negatively regulates PTEN activity through phosphorylation in endometrial cancer cells.

Hyperestrogenicity is a risk factor for endometrial cancer. 17ß-estradiol (E2) is known to stimulate both genomic and nongenomic estrogen receptor-α (ERα) actions in a number of reproductive tissues. However, the contributions of transcription-independent ERα signaling on normal and malignant endometrium are not fully understood. Phosphatase and tensin homolog (PTEN) is a tumor suppressor that decreases cellular mitosis primarily through negative regulation of the phosphoinositide 3-kinase/AKT signaling axis. PTEN levels are elevated during the E2 dominated, mitotically active, proliferative phase of the menstrual cycle, indicating possible hormonal regulation of PTEN in the uterus. In order to determine if rapid E2 signaling regulates PTEN, we used ERα-positive, PTEN-positive, endometrial cells. We show that cytosolic E2/ERα signaling leads to increased phosphorylation of PTEN at key regulatory residues. Importantly, E2 stimulation decreased PTEN lipid phosphatase activity and caused consequent increases in phospho-AKT. We further demonstrate that cytosolic ERα forms a complex with PTEN in an E2-dependent manner, and that ERα constitutively complexes with protein kinase2-α (CK2α), a kinase previously shown to phosphorylate the C-terminal tail of PTEN. These results provide mechanistic support for an E2-dependent, ERα cytosolic signaling complex that negatively regulates PTEN activity through carboxy terminus phosphorylation. Using an animal model, we show that sustained E2 signaling results in increased phospho-PTEN (S380, T382, and T383), total PTEN, and phospho-AKT (S473). Taken together, we provide a novel mechanism in which transcription-independent E2/ERα signaling may promote a pro-tumorigenic environment in the endometrium.

Hypoxia-inducible factor signaling provides protection in Clostridium difficile-induced intestinal injury.

BACKGROUND & AIMS: Clostridium difficile is the leading cause of nosocomial infectious diarrhea. Antibiotic resistance and increased virulence of strains have increased the number of C difficile-related deaths worldwide. The innate host response mechanisms to C difficile are not resolved; we propose that hypoxia-inducible factor (HIF-1) has an innate, protective role in C difficile colitis. We studied the impact of C difficile toxins on the regulation of HIF-1 and evaluated the role of HIF-1alpha in C difficile-mediated injury/inflammation. METHODS: We assessed HIF-1alpha mRNA and protein levels and DNA binding in human mucosal biopsy samples and Caco-2 cells following exposure to C difficile toxins. We used the mouse ileal loop model of C difficile toxin-induced intestinal injury. Mice with targeted deletion of HIF-1alpha in the intestinal epithelium were used to assess the effects of HIF-1alpha signaling in response to C difficile toxin. RESULTS: Mucosal biopsy specimens and Caco-2 cells exposed to C difficile toxin had a significant increase in HIF-1alpha transcription and protein levels. Toxin-induced DNA binding was also observed in Caco-2 cells. Toxin-induced HIF-1alpha accumulation was attenuated by nitric oxide synthase inhibitors. In vivo deletion of intestinal epithelial HIF-1alpha resulted in more severe, toxin-induced intestinal injury and inflammation. In contrast, stabilization of HIF-1alpha with dimethyloxallyl glycine attenuated toxin-induced injury and inflammation. This was associated with induction of HIF-1-regulated protective factors (such as vascular endothelial growth factor-alpha, CD73, and intestinal trefoil factor) and down-regulation of proinflammatory molecules such as tumor necrosis factor and Cxcl1. CONCLUSIONS: HIF-1alpha protects the intestinal mucosa from C difficile toxins. The innate protective actions of HIF-1alpha in response to C difficile toxins be developed as therapeutics for C difficile-associated disease.

Selective induction of integrin beta1 by hypoxia-inducible factor: implications for wound healing.

Because of localized vascular damage and increased tissue oxygen demand, wound healing occurs in a relatively hypoxic microenvironment. These features are particularly relevant to wound healing and fibrosis in chronic inflammatory conditions, such as Crohn's disease and ulcerative colitis. In these studies, we sought to identify the contribution of hypoxia to mechanisms of wound repair in a model of the intestinal submucosa. Initial studies revealed that hypoxia promotes wound healing, as modeled by an increase in intestinal fibroblast-mediated collagen gel contraction. Guided by results from transcriptional profiling, we identified the selective induction of fibroblast integrin beta1 (ITGB1) by hypoxia. Further analysis revealed that hypoxia, as well as pharmacological activators of hypoxia-inducible factor (HIF), induce fibroblast beta1 integrin mRNA, protein, and function by as much as 4-fold. Cloning and analysis of the beta1 integrin gene promoter revealed a 10 +/- 0.8-fold increase in promoter activity in response to hypoxia, and subsequent studies identified a functional DNA binding region for HIF in the ITGB1 gene promoter. Mutational analysis of the HIF binding site within the ITGB1 promoter resulted in a significant loss of ITGB1 hypoxia-inducibility. As proof of principle, studies in a murine model of colitis revealed a correlation between colitic disease severity and tissue ITGB1 expression (R(2)=0.80). Taken together, these results demonstrate that hypoxia induces fibroblast ITGB1 expression and function by transcriptional mechanisms dependent on HIF.