Role of site-directed mutagenesis and adjuvants in the stability and potency of anthrax protective antigen

Anthrax is a zoonotic infection caused by the gram-positive, aerobic, spore-forming bacterium Bacillus anthracis. Depending on the origin of the infection, serious health problems or mortality is possible. The virulence of B. anthracis is reliant on three pathogenic factors, which are secreted upon infection: protective antigen (PA), lethal factor (LF), and edema factor (EF). Systemic illness results from LF and EF entering cells through the formation of a complex with the heptameric form of PA, bound to the membrane of infected cells through its receptor. The currently available anthrax vaccines have multiple drawbacks, and recombinant PA is considered a promising second-generation vaccine candidate. However, the inherent chemical instability of PA through Asn deamidation at multiple sites prevents its use after long-term storage owing to loss of potency. Moreover, there is a distinct possibility of B. anthracis being used as a bioweapon; thus, the developed vaccine should remain efficacious and stable over the long-term. Second-generation anthrax vaccines with appropriate adjuvant formulations for enhanced immunogenicity and safety are desired. In this article, using protein engineering approaches, we have reviewed the stabilization of anthrax vaccine candidates that are currently licensed or under preclinical and clinical trials. We have also proposed a formulation to enhance recombinant PA vaccine potency via adjuvant formulation.     

Rociletinib (CO-1686) enhanced the efficacy of chemotherapeutic agents in ABCG2-overexpressing cancer cells in vitro and in vivo

Overexpression of adenosine triphosphate (ATP)-binding cassette subfamily G member 2 (ABCG2) in cancer cells is known to cause multidrug resistance (MDR), which severely limits the clinical efficacy of chemotherapy. Currently, there is no FDA-approved MDR modulator for clinical use. In this study, rociletinib (CO-1686), a mutant-selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), was found to significantly improve the efficacy of ABCG2 substrate chemotherapeutic agents in the transporter-overexpressing cancer cells in vitro and in MDR tumor xenografts in nude mice, without incurring additional toxicity. Mechanistic studies revealed that in ABCG2-overexpressing cancer cells, rociletinib inhibited ABCG2-mediated drug efflux and increased intracellular accumulation of ABCG2 probe substrates. Moreover, rociletinib, inhibited the ATPase activity, and competed with [¹²⁵I] iodoarylazidoprazosin (IAAP) photolabeling of ABCG2. However, ABCG2 expression at mRNA and protein levels was not altered in the ABCG2-overexpressing cells after treatment with rociletinib. In addition, rociletinib did not inhibit EGFR downstream signaling and phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK). Our results collectively showed that rociletinib reversed ABCG2-mediated MDR by inhibiting ABCG2 efflux function, thus increasing the cellular accumulation of the transporter substrate anticancer drugs. The findings advocated the combination use of rociletinib and other chemotherapeutic drugs in cancer patients with ABCG2-overexpressing MDR tumors.     

Flavin adenine dinucleotide synthase deficiency due to FLAD1 mutation presenting as multiple acyl-CoA dehydrogenation deficiency-like disease: A case report

Multiple acyl-CoA dehydrogenase deficiency (MADD), also known as glutaric acidemia type II, is classically caused by a congenital defect in electron transfer flavoprotein (ETF) or ETF dehydrogenase (ETFDH). Flavin adenine dinucleotide synthase (FADS) deficiency caused by mutations in FLAD1 was recently reported as a novel riboflavin metabolism disorder resembling MADD. Here, we describe a Japanese boy with FADS deficiency due to a novel mutation (p.R249*) in FLAD1. In the asymptomatic male infant born at full term, newborn screening showed positive results with elevated C5 and C14:1 acylcarnitine levels and an increased C14:1/C2 ratio. Biochemical studies were unremarkable except for lactic acidosis (pH 7.197, lactate 61 mg/dL). A diagnosis of MADD was suspected because of mild abnormalities of the acylcarnitine profile and apparent abnormalities of urinary organic acids, although mutations in the ETFA, ETFB, ETFDH, and riboflavin transporter genes (SLC52A1, SLC52A2, and SLC52A3) were not detected. Administration of riboflavin and L-carnitine was initiated at one month of age based on the diagnosis of “biochemical MADD” despite a lack of symptoms. Nevertheless, the acylcarnitine profile was not normalized. Symptoms resembling bulbar palsy, such as vocal cord paralysis and dyspnea with stridor, were present from 3 months of age. At 4 months of age, he became bedridden because of hypoxic-ischemic encephalopathy due to fulminant respiratory failure with aspiration pneumonia. At 2 years and 5 months of age, a homozygous c.745C > T (p.R249*) mutation in the FLAD1 gene was identified, confirming the diagnosis of FADS deficiency. His severe clinical course may be caused by this nonsense mutation associated with poor responsiveness to riboflavin. Persistent lactic acidosis and neuropathy, such as bulbar palsy, may be important for diagnosing FADS deficiency. Although the biochemical findings in FADS deficiency are similar to those in MADD, their clinical symptoms and severity may not be identical.     

Nicotinamide mononucleotide alters mitochondrial dynamics by SIRT3-dependent mechanism in male mice

Nicotinamide adenine dinucleotide (NAD⁺) is a central signaling molecule and enzyme cofactor that is involved in a variety of fundamental biological processes. NAD⁺ levels decline with age, neurodegenerative conditions, acute brain injury, and in obesity or diabetes. Loss of NAD⁺ results in impaired mitochondrial and cellular functions. Administration of NAD⁺ precursor, nicotinamide mononucleotide (NMN), has shown to improve mitochondrial bioenergetics, reverse age-associated physiological decline, and inhibit postischemic NAD⁺ degradation and cellular death. In this study, we identified a novel link between NAD⁺ metabolism and mitochondrial dynamics. A single dose (62.5 mg/kg) of NMN, administered to male mice, increases hippocampal mitochondria NAD⁺ pools for up to 24 hr posttreatment and drives a sirtuin 3 (SIRT3)-mediated global decrease in mitochondrial protein acetylation. This results in a reduction of hippocampal reactive oxygen species levels via SIRT3-driven deacetylation of mitochondrial manganese superoxide dismutase. Consequently, mitochondria in neurons become less fragmented due to lower interaction of phosphorylated fission protein, dynamin-related protein 1 (pDrp1 [S616]), with mitochondria. In conclusion, manipulation of mitochondrial NAD⁺ levels by NMN results in metabolic changes that protect mitochondria against reactive oxygen species and excessive fragmentation, offering therapeutic approaches for pathophysiologic stress conditions.     

Randomized Crossover Trial of Phosphate-binding Medication on Serum Phosphate Levels in Patients With Aortic Stenosis

PurposeAortic stenosis is a common cause of valvular heart disease with no means of prevention. The recognized association between aortic stenosis and serum phosphate raises the possibility of preventing progression of the disorder by using phosphate-binding drugs, but there is uncertainty whether such treatment lowers serum phosphate levels in patients without diagnosed renal failure. This pilot study was conducted to answer this question in patients with aortic stenosis.MethodsA randomized, double-blind, crossover trial of the phosphate-binding drug sevelamer was conducted in 72 patients. Patients were prescribed sevelamer 0.8 g (low-dose), sevelamer 2.4 g (high-dose), and matching placebo, 3 times daily with food; each regimen lasted 6 weeks and was allocated at random. Serum phosphate levels were measured at the end of each treatment period, and within-person levels were compared.FindingsSixty-one patients completed the 3 treatment periods. There was no significant difference in the mean end-treatment phosphate levels across all patients (3.38, 3.36, and 3.31 mg/dL with placebo, low-dose sevelamer, and high-dose sevelamer, respectively). Post hoc analysis showed a reduction in phosphate levels with increasing sevelamer dose in the highest baseline phosphate quartile group; a 0.3 mg/dL reduction (mean, 4.09 mg/dL with placebo, 3.95 mg/dL with low-dose sevelamer, and 3.79 mg/dL with high-dose sevelamer; P_trend = 0.027).ImplicationsSevelamer had no overall statistically significant effect in lowering serum phosphate levels, but a reduction was observed in patients with phosphate levels in the highest quartile group of the population distribution. This hypothesis-generating result requires confirmation in an independent study. If confirmed, a trial of sevelamer in preventing the progression of aortic stenosis may be justified in patients with high phosphate levels. ISRCTN Registry identifier: ISRCTN17365679.     

Faster bone union progression and less sclerosis at the osteotomy margin after medial opening-wedge high tibial osteotomy using highly porous β-tricalcium phosphate granules versus allogeneic bone chips: A matched case–control study

BackgroundThis study compared bone union progression using highly porous (80% porosity) β-tricalcium phosphate (β-TCP) granules or allogeneic bone chips in the gap created by medial opening-wedge high tibial osteotomy (MOWHTO).MethodsThe study population consisted of 54 patients who received MOWHTO with locking plate fixation: 27 patients using highly porous β-TCP granules, and 27 age- and sex-matched patients using allogeneic bone chips. Bone union progression was evaluated 1, 3, 6, and 12 months postoperatively. The presence of radiographic sclerosis at the osteotomy margin was also assessed.ResultsAmong all patients, the highest degree of bone union observed 12 months postoperatively was grade 4. As postoperative time passed, bone union progression of highly porous β-TCP granules increased linearly and was statistically significant compared with that of cancellous allogeneic bone chips (P = 0.014). The presence of radiographic sclerosis at the osteotomy margin was significantly less common in the β-TCP group than in the allograft group (P = 0.003) and was the strongest predictor of delayed progress of bone union (odds ratio = 6.16, P = 0.006).ConclusionsPatients who underwent MOWHTO using highly porous β-TCP granules had faster new bone remodeling, less radiographic sclerosis at the osteotomy margin, and no inferior clinical outcome compared with allogeneic bone chips, as determined at the 1-year follow up. The presence of radiographic sclerosis at the osteotomy margin in patients undergoing MOWHTO using allogeneic bone or synthetic bone substitute may indicate delayed progress of bone union.     

Single-dose combination nanovaccine induces both rapid and durable humoral immunity and toxin neutralizing antibody responses against Bacillus anthracis

Bacillus anthracis, the causative agent of anthrax, continues to be a prominent biological warfare and bioterrorism threat. Vaccination is likely to remain the most effective and user-friendly public health measure to counter this threat in the foreseeable future. The commercially available AVA BioThrax vaccine has a number of shortcomings where improvement would lead to a more practical and effective vaccine for use in the case of an exposure event. Identification of more effective adjuvants and novel delivery platforms is necessary to improve not only the effectiveness of the anthrax vaccine, but also enhance its shelf stability and ease-of-use. Polyanhydride particles have proven to be an effective platform at adjuvanting the vaccine-associated adaptive immune response as well as enhancing stability of encapsulated antigens. Another class of adjuvants, the STING pathway-targeting cyclic dinucleotides, have proven to be uniquely effective at inducing a beneficial inflammatory response that leads to the rapid induction of high titer antibodies post-vaccination capable of providing protection against bacterial pathogens. In this work, we evaluate the individual contributions of cyclic di-GMP (CDG), polyanhydride nanoparticles, and a combination thereof towards inducing neutralizing antibody (nAb) against the secreted protective antigen (PA) from B. anthracis. Our results show that the combination nanovaccine elicited rapid, high titer, and neutralizing IgG anti-PA antibody following single dose immunization that persisted for at least 108 DPI.