Multiple Envelope Research Articles

B cell repertoire sequencing of HIV-1 pediatric elite-neutralizers identifies multiple broadly neutralizing antibody clonotypes.

A limited subset of HIV-1 infected adult individuals typically after at least 2-3 years of chronic infection, develop broadly neutralizing antibodies (bnAbs), suggesting that highly conserved neutralizing epitopes on the HIV-1 envelope glycoprotein are difficult for B cell receptors to effectively target, during natural infection. Recent studies have shown the evolution of bnAbs in HIV-1 infected infants. We used bulk BCR sequencing (BCR-seq) to profile the B cell receptors from longitudinal samples (3 time points) collected from a rare pair of antiretroviralnaïve, HIV-1 infected pediatric monozygotic twins (AIIMS_329 and AIIMS_330) who displayed elite plasma neutralizing activity against HIV-1. BCR-seq of both twins revealed convergent antibody characteristics including V-gene use, CDRH3 lengths and somatic hypermutation (SHM). Further, antibody clonotypes with genetic features similar to highly potent bnAbs isolated from adults showed ongoing development in donor AIIMS_330 but not in AIIMS_329, corroborating our earlier findings based on plasma bnAbs responses. An increase in SHM was observed in sequences of the IgA isotype from AIIMS_330. This study suggests that children living with chronic HIV-1 can develop clonotypes of HIV-1 bnAbs against multiple envelope epitopes similar to those isolated from adults, highlighting that such B cells could be steered to elicit bnAbs responses through vaccines aimed to induce bnAbs against HIV-1 in a broad range of people including children.

EGFR core fucosylation, induced by hepatitis C virus, promotes TRIM40-mediated-RIG-I ubiquitination and suppresses interferon-I antiviral defenses

Aberrant N-glycosylation has been implicated in viral diseases. Alpha-(1,6)-fucosyltransferase (FUT8) is the sole enzyme responsible for core fucosylation of N-glycans during glycoprotein biosynthesis. Here we find that multiple viral envelope proteins, including Hepatitis C Virus (HCV)-E2, Vesicular stomatitis virus (VSV)-G, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-Spike and human immunodeficiency virus (HIV)-gp120, enhance FUT8 expression and core fucosylation. HCV-E2 manipulates host transcription factor SNAIL to induce FUT8 expression through EGFR-AKT-SNAIL activation. The aberrant increased-FUT8 expression promotes TRIM40-mediated RIG-I K48-ubiquitination and suppresses the antiviral interferon (IFN)-I response through core fucosylated-EGFR-JAK1-STAT3-RIG-I signaling. FUT8 inhibitor 2FF, N-glycosylation site-specific mutation (Q352AT) of EGFR, and tissue-targeted Fut8 silencing significantly increase antiviral IFN-I responses and suppress RNA viral replication, suggesting that core fucosylation mediated by FUT8 is critical for antiviral innate immunity. These findings reveal an immune evasion mechanism in which virus-induced FUT8 suppresses endogenous RIG-I-mediated antiviral defenses by enhancing core fucosylated EGFR-mediated activation.

OptEnvelope: A target point guided method for growth-coupled production using knockouts.

Finding the best knockout strategy for coupling biomass growth and production of a target metabolite using a mathematic model of metabolism is a challenge in biotechnology. In this research, a three-step method named OptEnvelope is presented based on finding minimal set of active reactions for a target point in the feasible solution space (envelope) using a mixed-integer linear programming formula. The method initially finds the reduced desirable solution space envelope in the product versus biomass plot by removing all inactive reactions. Then, with reinsertion of the deleted reactions, OptEnvelope attempts to reduce the number of knockouts so that the desirable production envelope is preserved. Additionally, OptEnvelope searches for envelopes with higher minimum production rates or fewer knockouts by evaluating different target points within the desired solution space. It is possible to limit the maximal number of knockouts. The method was implemented on metabolic models of E. coli and S. cerevisiae to test the method benchmarking the capability of these industrial microbes for overproduction of acetate and glycerol under aerobic conditions and succinate and ethanol under anaerobic conditions. The results illustrate that OptEnvelope is capable to find multiple strong coupled envelopes located in the desired solution space because of its novel target point oriented strategy of envelope search. The results indicate that E. coli is more appropriate to produce acetate and succinate while S. cerevisiae is a better host for glycerol production. Gene deletions for some of the proposed reaction knockouts have been previously reported to increase the production of these metabolites in experiments. Both organisms are suitable for ethanol production, however, more knockouts for the adaptation of E. coli are required. OptEnvelope is available at https://github.com/lv-csbg/optEnvelope.

Targeting herpesvirus entry complex and fusogen glycoproteins with prophylactic and therapeutic agents.

Herpesviruses are among the most successful viruses found in human populations. They establish lifelong latent infections, which are punctuated by recurrent reactivations. The entry process of herpesviruses into specific target cells requires a well-orchestrated teamwork involving multiple envelope glycoproteins. The conserved glycoprotein B (gB) is the membrane fusogen, of which conformational changes are induced by an entry complex (EC) consisting of at least gH and gL. Despite the high prevalence and heavy disease burdens associated with human herpesviruses (HHVs), vaccines against these pathogens are still lacking, except for varicella zoster virus (VZV). Recent advances in understanding the coordinated mechanisms of action of the key EC glycoproteins and fusogen will help to improve approaches for effective vaccine development and neutralizing antibody (nAb) screening.

Fusogenic structural changes in arenavirus glycoproteins are associated with viroporin activity.

Many enveloped viruses enter host cells by fusing with acidic endosomes. The fusion activity of multiple viral envelope glycoproteins does not generally affect viral membrane permeability. However, fusion induced by the Lassa virus (LASV) glycoprotein complex (GPc) is always preceded by an increase in viral membrane permeability and the ensuing acidification of the virion interior. Here, systematic investigation of this LASV fusion phenotype using single pseudovirus tracking in live cells reveals that the change in membrane barrier function is associated with the fusogenic conformational reorganization of GPc. We show that a small-molecule fusion inhibitor or mutations that impair viral fusion by interfering with GPc refolding into the post-fusion structure prevent the increase in membrane permeability. We find that the increase in virion membrane permeability occurs early during endosomal maturation and is facilitated by virus-cell contact. This increase is observed using diverse arenavirus glycoproteins, whether presented on lentivirus-based pseudoviruses or arenavirus-like particles, and in multiple different cell types. Collectively, these results suggest that conformational changes in GPc triggered by low pH and cell factor binding are responsible for virion membrane permeabilization and acidification of the virion core prior to fusion. We propose that this viroporin-like activity may augment viral fusion and/or post-fusion steps of infection, including ribonucleoprotein release into the cytoplasm.

Impact fault detection for marine current turbines blade via MEGK-means and PCA under variable marine conditions

The advancement of the marine current turbine (MCT) technology has the potential to aid China in achieving its carbon peak and carbon neutrality goals. The performance of MCTs is influenced by fluctuating water velocities and erratic turbulence. The blades of MCTs are susceptible to cracking due to prolonged exposure to seawater. Unpredictable changes in marine currents contribute to unstable working environments. This study presents multiple envelope geometrical K-means to categorize stator current readings and develop fault detection models. It first constructs an envelope geometric feature matrix by using the extracted modulus signal. Then, the matrix is used to select the initial center for clustering, and a multi-scale principal component analysis is performed under each working condition to decrease data dimensionality. Finally, T 2 and squared prediction error (SPE) serve as the projection of the sample vector on the principal element space and the remaining subspace, respectively, and can be utilized to monitor fault cases. The experimental findings demonstrate that the proposed method has excellent recognition capabilities and detection accuracy for the impact faults of MCTs under variable working conditions.

Reconstruction of a polyclonal ADCC antibody repertoire from an HIV-1 non-transmitting mother

Human natural history and vaccine studies support a protective role of antibody dependent cellular cytotoxicity (ADCC) activity against many infectious diseases. One setting where this has consistently been observed is in HIV-1 vertical transmission, where passively acquired ADCC activity in HIV-exposed infants has correlated with reduced acquisition risk and reduced pathogenesis in HIV+ infants. However, the characteristics of HIV-specific antibodies comprising a maternal plasma ADCC response are not well understood. Here, we reconstructed monoclonal antibodies (mAbs) from memory B cells from late pregnancy in mother MG540, who did not transmit HIV to her infant despite several high-risk factors. Twenty mAbs representing 14 clonal families were reconstructed, which mediated ADCC and recognized multiple HIV Envelope epitopes. In experiments using Fc-defective variants, only combinations of several mAbs accounted for the majority of plasma ADCC of MG540 and her infant. We present these mAbs as evidence of a polyclonal repertoire with potent HIV-directed ADCC activity.

Envelope-Fusion-Syncytium Formation in Microplitis bicoloratus bracovirus Maturation.

The viral envelope is essential for virus maturation. Virus-mediated syncytium formations are induced by viral envelope proteins that cause membrane fusion of the infected cells. Polydnaviridae (Polydnavirus) are enveloped viruses with multiple nucleocapsids, and virions mature in symbiotic parasitoid wasp ovaries. However, the mechanism governing the envelope packaging of multiple nucleocapsids remains unclear. In this study, we used transmission electron microscopy to examine the process whereby multiple nucleocapsids of Microplitis bicoloratus bracovirus are packaged into an envelope and observed envelope-fusion-syncytium formation in symbiotic wasp calyx cells during virus maturation. The virus maturation process in calyx cells comprised four stages: pre-virogenic stroma, virogenic stroma, assembly, and fusion. Each virus contained a single envelope with one nucleocapsid in the assembly stage; multiple envelopes then fused to form a viral envelope with multiple nucleocapsids (i.e., the envelope-fusion-syncytium) around the envelope fusion core in the fusion stage. The envelope-fusion-syncytium then stabilized the virions that were released into the lumen of the ovary across the calyx epithelial layer. The phagocytic calyx epithelial cells on the border of the calyx and ovary lumen cleared the majority of non-enveloped nucleocapsids. In contrast, non-phagocytic calyx epithelial cells with microvilli and a cuticular line between the ovary wall and the lumen remained intact in the ovary lumen. These results indicate that envelope-fusion-syncytium formation is important for packaging multiple nucleocapsids in bracovirus maturation.

Investigation on Pathological Aspects, Mode of Transmission, and Tissue Tropism of Antheraea proylei Nucleopolyhedrovirus Infecting Oak Tasar Silkworm.

The temperate oak tasar silkworm, Antheraea proylei, is frequently infested with Antheraea proylei nucleopolyhedrovirus (AnprNPV) causing tiger band disease. This disease is one of the key factors that obstructs production and productivity of oak tasar sericulture. The current study aimed to investigate the pathogenicity of AnprNPV, its mode of transmission, and detection of AnprNPV in different tissues. Transmission electron micrographs of AnprNPV showed single rod-shaped bodies and occlusion derived virus (ODV) enclosed within multiple envelopes. The infecting AnprNPV displayed tissue tropism with higher copy numbers detected in the insect fat body and ovary. The virus was observed to multiply in all developmental stages of the silkworm such as egg, larva, pupa, and moth, confirming its ability to spread throughout the silkworm lifecycle. Baculovirus isolated from infected A. proylei showed cross-infectivity in other Saturniidae wild silkworm species such as Antheraea pernyi, A. frithi, and Samia ricini, widening their probable host range for infection. Baculoviruses generally display a horizontal mode of transmission, mainly through ingestion of occlusion bodies (OBs); however, the present study revealed a trans-ovum vertical mode of transmission in addition to a horizontal mode. The observations made in this study aid a detailed understanding of the tiger band disease and its causative pathogen AnprNPV, which will support future studies and disease management in oak tasar sericulture.

Bearing failure impulse enhancement method using multiple resonance band centre positioning and envelope integration

Due to the complex structure of electromechanical systems, impulse components with a transient duration will excite multi-resonance phenomena and generate diverse peak-like bands in the spectra of vibration signals. Resonance bands excited by bearing failure-induced impulses that characterise failure area and form, are confused with frequency components caused by impulsive interference and background noise. Given the imperceptibility of the failure features of on-machine bearings, a better identification strategy is to extract the information of multiple predominate resonance bands excited by failure, referred to as effective resonance frequency bands (ERFBs), and to then integrate them, instead of locating only the optimal resonance frequency band (ORFB). In this paper, a multiple resonance band centre positioning and envelope integration method is proposed to enhance the bearing failure features. To this end, a robust indicator, ratio of cyclic content (RCC), is utilised to construct an Improved-Protrugram to position the centres of ERFBs correctly. Then, a dichotomy is introduced to establish an iterative search algorithm for improving position efficiency. Further, the positioned sub-band components are filtered and demodulated to obtain the envelopes of failure-induced multiple resonances. Finally, a variable weighting envelop integration strategy is proposed to eliminate interference that does not coexist between different sub-band components. The effectiveness of the proposed method is verified using both simulated and experimental bearing failure signals. The results indicate that the proposed method is superior to ORFB location methods for on-machine bearing failure identification.

Phosphorylation of the Autographa californica multiple nucleopolyhedrovirus polyhedron envelope protein plays an important role in the formation of the occlusion body envelope.

During the life cycle of a baculovirus, a crystallized protein matrix, formed by polyhedrin (POLH), is produced. The protein matrix is surrounded by a multilayered protein/carbohydrate envelope, and matrix and envelope together form a mature occlusion body (OB). The polyhedron envelope plays an important role in resistance against adverse external environments. The polyhedron envelope protein (PEP) is the main protein that forms the polyhedron envelope, but the mechanism of formation of the polyhedron envelope is unclear. Here, through immunofluorescence localization observations, we found that PEP interacted with both POLH and P10 during formation of the polyhedron envelope in the late stages of infection, and PEP was also required for P10 incorporation on the surface of OBs. In this process, the phosphorylation of PEP played an important role. PEP was determined to be a phosphorylated protein using the Phos-tag technique, and PK1 was determined to be the phosphokinase of PEP by co-immunoprecipitation and in vitro phosphorylation. Immunofluorescence localization revealed that PEP was continuously phosphorylated by PK1 after PEP entered the nucleus until PEP was correctly packaged on the OB surface. Multi-point mutations of PEP conservative potential phosphorylation sites showed that the simultaneous mutation of S85, T86 and Y92 caused changes in the location of PEP and P10 in the late stages of infection, and resulted in an OB surface that lacked the polyhedron envelope. These data suggested that the phosphorylation of PEP at particular sites, i.e. S85, T86 and Y92, plays an important role in the formation of the polyhedron envelope.

On-Chip RF Signal Shaping by Coherent Control of Optically Driven Acoustic Wave Interference

Photonic–phononic systems that mix photons and acoustic waves have been actively investigated for the development of next-generation integrated photonics for signal processing. Here, we propose a new method of arbitrary RF signal shaping using active control of optically driven acoustic-wave interference, offering new photonic–phononic–microwave signal processing schemes with MHz linewidths at GHz carrier frequencies. The demonstrated system consists of two suspended optical waveguides and an asymmetric Mach–Zehnder interferometer. The two waveguides generate acoustic waves at a 3.03 GHz carrier frequency with a 3.3 MHz bandwidth, and one of the paths of the asymmetric interferometer experiences phase modulation induced by the acoustic waves. Then, the interferometer converts the phase information to RF signals of complicated shapes using a linear combination of multiple optical envelopes. This RF signal-shaping method will open new applications of optomechanical systems for microwave photonics.

Gaseous NO2 induces various envelope alterations in Pseudomonas fluorescens MFAF76a

Anthropogenic atmospheric pollution and immune response regularly expose bacteria to toxic nitrogen oxides such as NO• and NO2. These reactive molecules can damage a wide variety of biomolecules such as DNA, proteins and lipids. Several components of the bacterial envelope are susceptible to be damaged by reactive nitrogen species. Furthermore, the hydrophobic core of the membranes favors the reactivity of nitrogen oxides with other molecules, making membranes an important factor in the chemistry of nitrosative stress. Since bacteria are often exposed to endogenous or exogenous nitrogen oxides, they have acquired protection mechanisms against the deleterious effects of these molecules. By exposing bacteria to gaseous NO2, this work aims to analyze the physiological effects of NO2 on the cell envelope of the airborne bacterium Pseudomonas fluorescens MFAF76a and its potential adaptive responses. Electron microscopy showed that exposure to NO2 leads to morphological alterations of the cell envelope. Furthermore, the proteomic profiling data revealed that these cell envelope alterations might be partly explained by modifications of the synthesis pathways of multiple cell envelope components, such as peptidoglycan, lipid A, and phospholipids. Together these results provide important insights into the potential adaptive responses to NO2 exposure in P. fluorescens MFAF76a needing further investigations.

Computational design of immunogenic peptide constructs comprising multiple human leukocyte antigen restricted dengue virus envelope epitopes.

Dengue virus (DENV) is endemic in 100 countries with the ability to impact nearly 50% of world population. DENV envelope (E) protein is responsible for viral attachment to host cells and has been target of various countermeasure development efforts. The current study focuses on a consensus computational approach to identify cross-reactive, immunogenic DENV-2 E peptides displaying promiscuity with a wide array of human leukocyte antigen (HLA) molecules. Four conserved peptides (FP-1, FP-2, FP-3 and FP-4) containing multiple CD8+ and CD4+ T cell epitopes were identified by employment of various immunoinformatics tools. FP-1, FP-2, FP-3 and FP-4 were estimated to bind with 227, 1787, 1008 and 834 HLA alleles, respectively. Root mean square deviation (RMSD) values obtained by molecular docking (CABS-Dock) with 20 HLA alleles (10 each of HLA classes I and II) resulted into comparable RMSD values of identified epitopes with native peptides, which represents the natural presentation of epitopes to HLA molecules. These peptides were also found to be part of previous experimentally validated immunogenic peptides. Further, a dengue immunogenic peptide construct was generated by linking the four peptides, an adjuvant and a 6× histidine tag. The construct showed strong binding and stability with Toll-like receptor. Collectively, these results provide strong evidence in the support of the immunogenic potential of the dengue immunogenic peptide construct.

Luciferase Immunosorbent Assay Based on Multiple E Antigens for the Detection of Chikungunya Virus-Specific IgG Antibodies.

ABSTRACTIn recent years, the chikungunya virus (CHIKV) has continued to spread from local epidemics to nonnative habitats until eventually reaching pandemic status. Nonendemic areas such as China have also emerged as potential epidemic areas of CHIKV. Serological detection of CHIKV is the key to diagnosing and controlling the prevalence of this virus. In this study, we review the progress of the serological detection of the envelope (E) protein in CHIKV, and we provide a novel research assay and ideas for the serological detection of CHIKV. The luciferase immunosorbent assay (LISA) does not require species-specific labeled secondary antibodies for detection, making it universally suitable for tracking samples from various animals or carriers. At present, most research on CHIKV antigen detection technology tends to combine two or more proteins to avoid the decrease in detection ability caused by antigen mutation. Our results indicate that two or more kinds of CHIKV E antigens combined with LISA detection can improve the detection rate of anti-CHIKV immunoglobulin G (IgG) antibodies in CHIKV-infected patient sera and detect antibodies in the early stage of infection accurately and sensitively. After 235 days of infection, the anti-CHIKV IgG antibodies could still be detected in CHIKV-infected patients. All serum samples were tested with a detection rate of 100% after combining various recombinant CHIKV E antigens. Our proposed CHIKV-specific LISA could be a useful tool for serum diagnosis of CHIKV infection and serum epidemic research in areas where CHIKV is endemic, which would help to manage potential epidemics in the future.IMPORTANCE At present, chikungunya virus (CHIKV) is still circulating in some parts of the world, and mutated strains have emerged, making it easier for the virus to spread among humans. With the continuous variation of CHIKV, its antigen variation leads to the decline of detection ability. In addition, the risk of transmission of CHIKV in areas where CHIKV is not endemic, such as China, has increased dramatically, which compels us to enhance the detection capacity of CHIKV and continuously monitor CHIKV antibody levels in the population. Real-time quantitative PCR (RT-PCR) detection technology will not be reliable when the infection time is chronic or in subclinical infection due to decreases in virus concentration, and an antibody detection technology must be adopted. In this study, multiple CHIKV envelope (E) antigens were used to detect anti-CHIKV IgG antibodies in serum for the first time. The new assay is characterized by convenient operation, high detection rate, and high sensitivity and has significance for early warning and monitoring. Moreover, it contributes to the prevention and control of CHIKV.

Epstein-Barr Virus Load Correlates with Multiple Sclerosis-Associated Retrovirus Envelope Expression.

pHERV-W ENV and syncytin-1, the envelope proteins of the human endogenous retrovirus W family (HERV-W), have been proposed as etiological factors for MS development. In addition, herpesviruses, such as the Epstein-Barr virus (EBV) and the human herpesvirus 6A/B (HHV-6A/B), have been also strongly associated with the disease. This work aims to study the possible link between viral loads and antibody titers against EBV and HHV-6A/B and the pHERV-W ENV/syncytin-1 protein/gene expression. For this purpose, we conducted a 12-month longitudinal study involving 98 RRMS patients. Peripheral blood samples were obtained from each patient. Serum antibody titers against EBV and HHV-6A/B were determined by ELISA, while viral loads were analyzed using qPCR. HLA MS-related alleles were also genotyped. pHERV-W ENV/syncytin-1 protein and gene expression levels in immune cells were assessed by flow cytometry and qPCR, respectively. We found that the 12-month variation of the pHERV-W ENV gene expression levels positively correlated with the variation of the EBV viral load, especially in those patients with high baseline EBV loads. Therefore, these results could support previous studies pointing to the transactivation of pHERV-W ENV by EBV. However, further studies are needed to better understand this possible relationship.

A pilot integrated qualification program to improve medication use in a long-term care facility: a hybrid effectiveness-implementation study

Objective: to describe the implementation and to assess the effectiveness of a pilot integrated qualification program to improve the medication use in a long-term care facility (LTCF). Methods: This was a type 1 hybrid effectiveness-implementation study. A pilot integrated qualification program to improve the medication use in a LTCF was carried out by implementing a new drug distribution system and a comprehensive medication management (CMM) service according to the following four steps: I) implementation of the drug distribution system followed by the evaluation of the health team’s opinion; II) prescription review with the identification of potential drug therapy problems (PDTPs); III) provision of the CMM service according to the framework of Pharmaceutical Care practice within one year; and, IV) evaluation of the effectiveness of the program through the comparison of clinical and laboratory parameters (blood pressure, glycated hemoglobin and lipid fractions) using the t-test or Wilcoxon signed-rank test. Results: In step I, the distribution system was fully outsourced to a company that furnished all solid oral dosage forms in individual boxes containing a plastic coil with multiple envelopes for 30 days. In step II, 180 PDTPs were identified, and all patients presented with at least one of them. In step III, after the first assessment of the CMM Service, 43 actual drug therapy problems (DTPs) were identified. After one year of service provision, 96 DTPs were identified and 75.8% of them were resolved (n=72). In step IV, a statistically significant difference was observed between the initial and final minimum and maximum systolic and diastolic blood pressure (p<0,05). Conclusions: The pilot integrated qualification program had a positive impact on the clinical parameters. The global population is rapidly aging, making this type of study important to exemplify a multifaceted strategy to improve the quality of drug therapy for institutionalized patients.

In vivo assessment of pulmonary fibrosis and edema in rodents using the backscatter coefficient and envelope statistics.

Quantitative ultrasound methods based on the backscatter coefficient (BSC) and envelope statistics have been used to quantify disease in a wide variety of tissues, such as prostate, lymph nodes, breast, and thyroid. However, to date, these methods have not been investigated in the lung. In this study, lung properties were quantified by BSC and envelope statistical parameters in normal, fibrotic, and edematous rat lungs in vivo. The average and standard deviation of each parameter were calculated for each lung as well as the evolution of each parameter with acoustic propagation time within the lung. The transport mean free path and backscattered frequency shift, two parameters that have been successfully used to assess pulmonary fibrosis and edema in prior work, were evaluated in combination with the BSC and envelope statistical parameters. Multiple BSC and envelope statistical parameters were found to provide contrast between control and diseased lungs. BSC and envelope statistical parameters were also significantly correlated with fibrosis severity using the modified Ashcroft fibrosis score as the histological gold standard. These results demonstrate the potential for BSC and envelope statistical parameters to improve the diagnosis of pulmonary fibrosis and edema as well as monitor pulmonary fibrosis.

Development of a platform-based approach for the clinical production of HIV gp120 envelope glycoprotein vaccine candidates

Preclinical development of vaccine candidates is an important link between the discovery and manufacture of vaccines for use in human clinical trials. Here, an exploratory clinical study utilizing multiple gp120 envelope proteins as vaccine antigens was pursued, which required a harmonized platform development approach for timely and efficient manufacture of the combined HIV vaccine product.Development of cell lines, processes, and analytical methods was initiated with a transmitted founder envelope protein (CH505TF), then applied to produce three subsequent gp120 Env (envelope) variants. Cell lines were developed using the commercially available Freedom CHO DG44 kit (Life Technologies). The fed-batch cell culture production process was based on a commercially-available medium with harmonized process parameters across the variants. A platform purification process was developed utilizing a mixed mode chromatography capture step, with ceramic hydroxyapatite and ion exchange polishing steps. A suite of analytical methods was developed to establish and monitor the Quality Target Profile (QTP), release and long-term stability testing of the vaccine products.The platform development strategy was successfully implemented to produce four gp120 envelope protein variants. In some cases, minor changes to the platform were required to optimize for a particular variant; however, baseline conditions for the processes (cell line type, media & feed system, chromatography resins, and analytical approaches) remained constant, leading to successful transfer and manufacture of all four proteins in a cGMP facility.This body of work demonstrates successful pursuit of a platform development approach to manufacture important vaccine candidates and can be used as a model for other vaccine glycoproteins, such as HIV gp140 trimers or other viral glycoproteins with global health implications.Clinical trial identifier.NCT03220724, NCT03856996.

Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope

A unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.