A phase 1 dose-sparing, randomized clinical trial of seasonal trivalent inactivated influenza vaccine combined with MAS-1, a novel water-in-oil adjuvant/delivery system.

BACKGROUND: New influenza vaccines are needed to increase vaccine efficacy. Adjuvants may allow hemagglutinin (HA) dose-sparing with enhanced immunogenicity. MAS-1 is an investigational low viscosity, free-flowing, water-in-oil emulsion-based adjuvant/delivery system comprised of stable nanoglobular aqueous droplets. METHODS: A phase 1, double-blind, safety and immunogenicity, HA dose escalation, randomized clinical trial was conducted. MAS-1 adjuvant with 1, 3, 5 or 9 µg per HA derived from licensed seasonal trivalent high dose inactivated influenza vaccine (IIV, Fluzone HD 60 µg per HA) in a 0.3 mL dose were compared to standard dose IIV (Fluzone SD, 15 µg per HA). Safety was measured by reactogenicity, adverse events, and clinical laboratory tests. Serum hemagglutination inhibition (HAI) antibody titers were measured for immunogenicity. RESULTS: Seventy-two subjects, aged 18-47 years, received one dose of either 0.3 mL adjuvanted vaccine or SD IIV intramuscularly. Common injection site and systemic reactions post-vaccination were mild tenderness, induration, pain, headache, myalgia, malaise and fatigue. All reactions resolved within 14 days post-vaccination. Safety laboratory measures were not different between groups. Geometric mean antibody titers, geometric mean fold increases in antibody titer, seroconversion rates and seroprotection rates against vaccine strains were in general higher and of longer duration (day 85 and 169 visits) with MAS-1-adjuvanted IIV at all doses of HA compared with SD IIV. Adjuvanted vaccine induced higher antibody responses against a limited number of non-study vaccine influenza B and A/H3N2 viruses including ones from subsequent years. CONCLUSION: MAS-1 adjuvant in a 0.3 mL dose volume provided HA dose-sparing effects without safety concerns and induced higher HAI antibody and seroconversion responses through at least 6 months, demonstrating potential to provide greater vaccine efficacy throughout an influenza season in younger adults. In summary, MAS-1 may provide enhanced, more durable and broader protective immunity compared with non-adjuvanted SD IIV. Clinical Trial Registry: ClinicalTrials.gov # NCT02500680.

MAS-1, a novel water-in-oil adjuvant/delivery system, with reduced seasonal influenza vaccine hemagglutinin dose may enhance potency, durability and cross-reactivity of antibody responses in the elderly.

BACKGROUND: Increased influenza vaccine efficacy is needed in the elderly at high-risk for morbidity and mortality due to influenza infection. Adjuvants may allow hemagglutinin (HA) dose-sparing with enhanced immunogenicity. MAS-1 is an investigational water-in-oil emulsion-based adjuvant/delivery system comprised of stable nanoglobular aqueous droplets. METHODS: A phase 1, randomized, double-blind, safety and immunogenicity, adjuvant dose escalation trial was conducted in persons aged 65 years and older. MAS-1 adjuvant dose volumes at 0.3 mL or 0.5 mL containing 9 µg per HA derived from licensed seasonal trivalent influenza vaccine (IIV, Fluzone HD 60 µg per HA, Sanofi Pasteur) were compared to high dose (HD) IIV (Fluzone HD). Safety was measured by reactogenicity, adverse events, and safety laboratory measures. Immunogenicity was assessed by serum hemagglutination inhibition (HAI) antibody titers. RESULTS: Forty-five subjects, aged 65-83 years, were randomly assigned to receive 9 µg per HA in 0.3 mL MAS-1 (15 subjects) or HD IIV (15 subjects) followed by groups randomly assigned to receive 9 µg per HA in 0.5 mL MAS-1 (10 subjects) or HD IIV (5 subjects). Injection site tenderness, induration, and pain, and headache, myalgia, malaise and fatigue were common, resolving before day 14 post-vaccination. Clinically significant late-onset injection site reactions occurred in four of ten subjects at the 0.5 mL adjuvant dose. Safety laboratory measures were within acceptable limits. MAS-1-adjuvanted IIV enhanced mean antibody titers, mean-fold increases in antibody titer, and seroconversion rates against vaccine strains for at least 168 days post-vaccination and enhanced cross-reactive antibodies against some non-study vaccine influenza viruses. CONCLUSION: MAS-1 adjuvant provided HA dose-sparing without safety concerns at the 0.3 mL dose, but the 0.5 mL dose caused late injection site reactions. MAS-1-adjuvanted IIV induced higher HAI antibody responses with prolonged durability including against historical strains, thereby providing greater potential vaccine efficacy in the elderly throughout an influenza season. Clinical Trial Registry: ClinicalTrials.gov # NCT02500680.

Active Amyloid-ß Vaccination Results in Epigenetic Changes in the Hippocampus of an Alzheimer's Disease-Like Mouse Model.

BACKGROUND: While evidence accumulates for a role of epigenetic modifications in the pathophysiological cascade of Alzheimer's disease (AD), amyloid-ß (Aß)-targeted active immunotherapy approaches are under investigation to prevent or slow the progression of AD. The impact of Aß active vaccines on epigenetic markers has not been studied thus far. OBJECTIVE: The current study aims to establish the relationship between active immunotherapy with a MER5101-based vaccine (consisting of Aß1-15 copies conjugated with a 7 aa spacer to the diphtheria toxoid carrier protein, formulated in a Th2-biased adjuvant) and epigenetic DNA modifications in the hippocampus of APPswe/PS1dE9 mice. METHODS: As we previously reported, immunotherapy started when the mice were 10 months of age and behavioral testing occurred at 14 months of age, after which the mice were sacrificed for further analysis of their brains. In this add-on study, global levels of DNA methylation and hydroxymethylation, and DNA methyltransferase 3A (DNMT3A) were determined using quantitative immunohistochemistry, and compared to our previously analyzed immunization-induced changes in AD-related neuropathology and cognition. RESULTS: Active immunization did not affect global DNA methylation levels but instead, resulted in decreased DNA hydroxymethylation and DNMT3A levels. Independent of immunization, inverse correlations with behavioral performance were observed for levels of DNA methylation and hydroxymethylation, as well as DNMT3A, while Aß pathology and synaptic markers did not correlate with DNA methylation levels but did positively correlate with DNA hydroxymethylation and levels of DNMT3A. CONCLUSION: Our results indicate that active Aß vaccination has significant effects on the epigenome in the hippocampus of APPswe/PS1dE9 mice, and suggest that DNA methylation and hydroxymethylation may be involved in cognitive functioning.

MAS-1 adjuvant immunotherapy generates robust Th2 type and regulatory immune responses providing long-term protection from diabetes in late-stage pre-diabetic NOD mice.

MAS-1, a nanoparticular, emulsion-based adjuvant, was evaluated for its ability to promote Th2 and regulatory immune responses and prevent type 1 diabetes progression when given alone or as antigen-specific immunotherapy (ASI) using insulin B chain (IBC; MER3101) and its analog B:9-23(19Ala) (MER3102). MAS-1 formulations were administered to NOD mice at age 9 and 13 weeks and followed through 52 weeks. MER3101 and MER3102 provided long-term protection with 60% and 73% of mice remaining diabetes-free at week 35, and 60% and 47% at week 52. MAS-1 adjuvant emulsion by itself also provided protection with 60% and 40% of mice diabetes-free at 35 and 52 weeks, respectively. Higher levels of interleukin (IL)-10 and IL-2 positive T cells were detected among splenocytes by week 15 in MER3101 and MER3102 immunized mice, whereas MAS-1 alone induced higher levels of IL-10-positive T cells. Diabetes-free 52-week-old mice expressed significant levels of antigen-specific IL-10-positive type 1 regulatory T cells and FoxP3-positive T cells when stimulated ex vivo with IBC. Antibodies targeting IBC and B:9-23(19Ala) induced by MER3101 and MER3102 were overwhelmingly Th2 type IgG1 and IgG2b isotypes. Splenocyte cultures from 52 week diabetes-free, MER3101-treated mice secreted significantly increased levels of IL-4 and IL-5 Th2 cytokines. Based on these pre-clinical results and its clinical safety profile, MAS-1 has the requisite qualities to be considered for use in prophylactic or early stage disease settings to augment ASI to prevent disease progression in type 1 diabetes.

MER5101, a novel Aß1-15:DT conjugate vaccine, generates a robust anti-Aß antibody response and attenuates Aß pathology and cognitive deficits in APPswe/PS1ΔE9 transgenic mice.

Active amyloid-ß (Aß) immunotherapy is under investigation to prevent or treat early Alzheimer's disease (AD). In 2002, a Phase II clinical trial (AN1792) was halted due to meningoencephalitis in ∼6% of the AD patients, possibly caused by a T-cell-mediated immunological response. Thus, generating a vaccine that safely generates high anti-Aß antibody levels in the elderly is required. In this study, MER5101, a novel conjugate of Aß1-15 peptide (a B-cell epitope fragment) conjugated to an immunogenic carrier protein, diphtheria toxoid (DT), and formulated in a nanoparticular emulsion-based adjuvant, was administered to 10-month-old APPswe/PS1ΔE9 transgenic (Tg) and wild-type (Wt) mice. High anti-Aß antibody levels were observed in both vaccinated APPswe/PS1ΔE9 Tg and Wt mice. Antibody isotypes were mainly IgG1 and IgG2b, suggesting a Th2-biased response. Restimulation of splenocytes with the Aß1-15:DT conjugate resulted in a strong proliferative response, whereas proliferation was absent after restimulation with Aß1-15 or Aß1-40/42 peptides, indicating a cellular immune response against DT while avoiding an Aß-specific T-cell response. Moreover, significant reductions in cerebral Aß plaque burden, accompanied by attenuated microglial activation and increased synaptic density, were observed in MER5101-vaccinated APPswe/PS1ΔE9 Tg mice compared with Tg adjuvant controls. Last, MER5101-immunized APPswe/PS1ΔE9 Tg mice showed improvement of cognitive deficits in both contextual fear conditioning and the Morris water maze. Our novel, highly immunogenic Aß conjugate vaccine, MER5101, shows promise for improving Aß vaccine safety and efficacy and therefore, may be useful for preventing and/or treating early AD.

The role of psychological factors in the fatigue of primary biliary cirrhosis.

Fatigue impairs the quality of life of primary biliary cirrhosis (PBC) patients. In this study, we explored the psychological factors and coping strategies in fatigued PBC patients. Patients participated in a semi-structured interview examining thoughts regarding the impact of fatigue and coping strategies. All completed the disease-specific quality-of-life tool, PBC-40, the Penn State Worry Questionnaire (PSWQ) (degree of habitual worry) and Hospital Anxiety and Depression Scale (HADS) (current anxiety and depression). PBC patients were allocated into high (>38, n=10) and low-fatigue (<38, n=14) groups. No differences were seen between high-fatigue and low-fatigue groups regarding age, marital status, employment status, PBC stage, years with diagnosis and years experiencing fatigue. High-fatigue participants were significantly more anxious (P=0.008), more depressed (P<0.001), and more likely to worry (<0.05). High-fatigue participants had more frequent thoughts about the impact of fatigue (P<0.005) and lower self-efficacy scores (P<0.001). In conclusion, PBC patients can experience profound distress associated with fatigue. PBC patients with high levels of fatigue seem to be more vulnerable to emotional distress, more likely to perceive that their quality of life has been negatively affected and are less confident to engage in everyday activities compared with those with low levels of fatigue.

Ribozyme to proliferating cell nuclear antigen to treat proliferative vitreoretinopathy.

PURPOSE: A DNA-RNA chimeric ribozyme was developed that targets the mRNA of a cell cycle regulatory protein, proliferating cell nuclear antigen (PCNA). The hypothesis was that inhibition of PCNA, essential in DNA replication, would decrease the proliferation of cells that are involved in formation of granuloma after surgical procedures in the eye. The ability of intravitreous injection of this ribozyme to prevent or inhibit development of proliferative vitreoretinopathy (PVR) was tested in a dispase-induced rabbit PVR model. METHODS: Rabbit genomic DNA encoding PCNA was cloned and sequenced. The cleavage of rabbit PCNA by the chimeric ribozyme was tested in vitro. Delivery of the ribozyme to rabbit retinal pigment epithelial (RPE) or fibroblast cells and its effects on proliferation of fibroblasts were examined. The stability of the ribozyme in vitreous fluid and serum was studied as well. In the dispase-induced rabbit model of PVR, the ability of the PCNA ribozyme to prevent or inhibit development of PVR and retinal detachment (RD) was tested. Experimental groups receiving intravitreous PCNA ribozyme, with or without a lipid vehicle, were compared with sham-treated control groups. Progression of PVR in rabbit eyes was followed by indirect ophthalmic examination and observations documented by fundoscopic photography, gross pathology, and histopathology. RESULTS: The chimeric ribozyme targeted a specific sequence in the rabbit PCNA that was identical with that in the human. In vitro cleavage assays confirmed the ability of the ribozyme to cleave the mRNA of PCNA. The catalytic efficiency in vitro, calculated as k(2)/K(m)(app), was 0.26 microM(-1) x min(-1). In vitro studies with fluoresceinated ribozyme indicated that lipid vehicles facilitated delivery of the ribozyme into cells causative of PVR (RPE and fibroblasts); however, the PCNA ribozyme decreased the proliferation of fibroblasts, with or without lipid vehicle. The ribozyme displayed good stability in vitreous fluid, whereas, it degraded quite rapidly in serum. In animal experiments, rabbits in sham-treated groups usually exhibited development of severe PVR characterized by focal traction or RD. Animals in the PCNA ribozyme-treated groups usually did not exhibit an RD. If they did have RD, it was small and localized, or focal tractions developed that did not progress to the degree that the sham-treated animal eyes did over the follow-up period. The in vivo use of a lipid delivery vehicle resulted in a precipitate; however, an effective naked ribozyme dose was identified that did not cause this side effect. CONCLUSIONS: In addition to validating the newly developed dispase PVR rabbit model, the results indicate that ribozyme targeted against the cell cycle agent PCNA is efficacious in the treatment or prevention of PVR in the rabbit eye. These experiments suggest that chimeric ribozyme targeted against PCNA may have a therapeutic or preventative role in humans.