A phase 3, single-arm, open-label study to evaluate the safety, tolerability, and immunogenicity of a 15-valent pneumococcal conjugate vaccine, V114, in a 3+1 regimen in healthy infants in South Korea (PNEU-PED-KOR).

There is an ongoing burden of pneumococcal disease in children despite the use of pneumococcal conjugate vaccines (PCVs). This phase 3, open-label, single-arm, multisite, descriptive study was designed to evaluate the safety and immunogenicity of a 3 + 1 regimen of V114 (VAXNEUVANCE™), a 15-valent PCV, in South Korean infants and toddlers. Adverse events (AEs) were reported for 14 d following any vaccination, and throughout the study period for serious AEs. Serotype-specific immunoglobulin G (IgG) response rates (proportion of participants meeting an IgG threshold value of ≥0.35 µg/mL) and geometric mean concentrations (GMCs) for the 15 serotypes at 30 d postdose 3 (PD3) and at 30 d postdose 4 (PD4) were evaluated as endpoints. Healthy infants enrolled at 42-90 d after birth were vaccinated with V114 (N = 57). The most commonly reported AEs were those solicited in the trial. The majority of reported AEs were transient and of mild or moderate intensity. Few serious AEs were reported; none were vaccine related. No participants died nor discontinued the study vaccine because of an AE. V114 was immunogenic for all 15 serotypes contained in the vaccine, as assessed by IgG response rates at 30 d PD3 and IgG GMCs at 30 d PD3 and at 30 d PD4. V114 was well tolerated and immunogenic when administered as a 3 + 1 regimen in healthy South Korean infants and toddlers.

Despite the use of pneumococcal vaccines, the burden of pneumococcal disease in children persists. V114, a 15-valent pneumococcal conjugate vaccine, was immunogenic and well-tolerated in healthy South Korean infants and toddlers.

Cost effective porous areca nut carbon nanospheres for adsorptive removal of dyes and their binary mixtures.

Novel porous nanospheres from areca nuts (ACNPs) were synthesized via one-step pyrolysis without the use of any chemical treatment and the materials were used as adsorbents for the removal of cationic methylene blue (MB) and anionic methyl orange (MO) as well as their binary mixtures. Around, 6-7 tonnes of areca nut biowaste is generated every year which are then burnt due to their slow rate of decomposition resulting in higher carbon footprints. Biosorbents are generally a preferable alternative for dye adsorption but involve chemical modification for surface enhancement and complex sample treatment. In this work, ACNPs, were investigated for their efficiency in the raw form and were characterized by SEM, EDS, FTIR, XRD, and BET techniques before and after subjecting to the dye adsorption studies. The BET analysis of the adsorbents showed a high specific surface area of 693.8 m2/g when prepared at 1000 °C, while the N2 adsorption-desorption plot showed type-IV isotherm, suggesting the microporous nature of the carbon matrix. Batch equilibrium studies showed the removal efficiency of >95% for both the dyes and their binary mixtures under the optimum conditions of 0.15 g/L dosage, 10 µM concentration and contact time of 70 min. Due to the synergistic effects of the binary dyes, higher removal efficiency of MB compared to MO was observed in the binary mixture. Adsorption results were tested using Langmuir, Freundlich, Temkin, Redlich-Peterson, and Elovich isotherms to assess the best fit of the models. The qm value of MB was found to be 97.37 mg/g, while that of MO was 71.22 mg/g which is higher compared to individual dye components having lower values of 86.12 mg/g and 50.35 mg/g, respectively. Extended Langmuir and Jain and Snoeyink isotherms were used for binary data interpretation. The kinetic results showed good agreement with the Pseudo-second order equation, indicating internal diffusion. The possible mechanism involved electrostatic and á´¨-á´¨ interactions between the dye molecules and ACNPs. This approach is comprehensible and cost effective and can be utilized for dye removal in textile industries.

Global Molecular Epidemiology of Respiratory Syncytial Virus from the 2017-2018 INFORM-RSV Study.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection among infants and young children, resulting in annual epidemics worldwide. INFORM-RSV is a multiyear clinical study designed to describe the global molecular epidemiology of RSV in children under 5 years of age by monitoring temporal and geographical evolution of current circulating RSV strains, F protein antigenic sites, and their relationships with clinical features of RSV disease. During the pilot season (2017-2018), 410 RSV G-F gene sequences were obtained from 476 RSV-positive nasal samples collected from 8 countries (United Kingdom, Spain, The Netherlands, Finland, Japan, Brazil, South Africa, and Australia). RSV B (all BA9 genotype) predominated over RSV A (all ON1 genotype) globally (69.0% versus 31.0%) and in all countries except South Africa. Geographic clustering patterns highlighted wide transmission and continued evolution with viral spread. Most RSV strains were from infants of <1 year of age (81.2%), males (56.3%), and patients hospitalized for >24 h (70.5%), with no differences in subtype distribution. Compared to 2013 reference sequences, variations at F protein antigenic sites were observed for both RSV A and B strains, with high-frequency polymorphisms at antigenic site Ø (I206M/Q209R) and site V (L172Q/S173L/K191R) in RSV B strains. The INFORM-RSV 2017-2018 pilot season establishes an important molecular baseline of RSV strain distribution and sequence variability with which to track the emergence of new strains and provide an early warning system of neutralization escape variants that may impact transmission or the effectiveness of vaccines and MAbs under development.

Antibacterial Monoclonal Antibodies Do Not Disrupt the Intestinal Microbiome or Its Function.

Antibiotics revolutionized the treatment of infectious diseases; however, it is now clear that broad-spectrum antibiotics alter the composition and function of the host's microbiome. The microbiome plays a key role in human health, and its perturbation is increasingly recognized as contributing to many human diseases. Widespread broad-spectrum antibiotic use has also resulted in the emergence of multidrug-resistant pathogens, spurring the development of pathogen-specific strategies such as monoclonal antibodies (MAbs) to combat bacterial infection. Not only are pathogen-specific approaches not expected to induce resistance in nontargeted bacteria, but they are hypothesized to have minimal impact on the gut microbiome. Here, we compare the effects of antibiotics, pathogen-specific MAbs, and their controls (saline or control IgG [c-IgG]) on the gut microbiome of 7-week-old, female, C57BL/6 mice. The magnitude of change in taxonomic abundance, bacterial diversity, and bacterial metabolites, including short-chain fatty acids (SCFA) and bile acids in the fecal pellets from mice treated with pathogen-specific MAbs, was no different from that with animals treated with saline or an IgG control. Conversely, dramatic changes were observed in the relative abundance, as well as alpha and beta diversity, of the fecal microbiome and bacterial metabolites in the feces of all antibiotic-treated mice. Taken together, these results indicate that pathogen-specific MAbs do not alter the fecal microbiome like broad-spectrum antibiotics and may represent a safer, more-targeted approach to antibacterial therapy.

Nanocarrier Mediated siRNA Delivery Targeting Stem Cell Differentiation.

Stem cell-based regenerative medicine holds exceptional therapeutic potential and hence the development of efficient techniques to enhance control over the rate of differentiation has been the focus of active research. One of the strategies to achieve this involves delivering siRNA into stem cells and exploiting the RNA interference (RNAi) mechanism. Transport of siRNA across the cell membrane is a challenge due to its anionic property, especially in primary human cells and stem cells. Moreover, naked siRNA incites immune responses, may cause off-target effects, exhibits low stability and is easily degraded by endonucleases in the bloodstream. Although siRNA delivery using viral vectors and electroporation has been used in stem cells, these methods demonstrate low transfection efficiency, cytotoxicity, immunogenicity, events of integration and may involve laborious customization. With the advent of nanotechnology, nanocarriers which act as novel gene delivery vehicles designed to overcome the problems associated with safety and practicality are being developed. The various nanomaterials that are currently being explored and discussed in this review include liposomes, carbon nanotubes, quantum dots, protein and peptide nanocarriers, magnetic nanoparticles, polymeric nanoparticles, etc. These nanodelivery agents exhibit advantages such as low immunogenic response, biocompatibility, design flexibility allowing for surface modification and functionalization, and control over the surface topography for achieving the desired rate of siRNA delivery and improved gene knockdown efficiency. This review also includes discussion on siRNA co-delivery with imaging agents, plasmid DNA, drugs etc. to achieve combined diagnostic and enhanced therapeutic functionality, both for in vitro and in vivo applications.

Towards a standard diet-induced and biopsy-confirmed mouse model of non-alcoholic steatohepatitis: Impact of dietary fat source.

BACKGROUND: The trans-fat containing AMLN (amylin liver non-alcoholic steatohepatitis, NASH) diet has been extensively validated in C57BL/6J mice with or without the Lepob/Lepob (ob/ob) mutation in the leptin gene for reliably inducing metabolic and liver histopathological changes recapitulating hallmarks of NASH. Due to a recent ban on trans-fats as food additive, there is a marked need for developing a new diet capable of promoting a compatible level of disease in ob/ob and C57BL/6J mice. AIM: To develop a biopsy-confirmed mouse model of NASH based on an obesogenic diet with trans-fat substituted by saturated fat. METHODS: Male ob/ob mice were fed AMLN diet or a modified AMLN diet with trans-fat (Primex shortening) substituted by equivalent amounts of palm oil [Gubra amylin NASH, (GAN) diet] for 8, 12 and 16 wk. C57BL/6J mice were fed the same diets for 28 wk. AMLN and GAN diets had similar caloric content (40% fat kcal), fructose (22%) and cholesterol (2%) level. RESULTS: The GAN diet was more obesogenic compared to the AMLN diet and impaired glucose tolerance. Biopsy-confirmed steatosis, lobular inflammation, hepatocyte ballooning, fibrotic liver lesions and hepatic transcriptome changes were similar in ob/ob mice fed the GAN or AMLN diet. C57BL/6J mice developed a mild to moderate fibrotic NASH phenotype when fed the same diets. CONCLUSION: Substitution of Primex with palm oil promotes a similar phenotype of biopsy-confirmed NASH in ob/ob and C57BL/6J mice, making GAN diet-induced obese mouse models suitable for characterizing novel NASH treatments.

Prevalence and Significance of Substitutions in the Fusion Protein of Respiratory Syncytial Virus Resulting in Neutralization Escape From Antibody MEDI8897.

Background: Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection among infants and young children. To date, no vaccine is approved for the broad population of healthy infants. MEDI8897, a potent anti-RSV fusion antibody with extended serum half-life, is currently under clinical investigation as a potential passive RSV vaccine for all infants. As a ribonucleic acid virus, RSV is prone to mutation, and the possibility of viral escape from MEDI8897 neutralization is a potential concern. Methods: We generated RSV monoclonal antibody (mAb)-resistant mutants (MARMs) in vitro and studied the effect of the amino acid substitutions identified on binding and viral neutralization susceptibility to MEDI8897. The impact of resistance-associated mutations on in vitro growth kinetics and the prevalence of these mutations in currently circulating strains of RSV in the United States was assessed. Results: Critical residues identified in MARMs for MEDI8897 neutralization were located in the MEDI8897 binding site defined by crystallographic analysis. Substitutions in these residues affected the binding of mAb to virus, without significant impact on viral replication in vitro. The frequency of natural resistance-associated polymorphisms was low. Conclusions: Results from this study provide insights into the mechanism of MEDI8897 escape and the complexity of monitoring for emergence of resistance.

Modulation of an ectodomain motif in the influenza A virus neuraminidase alters tetherin sensitivity and results in virus attenuation in vivo.

We previously demonstrated that ectodomain residue Asp286 in N2 neuraminidase (NA; Asp268 in N1 NA) present in budding-capable NA proteins contributes to productive NA plasma membrane transport partly by mediating escape from tetherin restriction [Yondola MA, Fernandes F, Belicha-Villanueva A, Uccelini M, Gao Q, Carter C, et al. (2011). Budding capability of the influenza virus neuraminidase can be modulated by tetherin. J Virol, 85, 2480-2491]. Budding-incapable NA proteins contain a G at this position and either co-expression of human immunodeficiency virus type 1 vpu or siRNA-mediated depletion of tetherin rescued budding capabilities in these proteins [Yondola MA, Fernandes F, Belicha-Villanueva A, Uccelini M, Gao Q, Carter C, et al. (2011). Budding capability of the influenza virus neuraminidase can be modulated by tetherin. J Virol, 85, 2480-2491]. Furthermore, replacement of D286 with G in budding-capable NA proteins caused loss of function, preventing release of NA virus-like particles (VLPs). Here, we show that mutation of this residue specifically modulates the ability of NA to escape tetherin restriction at the plasma membrane and results in virus attenuation in vivo. Based on immunogold electron microscopy and co-immunoprecipitation assays, both NAD286-containing and NAD286G-containing proteins associated with tetherin in the endoplasmic reticulum (ER). However, the NAD286G loss-of-function mutant also associated with the host factor outside the ER and in plasma-membrane-localized VLPs as visualized using immunogold electron microscopy. We conclude that the presence of aspartate at residue 286 liberates NA from tetherin-dependent restriction upon exit from the ER compartment thus preventing restriction at the plasma membrane. Underscoring the importance of these observations, knockdown of tetherin resulted in a 1-1.5 log increase in influenza virus growth. Additionally, the loss-of-function mutation conferred attenuation in a mouse model of influenza infection as evidenced by a 5-fold increase in LD50 and increases in either percent survival or time to death dependent on the administered dose in vivo.

Budding capability of the influenza virus neuraminidase can be modulated by tetherin.

We have determined that, in addition to its receptor-destroying activity, the influenza virus neuraminidase is capable of efficiently forming virus-like particles (VLPs) when expressed individually from plasmid DNA. This observation applies to both human subtypes of neuraminidase, N1 and N2. However, it is not found with every strain of influenza virus. Through gain-of-function and loss-of-function analyses, a critical determinant within the neuraminidase ectodomain was identified that contributes to VLP formation but is not sufficient to accomplish release of plasmid-derived VLPs. This sequence lies on the plasma membrane-proximal side of the neuraminidase globular head. Most importantly, we demonstrate that the antiviral restriction factor tetherin plays a role in determining the strain-specific limitations of release competency. If tetherin is counteracted by small interfering RNA knockdown or expression of the HIV anti-tetherin factor vpu, budding and release capability is bestowed upon an otherwise budding-deficient neuraminidase. These data suggest that budding-competent neuraminidase proteins possess an as-yet-unidentified means of counteracting the antiviral restriction factor tetherin and identify a novel way in which the influenza virus neuraminidase can contribute to virus release.

Phosphoinositides direct equine infectious anemia virus gag trafficking and release.

Phosphatidylinositol 4,5-biphosphate [PI(4,5)P(2) ], the predominant phosphoinositide (PI) on the plasma membrane, binds the matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) with similar affinities in vitro. Interaction with PI(4,5)P(2) is critical for HIV-1 assembly on the plasma membrane. EIAV has been shown to localize in internal compartments; hence, the significance of its interaction with PI(4,5)P(2) is unclear. We therefore investigated the binding in vitro of other PIs to EIAV MA and whether intracellular association with compartments bearing these PIs was important for assembly and release of virus-like particles (VLPs) formed by Gag. In vitro, EIAV MA bound phosphatidylinositol 3-phosphate [PI(3)P] with higher affinity than PI(4,5)P(2) as revealed by nuclear magnetic resonance (NMR) spectra upon lipid titration. Gag was detected on the plasma membrane and in compartments enriched in phosphatidylinositol 3,5-biphosphate [PI(3,5)P(2) ]. Treatment of cells with YM201636, a kinase inhibitor that blocks production of PI(3,5)P(2) from PI(3)P, caused Gag to colocalize with aberrant compartments and inhibited VLP release. In contrast to HIV-1, release of EIAV VLPs was not significantly diminished by coexpression with 5-phosphatase IV, an enzyme that specifically depletes PI(4,5)P(2) from the plasma membrane. However, coexpression with synaptojanin 2, a phosphatase with broader specificity, diminished VLP production. PI-binding pocket mutations caused striking budding defects, as revealed by electron microscopy. One of the mutations also modified Gag-Gag interaction, as suggested by altered bimolecular fluorescence complementation. We conclude that PI-mediated targeting to peripheral and internal membranes is a critical factor in EIAV assembly and release.

Cyclosporine inhibits a direct interaction between cyclophilins and hepatitis C NS5A.

BACKGROUND: Hepatitis C Virus (HCV) infection is a leading indication for liver transplantation. HCV infection reoccurs almost universally post transplant, decreasing both graft longevity and patient survival. The immunosuppressant, cyclosporine A (CsA) has potent anti-HCV activity towards both HCV replicons and the genotype 2a cell culture infectious virus. Previously, we isolated mutations in the 1bN replicon with less sensitivity to CsA that mapped to both NS5A and NS5B regions of the virus. Mutations in NS5A alone conferred decreased CsA susceptibility regardless of NS5B mutations. METHODOLOGY/PRINCIPAL FINDINGS: We examined the mechanisms by which NS5A mutations contribute to CsA resistance and if they are strain dependent. Using in vitro mutagenesis, the amino acid position 321 mutation of NS5A was restored to the wild-type tyrosine residue conferring partial CsA susceptibility on the mutant replicon. The 321 mutation also alters CsA susceptibility of the JFH cell culture virus. Additionally, we demonstrated a novel CsA-sensitive interaction between NS5A and both cyclophilin A and B. Both the mutant NS5A and wild type NS5A bind cyclophilin in vitro. The NS5A: cyclophilin interaction requires both the NS5A region identified by the resistance mutants and cyclophilin catalytic residues. In cell culture, NS5A from CsA resistant mutant has an enhanced interaction with cyclophilin B. Additionally; NS5B facilitates a stronger binding of mutant NS5A to endogenous cyclophilin B than wild-type in cell culture. CONCLUSIONS/SIGNIFICANCE: Collectively, this data suggests direct interactions between cyclophilins and NS5A are critical to understand for optimal use of cyclophilin inhibitors in anti-HCV therapy.

Sensitivity of hepatitis C virus to cyclosporine A depends on nonstructural proteins NS5A and NS5B.

UNLABELLED: HCV re-occurs after liver transplantation and increases mortality. Cyclosporine, but not tacrolimus, has potent antiviral effects against HCV replication in cell culture. To determine the conditions, if any, under which HCV is susceptible to cyclosporine in vivo, we selected for cyclosporine-resistant mutant HCV in vitro. The resulting mutations were mapped to x-ray crystallographic structures and sequence databases. Mutations selected by cyclosporine were clustered in the nonstructural (NS) proteins NS5A and NS5B. Different sets of mutations in NS5A, paired with the same 2 NS5B mutations, conferred different levels of cyclosporine resistance when engineered back into the HCV replicon. Mutations in NS5B are structurally consistent with a proposed model of regulation of RNA binding by cyclophilin B (CyPB). These mutations also highlight a natural polymorphism between different HCV genotypes that correlates with the variation in response to cyclosporine A (CsA) noted in some clinical trials. Replicons engineered to have mutations in only NS5A (P < or = 0.0001) or only NS5B (P = 0.002) suggest that while both NS5A or NS5B variants alter cyclosporine susceptibility, NS5A has the largest effect. CONCLUSION: Preexisting sequence variation could alter the effect of cyclosporine on HCV in vivo.

Evaluation of attenuation, immunogenicity and efficacy of a bovine parainfluenza virus type 3 (PIV-3) vaccine and a recombinant chimeric bovine/human PIV-3 vaccine vector in rhesus monkeys.

Restricted replication in the respiratory tract of rhesus monkeys is an intrinsic property of bovine parainfluenza virus type 3 (bPIV-3) strains. This host range phenotype of bPIV-3 has been utilized as a marker to evaluate the attenuation of bPIV-3 vaccines for human use. Two safety, immunogenicity and efficacy studies in primates evaluated and compared three human parainfluenza virus type 3 (hPIV-3) vaccine candidates: biologically derived bPIV-3, a plasmid-derived bPIV-3 (r-bPIV-3) and a chimeric bovine/human PIV-3 (b/hPIV-3). These studies also examined the feasibility of substituting Vero cells, cultured in the presence or absence of foetal bovine serum, for foetal rhesus lung-2 (FRhL-2) cells as the tissue culture substrate for the production of bPIV-3 vaccine. The results demonstrated that (i) Vero cell-produced bPIV-3 was as attenuated, immunogenic and efficacious as bPIV-3 vaccine grown in FRhL-2 cells, (ii) plasmid-derived bPIV-3 was as attenuated, immunogenic and efficacious as the biologically derived bPIV-3 and (iii) the b/hPIV-3 chimera displayed an intermediate attenuation phenotype and protected animals completely from hPIV-3 challenge. These results support the use of bPIV-3 vaccines propagated in Vero cells in human clinical trials and the use of b/hPIV-3 as a virus vaccine vector to express foreign viral antigens.

Effects of human metapneumovirus and respiratory syncytial virus antigen insertion in two 3' proximal genome positions of bovine/human parainfluenza virus type 3 on virus replication and immunogenicity.

A live attenuated bovine parainfluenza virus type 3 (PIV3), harboring the fusion (F) and hemagglutinin-neuraminidase (HN) genes of human PIV3, was used as a virus vector to express surface glycoproteins derived from two human pathogens, human metapneumovirus (hMPV) and respiratory syncytial virus (RSV). RSV and hMPV are both paramyxoviruses that cause respiratory disease in young children, the elderly, and immunocompromised individuals. RSV has been known for decades to cause acute lower respiratory tract infections in young children, which often result in hospitalization, while hMPV has only been recently identified as a novel human respiratory pathogen. In this study, the ability of bovine/human PIV3 to express three different foreign transmembrane surface glycoproteins and to induce a protective immune response was evaluated. The RNA-dependent RNA polymerase of paramyxoviruses binds to a single site at the 3' end of the viral RNA genome to initiate transcription of viral genes. The genome position of the viral gene determines its level of gene expression. The promoter-proximal gene is transcribed with the highest frequency, and each downstream gene is transcribed less often due to attenuation of transcription at each gene junction. This feature of paramyxoviruses was exploited using the PIV3 vector by inserting the foreign viral genes at the 3' terminus, at position 1 or 2, of the viral RNA genome. These locations were expected to yield high levels of foreign viral protein expression stimulating a protective immune response. The immunogenicity and protection results obtained with a hamster model showed that bovine/human PIV3 can be employed to generate bivalent PIV3/RSV or PIV3/hMPV vaccine candidates that will be further evaluated for safety and efficacy in primates.

Stable replication of papillomavirus genomes in Saccharomyces cerevisiae.

Papillomaviruses normally replicate in stratified squamous epithelial tissues of their mammalian hosts, in which the viral genome is found as a nuclear plasmid. Two viral proteins, E1, a helicase, and E2, a transcriptional activator and plasmid maintenance factor, are known to contribute to the episomal replication of the viral genome. Recently, our laboratory discovered that papillomaviruses can also replicate in an E1-independent manner in mammalian cells (K. Kim and P. F. Lambert, Virology, in press; K. Kim and P. F. Lambert, submitted for publication). In this study, we describe experiments investigating the capacity of the human papillomavirus type 16 (HPV16) genome to replicate in yeast (Saccharomyces cerevisiae). The full-length HPV16 genome, when linked in cis to a selectable yeast marker gene, either TRP1 or URA3, could replicate stably as an episome in yeast. The replication of papillomavirus genomes in yeast is not limited to HPV16. Bovine papillomavirus type 1 and HPV6b, -11, -16, -18, and -31 were all capable of replicating in short-term assays over a period of 20 cell doublings. The long-term persistence of viral episomes did not require any one viral gene, as mutant genomes defective in single genes also replicated episomally. These results indicate that the viral episome can replicate in the absence of the E1 DNA helicase. Similarly, E2 was also not required for replication in yeast, and E2 mutant viral genomes were stably maintained in the absence of selection, indicating the existence of an E2-independent mechanism for plasmid maintenance. The episomal replication of papillomavirus genomes in yeast provides a genetically manipulatable system in which to investigate cellular factors required for episomal replication and may provide a novel means for generating infectious papillomavirus.