Characterization of Host-Cell Line Specific Glycosylation Profiles of Early Transmitted/Founder HIV-1 gp120 Envelope Proteins

Glycosylation plays an essential role in regulating protein function by modulating biological, structural, and therapeutic properties. However, due to its inherent heterogeneity and diversity, the comprehensive analysis of protein glycosylation remains a challenge. As part of our continuing effort in the analysis of glycosylation profiles of recombinant HIV-1 envelope-based immunogens, we evaluated and compared the host-cell specific glycosylation pattern of recombinant HIV-1 surface glycoprotein, gp120, derived from clade C transmitted/founder virus 1086.C expressed in Chinese hamster ovary (CHO) and human embryonic kidney containing T antigen (293T) cell lines. We used an integrated glycopeptide-based mass mapping workflow that includes a partial deglycosylation step described in our previous study1 with the inclusion of the fragmentation technique, electron transfer dissociation (ETD), to complement collision induced dissociation (CID). The inclusion of ETD facilitated the analysis by providing additional validation for glycopeptide identification and expanding the identified glycopeptides to include coverage of O-linked glycosylation. The site-specific glycosylation analysis shows that the transmitted/founder 1086.C gp120 expressed in CHO and 293T displayed distinct similarities and differences. For N-linked glycosylation, two sites (N386 and N392), in the V4 region were populated with high mannose glycans in the CHO cell-derived 1086.C gp120, while these sites had a mixture of high mannose and processed glycans in the 293T cell-derived 1086.C gp120. Compositional analysis of O-linked glycans revealed that 293T cell-derived 1086.C gp120 consisted of cores 1, 2, and 4 type O-linked glycans while CHO cell-derived 1086.C exclusively consisted of core 1 type O-linked glycans. Overall, glycosylation site occupancy of the CHO and 293T cell-derived 1086.C gp120 show high degree of similarity except for one site at N88 in the C1 region. This site was partially occupied in 293T-gp120 but fully occupied in CHO-gp120. Site-specific glycopeptide analysis of transmitted/founder 1086.C gp120 expressed in CHO cells revealed the presence of phosphorylated glycans while 293T cell produced 1086.C gp120 glycans were not phosphorylated. While the influence of phosphorylated glycans on immunogenicity is unclear, distinguishing host-cell specific variations in glycosylation profiles provides insights into the similarity (or difference) in recombinant vaccine products. While these differences had minimal effect on envelope antigenicity, they may be important in considering immunogenicity and functional capacities of recombinant envelope proteins produced in different expression systems

Glycosylation Site-Specific Analysis of Clade C HIV-1 Envelope Proteins

The extensive glycosylation of HIV-1 envelope proteins (Env), gp120/gp41, is known to play an important role in evasion of host immune response by masking key neutralization epitopes and presenting the Env glycosylation as “self” to the host immune system. The Env glycosylation is mostly conserved but continues to evolve to modulate viral infectivity. Thus, profiling Env glycosylation and distinguishing interclade and intraclade glycosylation variations are necessary components in unraveling the effects of glycosylation on Env’s immunogenicity. Here, we describe a mass spectrometry-based approach to characterize the glycosylation profiles of two rVV-expressed clade C Envs by identifying the glycan motifs on each glycosylation site and determining the degree of glycosylation site occupancy. One Env is a wild-type Env, while the other is a synthetic “consensus” sequence (C.CON). The observed differences in the glycosylation profiles between the two clade C Envs show that C.CON has more unutilized sites and high levels of high mannose glycans; these features mimic the glycosylation profile of a Group M consensus immunogen, CONS. Our results also reveal a clade-specific glycosylation pattern. Discerning interclade and intraclade glycosylation variations could provide valuable information in understanding the molecular differences among the different HIV-1 clades and in designing new Env-based immunogens

Environmental Factors Influencing Epidemic Cholera

Cholera outbreak following the earthquake of 2010 in Haiti has reaffirmed that the disease is a major public health threat. Vibrio cholerae is autochthonous to aquatic environment, hence, it cannot be eradicated but hydroclimatology-based prediction and prevention is an achievable goal. Using data from the 1800s, we describe uniqueness in seasonality and mechanism of occurrence of cholera in the epidemic regions of Asia and Latin America. Epidemic regions are located near regional rivers and are characterized by sporadic outbreaks, which are likely to be initiated during episodes of prevailing warm air temperature with low river flows, creating favorable environmental conditions for growth of cholera bacteria. Heavy rainfall, through inundation or breakdown of sanitary infrastructure, accelerates interaction between contaminated water and human activities, resulting in an epidemic. This causal mechanism is markedly different from endemic cholera where tidal intrusion of seawater carrying bacteria from estuary to inland regions, results in outbreaks

P20-08. Glycosylation: an important factor in Env diversity

Supported by a CAVD Grant from the Bill and Melinda Gates Foundation

Uptake and clearance analysis of Technetium(99m) labelled iron oxide nanoparticles in a rabbit brain

Nanoparticles as solid colloidal particles are extensively studied and used as anticancer drug delivery agents because of their physical properties. This current research aims to prepare water base suspension of uncoated iron oxide nanoparticles and their biodistribution study to different organs, especially the brain, by using a single photon emission computed tomography gamma camera. The water-based suspension of iron oxide nanoparticles was synthesised by a reformed version of the co-precipitation method and labelled with Tc99m for intravenous injection. The nanoparticles were injected without surface modification. X-ray diffraction (XRD), energy dispersive spectrometry (EDS) and transmission electron microscope (TEM) techniques were used for characterisation. Peaks of XRD and EDS indicate that the particles are magnetite and exist in aqueous suspension. The average diameter of iron oxide nanoparticles without any surface coating determined by TEM is 10 nm. These particles are capable of evading the reticuloendothelial system and can cross the blood-brain barrier in the rabbit. The labelling efficiency of iron oxide nanoparticles labelled with Tc99m is 85%, which is good for the biodistribution study. The sufficient amount of iron oxide nanoparticles concentration in the brain as compared with the surrounding soft tissues and their long blood retention time indicates that the water-based suspension of iron oxide nanoparticles may be an option for drug delivery into the brain

TitrationAnalysis: a tool for high throughput binding kinetics data analysis for multiple label-free platforms [version 1; peer review: 3 approved]

Label-free techniques including Surface Plasmon Resonance (SPR) and Biolayer Interferometry (BLI) are biophysical tools widely used to collect binding kinetics data of bimolecular interactions. To efficiently analyze SPR and BLI binding kinetics data, we have built a new high throughput analysis tool named the TitrationAnalysis. It can be used as a package in the Mathematica scripting environment and ultilize the non-linear curve-fitting module of Mathematica for its core function. This tool can fit the binding time course data and estimate association and dissociation rate constants (ka and kd respectively) for determining apparent dissociation constant (KD) values. The high throughput fitting process is automatic, requires minimal knowledge on Mathematica scripting and can be applied to data from multiple label-free platforms. We demonstrate that the TitrationAnalysis is optimal to analyze antibody-antigen binding data acquired on Biacore T200 (SPR), Carterra LSA (SPR imaging) and ForteBio Octet Red384 (BLI) platforms. The ka, kd and KD values derived using TitrationAnalysis very closely matched the results from the commercial analysis software provided specifically for these instruments. Additionally, the TitrationAnalysis tool generates user-directed customizable results output that can be readily used in downstream Data Quality Control associated with Good Clinical Laboratory Practice operations. With the versatility in source of data input source and options of analysis result output, the TitrationAnalysis high throughput analysis tool offers investigators a powerful alternative in biomolecular interaction characterization

A computationally efficient symmetric diagonally dominant matrix projection-based Gaussian process approach

Although kernel approximation methods have been widely applied to mitigate the O(n3) cost of the n × n kernel matrix inverse in Gaussian process methods, they still face computational challenges. The ‘residual’ matrix between the covariance and the approximating component is often discarded as it prevents the computational cost reduction. In this paper, we propose a computationally efficient Gaussian process approach that achieves better computational efficiency, O(mn2), compared with standard Gaussian process methods, when using m n data. The proposed approach incorporates the ‘residual’ matrix in its symmetric diagonally dominant form which can be further approximated by the Neumann series. We have validated and compared the approach with full Gaussian process approaches and kernel approximation based Gaussian process variants, both on synthetic and real air quality data

Microsecond dynamics control the HIV-1 Envelope conformation

The HIV-1 Envelope (Env) glycoprotein facilitates host cell fusion through a complex series of receptor-induced structural changes. Although remarkable progress has been made in understanding the structures of various Env conformations, microsecond timescale dynamics have not been studied experimentally. Here, we used time-resolved, temperature-jump small-angle x-ray scattering to monitor structural rearrangements in an HIV-1 Env SOSIP ectodomain construct with microsecond precision. In two distinct Env variants, we detected a transition that correlated with known Env structure rearrangements with a time constant in the hundreds of microseconds range. A previously unknown structural transition was also observed, which occurred with a time constant below 10 μs, and involved an order-to-disorder transition in the trimer apex. Using this information, we engineered an Env SOSIP construct that locks the trimer in the prefusion closed state by connecting adjacent protomers via disulfides. Our findings show that the microsecond timescale structural dynamics play an essential role in controlling the Env conformation with impacts on vaccine design

Analyses of structural and electrical properties of aluminium doped ZnO-NPs by experimental and mathematical approaches

Pure and aluminium doped ZnO-NPs were played the central role in every field of life due to extraordi-nary physical, chemical and electrical properties. The main objective of the present research was used to enhance the electrical conductivity and reduce the electrical resistivity of aluminium doped zinc oxide-NPs. Synthesis of pure and aluminium doped zinc oxide-NPs (Zn1-xAlxO) at x = 0, 2.5, 5, 7.5 and 10 wt% was carried out by co-precipitation method. The XRD results depicted that hexagonal wurtzite crystal structure and crystallite size in the range of 13-25 nm were calculated by using Debye-Scherrer's equa-tion. Likewise, the non-uniform, irregular and pore like surface morphology of the prepared NPs was evi-dent from SEM micrographs. Various functional groups (CH, CO, OH and ZnO) attached to the surface of aluminium doped zinc oxide-NPs were identified by FTIR analysis. The UV-VIS spectra also depicted a shift towards the blue region of the visible spectrum. In terms of electrical properties with the help of experimental and mathematical analyses of aluminum doped zinc oxide-NPs exhibited higher conductiv-ity (1.34 x 10(-6) to 1.43 x 10(-3) S/cm) and lower resistivity (5.46 x 10(5) to 6.99 x 10(2) Omega-cm). The present results suggest that the aluminum doped zinc oxide-NPs have been improved the structural and electrical properties which make it a good candidate for optoelectronic devices. (c) 2021 The Authors. Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).</p

Developmental Pathway of the MPER-Directed HIV-1-Neutralizing Antibody 10E8

Antibody 10E8 targets the membrane-proximal external region (MPER) of HIV-1 gp41, neutralizes >97% of HIV-1 isolates, and lacks the auto-reactivity often associated with MPER-directed antibodies. The developmental pathway of 10E8 might therefore serve as a promising template for vaccine design, but samples from time-of-infection—often used to infer the B cell record—are unavailable. In this study, we used crystallography, next-generation sequencing (NGS), and functional assessments to infer the 10E8 developmental pathway from a single time point. Mutational analysis indicated somatic hypermutation of the 2nd-heavy chain-complementarity determining region (CDR H2) to be critical for neutralization, and structures of 10E8 variants with V-gene regions reverted to genomic origin for heavy-and-light chains or heavy chain-only showed structural differences >2 Å relative to mature 10E8 in the CDR H2 and H3. To understand these developmental changes, we used bioinformatic sieving, maximum likelihood, and parsimony analyses of immunoglobulin transcripts to identify 10E8-lineage members, to infer the 10E8-unmutated common ancestor (UCA), and to calculate 10E8-developmental intermediates. We were assisted in this analysis by the preservation of a critical D-gene segment, which was unmutated in most 10E8-lineage sequences. UCA and early intermediates weakly bound a 26-residue-MPER peptide, whereas HIV-1 neutralization and epitope recognition in liposomes were only observed with late intermediates. Antibody 10E8 thus develops from a UCA with weak MPER affinity and substantial differences in CDR H2 and H3 from the mature 10E8; only after extensive somatic hypermutation do 10E8-lineage members gain recognition in the context of membrane and HIV-1 neutralization