Emily J. Lombardi, Candice L. Bywater, and Craig R. White

The Oxygen and Capacity-Limited Thermal Tolerance (OCLTT) hypothesis proposes that the thermal tolerance of an animal is shaped by its capacity to deliver oxygen in relation to oxygen demand. Studies testing this hypothesis have largely focused on measuring short-term performance responses in animals under acute exposure to critical thermal maximums. The OCLTT hypothesis, however, emphasises the importance of sustained animal performance over acute tolerance. The present study tested the effect of chronic hypoxia and hyperoxia during development on medium to long-term performance indicators at temperatures spanning the optimal temperature for growth in the speckled cockroach, Nauphoeta cinerea. In contrast to the predictions of the OCLTT hypothesis, development under hypoxia did not significantly reduce growth rate or running performance, and development under hyperoxia did not significantly increase growth rate or running performance. The effect of developmental temperature and oxygen on tracheal morphology and metabolic rate were also not consistent with OCLTT predictions, suggesting that oxygen delivery capacity is not the primary driver shaping thermal tolerance in this species. Collectively, these findings suggest that the OCLTT hypothesis does not explain moderate-to-long-term thermal performance in Nauphoeta cinerea, which raises further questions about the generality of the hypothesis.

Glycine riboswitches utilize both single- and tandem-aptamer architectures. In the tandem system, the relative contribution of each aptamer toward gene regulation is not well understood. To dissect these contributions, the effects of 684 single mutants of a tandem ON switch from Bacillus subtilis were characterized for the wild-type construct and binding site mutations that selectively restrict ligand binding to either the first or second aptamer. Despite the structural symmetry of tandem aptamers, the response to these mutations was frequently asymmetrical. Mutations in the first aptamer often significantly weakened the K_1/2, while several mutations in the second aptamer improved the amplitude. These results demonstrate that this ON switch favors ligand binding to the first aptamer. This is in contrast to the tandem OFF switch variant from Vibrio cholerae, which was previously shown to have preferential binding to its second aptamer. A bioinformatic analysis of tandem glycine riboswitches revealed that the two binding pockets are differentially conserved between ON and OFF switches. Altogether, this indicates that tandem ON switch variants preferentially utilize binding to the first aptamer to promote helical switching, while OFF switch variants favor binding to the second aptamer. The data set also revealed a cooperative glycine response when both binding pockets were maximally stabilized with three GC base pairs. This indicates a cooperative response may sometimes be obfuscated by a difference in the affinities of the two aptamers. This conditional cooperativity provides an additional layer of tunability to tandem glycine riboswitches that adds to their versatility as genetic switches.

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on kcat and K0.5 for catalysis, K0.5 for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding, and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants, enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.

Virus-like-particle (VLP) influenza vaccines can be given intramuscularly (i.m.) or intranasally (i.n.) and may have advantages over split-virion formulations in the elderly. We tested a plant-made VLP vaccine candidate bearing the viral hemagglutinin (HA) delivered either i.m. or i.n. in young and aged mice. Young adult (5- to 8-week-old) and aged (16- to 20-month-old) female BALB/c mice received a single 3-μg dose based on the HA (A/California/07/2009 H1N1) content of a plant-made H1-VLP (i.m. or i.n.) split-virion vaccine (i.m.) or were left naive. After vaccination, humoral and splenocyte responses were assessed, and some mice were challenged. Both VLP and split vaccines given i.m. protected 100% of the young animals, but the VLP group lost the least weight and had stronger humoral and cellular responses. Compared to split-vaccine recipients, aged animals vaccinated i.m. with VLP were more likely to survive challenge (80% versus 60%). The lung viral load postchallenge was lowest in the VLP i.m. groups. Mice vaccinated with VLP i.n. had little detectable immune response, but survival was significantly increased. In both age groups, i.m. administration of the H1-VLP vaccine elicited more balanced humoral and cellular responses and provided better protection from homologous challenge than the split-virion vaccine.

A vaccine against congenital human cytomegalovirus (CMV) infection is a major public health priority. Congenital CMV causes substantial long-term morbidity, particularly sensorineural hearing loss (SNHL), in newborns, and the public health impact of this infection on maternal and child health is underrecognized. Although progress toward development of a vaccine has been limited by an incomplete understanding of the correlates of protective immunity for the fetus, knowledge about some of the key components of the maternal immune response necessary for preventing transplacental transmission is accumulating. Moreover, although there have been concerns raised about observations indicating that maternal seropositivity does not fully prevent recurrent maternal CMV infections during pregnancy, it is becoming increasing clear that preconception immunity does confer some measure of protection against both CMV transmission and CMV disease (if transmission occurs) in the newborn infant. Although the immunity to CMV conferred by both infection and vaccination is imperfect, there are encouraging data emerging from clinical trials demonstrating the immunogenicity and potential efficacy of candidate CMV vaccines. In the face of the knowledge that between 20,000 and 30,000 infants are born with congenital CMV in the United States every year, there is an urgent and compelling need to accelerate the pace of vaccine trials. In this minireview, we summarize the status of CMV vaccines in clinical trials and provide a perspective on what would be required for a CMV immunization program to become incorporated into clinical practice.

Reactive immunization with a single-dose cholera vaccine that could rapidly (within days) protect immunologically naive individuals during virgin soil epidemics, when cholera reaches immunologically naive populations that have not experienced cholera for decades, would facilitate cholera control. One dose of attenuated Vibrio cholerae O1 classical Inaba vaccine CVD 103-HgR (Vaxchora) containing ≥2 x 10⁸ CFU induces vibriocidal antibody seroconversion (a correlate of protection) in >90% of U.S. adults. A previous CVD 103-HgR commercial formulation required ≥2 x 10⁹ CFU to elicit high levels of seroconversion in populations in developing countries. We compared the vibriocidal responses of Malians (individuals 18 to 45 years old) randomized to ingest a single ≥2 x 10⁸-CFU standard dose (n = 50) or a ≥2 x 10⁹-CFU high dose (n = 50) of PaxVax CVD 103-HgR with buffer or two doses (n = 50) of Shanchol inactivated cholera vaccine (the immunologic comparator). To maintain blinding, participants were dosed twice 2 weeks apart; CVD 103-HgR recipients ingested placebo 2 weeks before or after ingesting vaccine. Seroconversion (a ≥4-fold vibriocidal titer rise) between the baseline and 14 days after CVD 103-HgR ingestion and following the first and second doses of Shanchol were the main outcomes measured. By day 14 postvaccination, the rates of seroconversion after ingestion of a single standard dose and a high dose of CVD 103-HgR were 71.7% (33/46 participants) and 83.3% (40/48 participants), respectively. The rate of seroconversion following the first dose of Shanchol, 56.0% (28/50 participants), was significantly lower than that following the high dose of CVD 103-HgR (P = 0.003). The vibriocidal geometric mean titer (GMT) of the high dose of CVD 103-HgR exceeded the GMT of the standard dose at day 14 (214 versus 95, P = 0.045) and was ~2-fold higher than the GMT on day 7 and day 14 following the first Shanchol dose (P > 0.05). High-dose CVD 103-HgR is recommended for accelerated evaluation in developing countries to assess its efficacy and practicality in field situations. (This study has been registered at ClinicalTrials.gov under registration no. NCT02145377.)

As yet, very few vaccine candidates with activity in animals against Mycobacterium tuberculosis infection have been tested as therapeutic postexposure vaccines. We recently described two pools of mycobacterial proteins with this activity, and here we describe further studies in which four of these proteins (Rv1738, Rv2032, Rv3130, and Rv3841) were generated as a fusion polypeptide and then delivered in a novel yeast-based platform (Tarmogen) which itself has immunostimulatory properties, including activation of Toll-like receptors. This platform can deliver antigens into both the class I and class II antigen presentation pathways and stimulate strong Th1 and Th17 responses. In mice this fusion vaccine, designated GI-19007, was immunogenic and elicited strong gamma interferon (IFN-) and interleukin-17 (IL-17) responses; despite this, they displayed minimal prophylactic activity in mice that were subsequently infected with a virulent clinical strain. In contrast, in a therapeutic model in the guinea pig, GI-19007 significantly reduced the lung bacterial load and reduced lung pathology, particularly in terms of secondary lesion development, while significantly improving survival in one-third of these animals. In further studies in which guinea pigs were vaccinated with BCG before challenge, therapeutic vaccination with GI-19007 initially improved survival versus that of animals given BCG alone, although this protective effect was gradually lost at around 400 days after challenge. Given its apparent ability to substantially limit bacterial dissemination within and from the lungs, GI-19007 potentially can be used to limit lung damage as well as facilitating chemotherapeutic regimens in infected individuals.

RiVax is a promising recombinant ricin toxin A subunit (RTA) vaccine antigen that has been shown to be safe and immunogenic in humans and effective at protecting rhesus macaques against lethal-dose aerosolized toxin exposure. We previously used a panel of RTA-specific monoclonal antibodies (MAbs) to demonstrate, by competition enzyme-linked immunosorbent assay (ELISA), that RiVax elicits similar serum antibody profiles in humans and macaques. However, the MAb binding sites on RiVax have yet to be defined. In this study, we employed hydrogen exchange-mass spectrometry (HX-MS) to localize the epitopes on RiVax recognized by nine toxin-neutralizing MAbs and one nonneutralizing MAb. Based on strong protection from hydrogen exchange, the nine MAbs grouped into four spatially distinct epitope clusters (namely, clusters I to IV). Cluster I MAbs protected RiVax's α-helix B (residues 94 to 107), a protruding immunodominant secondary structure element known to be a target of potent toxin-neutralizing antibodies. Cluster II consisted of two subclusters located on the "back side" (relative to the active site pocket) of RiVax. One subcluster involved α-helix A (residues 14 to 24) and α-helices F-G (residues 184 to 207); the other encompassed β-strand d (residues 62 to 69) and parts of α-helices D-E (154 to 164) and the intervening loop. Cluster III involved α-helices C and G on the front side of RiVax, while cluster IV formed a sash from the front to back of RiVax, spanning strands b, c, and d (residues 35 to 59). Having a high-resolution B cell epitope map of RiVax will enable the development and optimization of competitive serum profiling assays to examine vaccine-induced antibody responses across species.

We have been exploring the use of the live attenuated Salmonella enterica serovar Typhi Ty21a vaccine strain as a versatile oral vaccine vector for the expression and delivery of multiple foreign antigens, including Shigella O-antigens. In this study, we separately cloned genes necessary for the biosynthesis of the Shigella flexneri serotype 2a and 3a O-antigens, which have been shown to provide broad cross-protection to multiple disease-predominant S. flexneri serotypes. The cloned S. flexneri 2a rfb operon, along with bgt and gtrII, contained on the SfII bacteriophage, was sufficient in Ty21a to express the heterologous S. flexneri 2a O-antigen containing the 3,4 antigenic determinants. Further, this rfb operon, along with gtrA, gtrB, and gtrX contained on the Sfx bacteriophage and oac contained on the Sf6 bacteriophage, was sufficient to express S. flexneri 3a O-antigen containing the 6, 7, and 8 antigenic determinants. Ty21a, with these plasmid-carried or chromosomally inserted genes, demonstrated simultaneous and stable expression of homologous S. Typhi O-antigen plus the heterologous S. flexneri O-antigen. Candidate Ty21a vaccine strains expressing heterologous S. flexneri 2a or 3a lipopolysaccharide (LPS) elicited significant serum antibody responses against both homologous S. Typhi and heterologous Shigella LPS and protected mice against virulent S. flexneri 2a or 3a challenges. These new S. flexneri 2a and 3a O-antigen-expressing Ty21a vaccine strains, together with our previously constructed Ty21a strains expressing Shigella sonnei or Shigella dysenteriae 1 O-antigens, have the potential to be used together for simultaneous protection against the predominant causes of shigellosis worldwide as well as against typhoid fever.

The global burden of disease caused by extraintestinal pathogenic Escherichia coli (ExPEC) is increasing as the prevalence of multidrug-resistant strains rises. A multivalent ExPEC O-antigen bioconjugate vaccine could have a substantial impact in preventing bacteremia and urinary tract infections. Development of an ExPEC vaccine requires a readout to assess the functionality of antibodies. We developed an opsonophagocytic killing assay (OPA) for four ExPEC serotypes (serotypes O1A, O2, O6A, and O25B) based on methods established for pneumococcal conjugate vaccines. The performance of the assay was assessed with human serum by computing the precision, linearity, trueness, total error, working range, and specificity. Serotypes O1A and O6A met the acceptance criteria for precision (coefficient of variation for repeatability and intermediate precision, ≤50%), linearity (90% confidence interval of the slope of each strain, 0.80, 1.25), trueness (relative bias range, –30% to 30%), and total error (total error range, –65% to 183%) at five serum concentrations and serotypes O2 and O25B met the acceptance criteria at four concentrations (the lowest concentration for serotypes O2 and O25B did not meet the system suitability test of maximum killing of ≥85% of E. coli cells). All serotypes met the acceptance criteria for specificity (opsonization index value reductions of ≤20% for heterologous serum preadsorption and ≥70% for homologous serum preadsorption). The assay working range was defined on the basis of the lowest and highest concentrations at which the assay jointly fulfilled the target acceptance criteria for linearity, precision, and accuracy. An OPA suitable for multiple E. coli serotypes has been developed, qualified, and used to assess the immunogenicity of a 4-valent E. coli bioconjugate vaccine (ExPEC4V) administered to humans.

It has been identified that arginine vasopressin (AVP), vasopressin receptor 2(V2R), and the aquaporin 2 (AQP2) signaling pathway in the inner ear play important roles in hearing and balance functions through regulating the endolymph equilibrium; however, the contributions of this signaling pathway to the development of motion sickness are unclear. The present study was designed to investigate whether the activation of the AVP-V2R-AQP2 signaling pathway in the inner ear is involved in the induction of motion sickness and whether mozavaptan, a V2R antagonist, could reduce motion sickness. We found that both rotatory stimulus and intraperitoneal AVP injection induced conditioned taste aversion (a confirmed behavioral index for motion sickness) in rats and activated the AVP-V2R-AQP2 signaling pathway with a responsive V2R downregulation in the inner ears, and AVP perfusion in cultured epithelial cells from rat endolymphatic sacs induced similar changes in this pathway signaling. Vestibular training, V2R antagonist mozavaptan, or PKA inhibitor H89 blunted these changes in the V2R-AQP2 pathway signaling while reducing rotatory stimulus– or DDAVP (a V2R agonist)-induced motion sickness in rats and dogs. Therefore, our results suggest that activation of the inner ear AVP-V2R-AQP2 signaling pathway is potentially involved in the development of motion sickness; thus, mozavaptan targeting AVP V2Rs in the inner ear may provide us with a new application option to reduce motion sickness.

Metastatic breast cancer is prevalent worldwide, and one of the most common sites of metastasis is long bones. Of patients with disease, the major symptom is pain, yet current medications fail to adequately result in analgesic efficacy and present major undesirable adverse effects. In our study, we investigate the potential of a novel monoacylglycerol lipase (MAGL) inhibitor, MJN110, in a murine model of cancer-induced bone pain. Literature has previously demonstrated that MAGL inhibitors function to increase the endogenous concentrations of 2-arachydonylglycerol, which then activates CB1 and CB2 receptors to inhibit inflammation and pain. We demonstrate that administration of MJN110 significantly and dose dependently alleviates spontaneous pain behavior during acute administration compared with vehicle control. In addition, MJN110 maintains its efficacy in a chronic-dosing paradigm over the course of 7 days without signs of receptor sensitization. In vitro analysis of MJN110 demonstrated a dose-dependent and significant decrease in cell viability and proliferation of 66.1 breast adenocarcinoma cells to a greater extent than KML29, an alternate MAGL inhibitor, or the CB2 agonist JWH015. Chronic administration of the compound did not appear to affect tumor burden, as evidenced by radiograph or histologic analysis. Together, these data support the application for MJN110 as a novel therapeutic for cancer-induced bone pain.

PF06821497 has been identified as an orally available small-molecule enhancer of zeste homolog 2 inhibitor. The objectives of the present study were to characterize pharmacokinetic-pharmacodynamic-disease relationships of PF06821497 in xenograft mouse models with diffuse large B-cell lymphoma (Karpas422). An indirect-response model reasonably fit dose-dependent pharmacodynamic responses [histone H3 on lysine 27 (H3K27) me3 inhibition] with an unbound EC₅₀ of 76 nM, whereas a signal-transduction model sufficiently fit dose-dependent disease responses (tumor growth inhibition) with an unbound tumor stasis concentration (T_sc) of 168 nM. Thus, effective concentration for 70% of maximal effect (EC₇₀) for H3K27me3 inhibition was roughly comparable to T_sc, suggesting that 70% H3K27me3 inhibition could be required for tumor stasis. Consistently, an integrated pharmacokinetic-pharmacodynamic-disease model adequately describing tumor growth inhibition also suggested that ~70% H3K27me3 inhibition was associated with tumor stasis. Based on these results, we would propose that an EC₇₀ estimate for H3K27me3 inhibition corresponding to tumor stasis could be considered a minimum target efficacious concentration of PF06821497 in cancer patients.

Glucagon-like peptide-2 (GLP-2) agonists have therapeutic potential in clinical indications in which the integrity or absorptive function of the intestinal mucosa is compromised, such as in short bowel syndrome (SBS). Native hGLP-2, a 33–amino acid peptide secreted from the small intestine, contributes to nutritional absorption but has a very short half-life because of enzymatic cleavage and renal clearance and thus is of limited therapeutic value. The GLP-2 analog teduglutide (Revestive/Gattex; Shire Inc.) has been approved for use in SBS since 2012 but has a once-daily injection regimen. Pharmacokinetic (PK) and pharmacodynamic studies confirm that apraglutide, a novel GLP-2 analog, has very low clearance, long elimination half-life, and high plasma protein binding compared with GLP-2 analogs teduglutide and glepaglutide. Apraglutide and teduglutide retain potency and selectivity at the GLP-2 receptor comparable to native hGLP-2, whereas glepaglutide was less potent and less selective. In rat intravenous PK studies, hGLP-2, teduglutide, glepaglutide, and apraglutide had clearances of 25, 9.9, 2.8, and 0.27 ml/kg per minute, respectively, and elimination half-lives of 6.4, 19, 16, and 159 minutes, respectively. The unique PK profile of apraglutide administered via intravenous and subcutaneous routes was confirmed in monkey and minipig and translated into significantly greater in vivo pharmacodynamic activity, measured as small intestinal growth in rats. Apraglutide showed greater intestinotrophic activity than the other peptides when administered at less-frequent dosing intervals because of its prolonged half-life. We postulate that apraglutide offers several advantages over existing GLP-2 analogs and is an excellent candidate for the treatment of gastrointestinal diseases, such as SBS.

Abicipar pegol (abicipar) is a novel DARPin therapeutic and highly potent vascular endothelial growth factor (VEGF) inhibitor intended for the treatment of neovascular age-related macular degeneration (nAMD). Here we develop a translational pharmacokinetic/pharmacodynamic (PK/PD) model for abicipar to guide dosing regimens in the clinic. The model incorporated abicipar-VEGF binding kinetics, VEGF expression levels, and VEGF turnover rates to describe the ocular and systemic PK data collected from the vitreous, aqueous humor (AH), choroid, retina, and serum of rabbits after a 1-mg abicipar intravitreal (IVT) dose. The model was translated to humans using human-specific mechanistic parameters and refitted to human serum and AH concentrations from patients with diabetic macular edema and nAMD. The model was then used to simulate 8-, 12- (quarterly), and 16-week dosing intervals in the clinic. Simulations of 2 mg abicipar IVT at 8-week or quarterly dosing in humans indicates minimum steady-state vitreal concentrations are maintained above both in vitro IC₅₀ and in vivo human IC₅₀ values. The model predicted virtually complete VEGF inhibition for the 8-week and quarterly dosing schedule during the 52-week treatment period. In the 16-week schedule, clinically significant VEGF inhibition was maintained during the 52-week period. The model quantitatively described abicipar-VEGF target engagement leading to rapid reduction of VEGF and a long duration of VEGF inhibition demonstrating the clinical feasibility of up to a 16-week dosing interval. Abicipar is predicted to reduce IVT dosing compared with other anti-VEGF therapies with the potential to lessen patient treatment burden.

Infection with the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may remain asymptomatic, leading to under-recognition of the related disease, coronavirus disease, 2019 (COVID-19), and to incidental findings in nuclear imaging procedures performed for standard clinical indications. Here, we report about our local experience in a region with high COVID-19 prevalence and dynamically increasing infection rates. Methods: Within the 8-d period of March 16–24, 2020, hybrid imaging studies of asymptomatic patients who underwent ¹⁸F-FDG PET/CT or ¹³¹I SPECT/CT for standard oncologic indications at our institution in Brescia, Italy, were analyzed for findings suggestive of COVID-19. The presence, radiologic features, and metabolic activity of interstitial pneumonia were identified, correlated with the subsequent short-term clinical course, and described in a case series. Results: Six of 65 patients (9%) who underwent PET/CT for various malignancies showed unexpected signs of interstitial pneumonia on CT and elevated regional ¹⁸F-FDG avidity. Additionally, 1 of 12 patients who received radioiodine for differentiated thyroid carcinoma also showed interstitial pneumonia on SPECT/CT. Five of 7 patients had subsequent proof of COVID-19 by reverse-transcriptase polymerase chain reaction. The remaining 2 patients were not tested immediately but underwent quarantine and careful monitoring. Conclusion: Incidental findings suggestive of COVID-19 may not be infrequent in hybrid imaging of asymptomatic patients in regions with an expansive spread of SARS-CoV-2. Nuclear medicine services should prepare accordingly.

ABSTRACT

Members of family Coronaviridae cause a variety of diseases in birds and mammals. Porcine hemagglutinating encephalomyelitis virus (PHEV), a lesser-researched coronavirus, can infect naive pigs of any age, but clinical disease is observed in pigs ≤4 weeks of age. No commercial PHEV vaccines are available, and neonatal protection from PHEV-associated disease is presumably dependent on lactogenic immunity. Although subclinical PHEV infections are thought to be common, PHEV ecology in commercial swine herds is unknown. To begin to address this gap in knowledge, a serum IgG antibody enzyme-linked immunosorbent assay (ELISA) based on the S1 protein was developed and evaluated on known-status samples and then used to estimate PHEV seroprevalence in U.S. sow herds. Assessment of the diagnostic performance of the PHEV S1 ELISA using serum samples (n = 924) collected from 7-week-old pigs (n = 84; 12 pigs per group) inoculated with PHEV, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine respiratory coronavirus, or porcine deltacoronavirus showed that a sample-to-positive cutoff value of ≥0.6 was both sensitive and specific, i.e., all PHEV-inoculated pigs were seropositive from days postinoculation 10 to 42, and no cross-reactivity was observed in samples from other groups. The PHEV S1 ELISA was then used to estimate PHEV seroprevalence in U.S. sow herds (19 states) using 2,756 serum samples from breeding females (>28 weeks old) on commercial farms (n = 104) with no history of PHEV-associated disease. The overall seroprevalence was 53.35% (confidence interval [CI], ±1.86%) and herd seroprevalence was 96.15% (CI, ±3.70%).

IMPORTANCE There is a paucity of information concerning the ecology of porcine hemagglutinating encephalomyelitis virus (PHEV) in commercial swine herds. This study provided evidence that PHEV infection is endemic and highly prevalent in U.S. swine herds. These results raised questions for future studies regarding the impact of endemic PHEV on swine health and the mechanisms by which this virus circulates in endemically infected populations. Regardless, the availability of the validated PHEV S1 enzyme-linked immunosorbent assay (ELISA) provides the means for swine producers to detect and monitor PHEV infections, confirm prior exposure to the virus, and to evaluate the immune status of breeding herds.

Trumenba (MenB-FHbp; bivalent rLP2086), the first meningococcal serogroup B vaccine approved in the United States and subsequently approved in Europe, Canada, and Australia, is well-characterized. Pfizer devised a control strategy approach by using a simplified control strategy wheel for Trumenba based on International Council for Harmonisation (ICH) Q8 (R2), Q9, Q10, and Q11 guidelines, which provide complementary guidance on pharmaceutical development, quality risk management, quality systems, and development and manufacture of drug substances, respectively. These guidelines ensure product quality using a scientific and risk-based approach. Trumenba contains two factor H binding proteins (FHbps), one from each of the two FHbp subfamilies (A and B), adsorbed onto aluminum phosphate. Trumenba manufacturing processes are complicated by the recombinant protein expression of Subfamily A and B proteins and the nature of the drug product (suspension in syringes); the latter also introduces challenges in controlling product critical quality attributes during the development process. In such complex systems, the control strategy is critical to ensuring consistent desired product quality; it also supports the regulatory requirement of continued improvement through continuous process verification and aids regulatory filing. This article describes Pfizer's approach toward robust control strategy development, built on product and process understanding, and links control strategy to regulatory document sections and flow of controls. Specifically, an approach is presented on product quality attribute criticality determination based on safety and efficacy and on an understanding of process parameter criticality. This was achieved by studying the impact of the approach on product quality attributes to define process parameter and in-process controls. This approach is further explained through Trumenba case studies, highlighting specific quality attributes and the associated controls implemented, and provides a holistic view of controls employed for both drug substance and drug product.

We are already a couple of months into 2020 and I hope you had a good start to the new year. I wish you, our readers, reviewers, authors and editors, happiness, success and health in this new decade.

Tigecycline serves as one of the antibiotics of last resort to treat multidrug-resistant (including carbapenem-resistant) pathogens. However, the recently emerged plasmid-mediated tigecycline resistance mechanism, Tet(X), challenges the clinical efficacy of this class of antibiotics. In this study, we detected 180 tet(X)-harboring Acinetobacter isolates (8.9%, n = 180) from 2,018 samples collected from avian farms and adjacent environments in China. Eighteen tet(X)-harboring isolates (10.0%) were found to cocarry the carbapenemase gene bla_NDM-1, mostly from waterfowl samples (94.4%, 17/18). Interestingly, among six Acinetobacter strains, tet(X) and bla_NDM-1 were found to colocalize on the same plasmids. Moreover, whole-genome sequencing (WGS) revealed a novel orthologue of tet(X) in the six isolates coharboring tet(X) and bla_NDM-1. Inverse PCR suggested that the two tet(X) genes form a single transposable unit and may be cotransferred. Sequence comparison between six tet(X)- and bla_NDM-1-coharboring plasmids showed that they shared a highly homologous plasmid backbone even though they were isolated from different Acinetobacter species (three from Acinetobacter indicus, two from Acinetobacter schindleri, and one from Acinetobacter lwoffii) from various sources and from different geological regions, suggesting the horizontal genetic transfer of a common tet(X)- and bla_NDM-1-coharboring plasmid among Acinetobacter species in China. Emergence and spread of such plasmids and strains are of great clinical concern, and measures must be implemented to avoid their dissemination.

KBP-7072 is a novel third-generation tetracycline (aminomethylcycline) antibacterial that overcomes common efflux and ribosomal protection resistance mechanisms that cause resistance in older-generation tetracyclines. KBP-7072 completed phase 1 clinical development studies for safety, tolerability, and pharmacokinetics (ClinicalTrials.gov identifier NCT02454361) and multiple ascending doses in healthy subjects (ClinicalTrials.gov identifier NCT02654626) in December 2015. Both oral and intravenous formulations of KBP-7072 are being developed. In this study, we evaluated the in vitro activities of KBP-7072 and comparator agents by CLSI document M07 (2018) broth microdilution against 531 recent geographically diverse and/or molecularly characterized Acinetobacter baumannii-A. calcoaceticus species complex (A. baumannii) isolates from the United States, Europe, Asia-Pacific (excluding China), and Latin America. A. baumannii isolates included carbapenem-resistant, colistin-resistant, tetracycline-resistant, and extended-spectrum-β-lactamase (ESBL)- and metallo-β-lactamase (MBL)-producing isolates. Overall, KBP-7072 (MIC_50/90, 0.25/1 mg/liter) was comparable in activity to colistin (92.8%/92.8% susceptible [S] [CLSI/EUCAST]) against A. baumannii isolates, inhibiting 99.2% of isolates at ≤2 mg/liter and 97.6% of isolates at ≤1 mg/liter. KBP-7072 was equally active against A. baumannii isolates, including carbapenem-resistant, colistin-resistant, and tetracycline-resistant isolates, regardless of geographic location, and maintained activity against ESBL- and MBL-producing isolates. KBP-7072 outperformed comparator agents, including ceftazidime (40.3% S [CLSI]), gentamicin (48.2%/48.2% S [CLSI/EUCAST]), levofloxacin (39.5%/37.9% S [CLSI/EUCAST]), meropenem (42.0%/42.0% S [CLSI/EUCAST]), piperacillin-tazobactam (33.3% S [CLSI]), and all tetracycline-class comparator agents, which include doxycycline (67.3% S [CLSI]), minocycline (73.8% S [CLSI]), tetracycline (37.2% S [CLSI]), and tigecycline (79.5% inhibited by ≤2 mg/liter). The potent in vitro activity of KBP-7072 against recent geographically diverse, molecularly characterized, and drug-resistant A. baumannii isolates supports continued clinical development for the treatment of serious infections, including those caused by A. baumannii.

Balamuthia mandrillaris is an under-reported, pathogenic free-living amoeba that causes Balamuthia amoebic encephalitis (BAE) and cutaneous skin infections. Although cutaneous infections are not typically lethal, BAE with or without cutaneous involvement is usually fatal. This is due to the lack of drugs that are both efficacious and can cross the blood-brain barrier. We aimed to discover new leads for drug discovery by screening the open-source Medicines for Malaria Venture (MMV) Malaria Box and MMV Pathogen Box, with 800 compounds total. From an initial single point screen at 1 and 10 μM, we identified 54 hits that significantly inhibited the growth of B. mandrillaris in vitro. Hits were reconfirmed in quantitative dose-response assays and 23 compounds (42.6%) were confirmed with activity greater than miltefosine, the current standard of care.

We report a systematic, cellular phenotype-based antimalarial screening of the Medicines for Malaria Venture Pathogen Box collection, which facilitated the identification of specific blockers of late-stage intraerythrocytic development of Plasmodium falciparum. First, from standard growth inhibition assays, we identified 173 molecules with antimalarial activity (50% effective concentration [EC₅₀] ≤ 10 μM), which included 62 additional molecules over previously known antimalarial candidates from the Pathogen Box. We identified 90 molecules with EC₅₀ of ≤1 μM, which had significant effect on the ring-trophozoite transition, while 9 molecules inhibited the trophozoite-schizont transition and 21 molecules inhibited the schizont-ring transition (with ≥50% parasites failing to proceed to the next stage) at 1 μM. We therefore rescreened all 173 molecules and validated hits in microscopy to prioritize 12 hits as selective blockers of the schizont-ring transition. Seven of these molecules inhibited the calcium ionophore-induced egress of Toxoplasma gondii, a related apicomplexan parasite, suggesting that the inhibitors may be acting via a conserved mechanism which could be further exploited for target identification studies. We demonstrate that two molecules, MMV020670 and MMV026356, identified as schizont inhibitors in our screens, induce the fragmentation of DNA in merozoites, thereby impairing their ability to egress and invade. Further mechanistic studies would facilitate the therapeutic exploitation of these molecules as broadly active inhibitors targeting late-stage development and egress of apicomplexan parasites relevant to human health.

A 4-year surveillance of carbapenem-resistant Acinetobacter spp. isolates in Argentina identified 40 strains carrying bla_NDM-1. Genome sequencing revealed that most were Acinetobacter baumannii, whereas seven represented other Acinetobacter spp. The A. baumannii genomes were closely related, suggesting recent spread. bla_NDM-1 was located in the chromosome of A. baumannii strains and on a plasmid in non-A. baumannii strains. A resistance gene island carrying bla_PER-7 and other resistance determinants was found on a plasmid in some A. baumannii strains.

Patients with HPV16⁺ cervical cancer and high T-cell responses to an HPV16 vaccine survived longer.

Quality Improvement Success Stories are published by the American Diabetes Association in collaboration with the American College of Physicians, Inc. (ACP), and the National Diabetes Education Program. This series is intended to highlight best practices and strategies from programs and clinics that have successfully improved the quality of care for people with diabetes or related conditions. Each article in the series is reviewed and follows a standard format developed by the editors of Clinical Diabetes. The following article describes an initiative of the Cleveland Clinic’s internal medicine residents to improve diabetes care and outcomes within an underserved patient population at an East Cleveland, OH, health center.

Consistently, the field of genetic counseling has advocated that parents be advised to defer elective genetic testing of minors until adulthood to prevent a range of potential harms, including stigma, discrimination, and the loss of the child's ability to decide for him- or herself as an adult. However, consensus around the policy of "defer-when-possible" obscures the extent to which this norm is currently under siege. Increasingly, routine use of full or partial genome sequencing challenges our ability to control what is discovered in childhood or, when applied in a prenatal context, even before birth. The expansion of consumer-initiated genetic testing services challenges our ability to restrict what is available to minors. As the barriers to access crumble, medical professionals should proceed with caution, bearing in mind potential risks and continuing to assess the impact of genetic testing on this vulnerable population.

As a very ugly standoff develops between legacy cinemas and legacy studios, Disney appears to be playing its cards close to its chest.

From his fateful game of chess to a moving turn in The Diving Bell and the Butterfly, Von Sydow was the last standard bearer of Bergman’s high-minded movie idiom

•Max von Sydow dies aged 90
•A life in pictures

The opening of the seventh seal in the Book of Revelation, disclosing the truth of God’s existence and the second coming, will result in a mysterious silence in the kingdom of heaven – then the sound of trumpets and the thunderous uproar of Earth’s apocalyptic ending. In the movies, no actor has ever represented these ideas more seriously, nor shown humanity’s anguish in the face of God’s implacable silence or unassuageable anger more clearly, than Max von Sydow. He was virtually a book of revelation in himself.

The passionate severity of Von Sydow – and his ability to impersonate the ascetic nobility of some impossibly remote priestly or knightly order but with very human flaws – formed the bedrock of Ingmar Bergman’s The Seventh Seal and the staggering series of films he was to make with Bergman in the 1950s and 1960s. Beyond that, he virtually epitomised an entire, distinctively high-minded attitude to cinematic art in Europe. His films for Bergman were composed in a movie idiom that drew on Ibsen and Strindberg, Sjöström and Dreyer – and of which, since Bergman’s death in 2007, Von Sydow could be said to be the final standard bearer.

The UK Cinema Association aims to resume business before July release of Christopher Nolan’s blockbuster Tenet, as studios and distributors scramble to protect theatrical business model

The UK cinema industry is understood to be lobbying the government to approve a proposed reopening scheme that would see venues welcome customers by the end of June.

Phil Clapp, the chief executive of the UK Cinema Association said: “We’ve made representations to government on the safeguards which UK cinemas would look to have in place for audiences and staff alike upon re-opening, and have asked that consideration be given – with these in mind – to allow cinemas to open by the end of June.”

A long-standing hypothesis suggests the BCG vaccine also serves to generally enhance the immune system, meaning it could protect against covid-19, and trials are under way to find out

Trials of experimental coronavirus vaccines are already under way, but it’s still likely to be years before one is ready and vaccination may not even be possible

The BCG tuberculosis vaccine boosts the production of immune cells and this may explain how it protects newborns from dying of sepsis

Scientists are working hard to understand how age changes the immune system so that they can better protect seniors against current and future infectious diseases