Analytical Characterization of Heterogeneities in mRNA-Lipid Nanoparticles Using Sucrose Density Gradient Ultracentrifugation.

Rational design and robust formulation processes are critical for optimal delivery of mRNA by lipid nanoparticles (LNPs). Varying degrees of heterogeneity in mRNA-LNPs can affect their biophysical and functional properties. Given the profound complexity of mRNA-LNPs, it is critical to develop comprehensive and orthogonal analytical techniques for a better understanding of these formulations. To this end, we developed a robust ultracentrifugation method for density-based separation of subpopulations of mRNA-LNPs. Four LNP formulations encapsulating human erythropoietin (hEPO) with varying functionalities were synthesized using two ionizable lipids, A and B, and two helper lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), along with cholesterol and DMG-PEG-2K. Upon ultracentrifugation on a sucrose gradient, a distinct pattern of "fractions" was observed across the gradient, from the less dense topmost fraction to the increasingly denser bottom fractions, which were harvested for comprehensive analyses. Parent LNPs, A-DOPE and B-DOPE, were resolved into three density-based fractions, each differing significantly in the hEPO expression following intravenous and intramuscular routes of administration. Parent B-DEPE LNPs resolved into two density-based fractions, with most of the payload and lipid content being attributed to the topmost fraction compared to the lower one, indicating some degree of heterogeneity, while parent A-DEPE LNPs showed remarkable homogeneity, as indicated by comparable in vivo potency, lipid numbers, and particle count among the three density-based fractions. This study is the first to demonstrate the application of density gradient-based ultracentrifugation (DGC) for a head-to-head comparison of heterogeneity as a function of biological performance and biophysical characteristics of parent mRNA-LNPs and their subpopulations.

Analysis of highly frequent point mutations in glycoprotein C, glycoprotein N, and nucleoprotein of CCHFV.

Crimean-Congo hemorrhagic fever virus (CCHFV) is classified among top 10 priority pathogens by World Health Organization. CCHFV belongs to Bunyaviridae family and negative sense ssRNA genome composed of three RNA segments: L, M, and S. RNA viruses show higher mutation rate as compared to DNA viruses. To gain deeper understanding of impact of point mutations in CCHFV M and S segment, mutation profiling, homology modeling, and molecular dynamic (MD) simulation were performed. Structural glycoproteins (glycoprotein C [Gc] and glycoprotein N [Gn]) of CCHFV are important for host-virus interaction and genome packaging, whereas CCHFV nucleoprotein (NP) is crucial for viral replication. Hence, current study is focused on evaluation of eight mutations in structural glycoproteins (Gc: 7 and Gn: 1) of M segment and seven mutations in NP of S segment. All these mutations were highly frequent, with mutation frequency between 0.81 and 1.0 and found to be persistent in the recent strains of CCHFV. Solubility analysis predicted that selected point mutations reduce solubility of Gc protein and increase solubility of Gn and NP proteins. MD simulation study deciphered that A1046V and G1158E in Gc protein, I778T in Gn protein, and H195R in NP protein displayed large deviation and fluctuation, and affected intramolecular interactions. In conclusion, we observed that point mutations could impact structure, stability, and host-virus interaction of protein, and might lead to evolution of new strains for better survival and drug resistance.

An Innovative Approach for Biocontrol of Meloidogyne incognita in Ginger Using Potential Bacteria Isolated from Indian Himalayas.

The prevalence of Meloidogyne incognita, a severe root-knot nematode, is alarmingly high in the production of ginger-a main cash crop of Himachal Pradesh, a Himalayan state of India. In order to control this through natural means, the nematicidal potential of plant growth-promoting rhizobacteria (PGPR) against M. incognita was analyzed. This is an effective alternative solution to manage nematode incidence as compared to hazardous chemicals under protected and field cultivation of ginger. In the present study an attempt has been made to isolate, characterize, and identify potential rhizobacteria associated with ginger rhizosphere and endosphere. In total, 169 bacterial isolates were isolated from ginger (Zingiber officinale) rhizosphere and endosphere of 4 different sites of Sirmaur district, screened out for multifarious PGP traits showing positive results. The combined cluster analysis and 16S rRNA genotypic analysis of selected bacterial isolates revealed that Serratia marcescens FS-23, Pseudochrobacter sp. GS-15, Stonotrophomonas pavanii HER-9, Pseudomonas brassicacearum HER-20 and Serratia marcescens IS-2 exhibited highest PGP traits. All tested bacterial isolates were capable of exerting a significant effect on mortality of juvenile M. incognita ranging upto 40-90% in laboratory experiments. Further a consortium of these screened isolates showed 86.67% reduction in gall formation by M. incognita in lab conditions. The remarkable increase to 93.24% with 138.73 q/ha with application of charcoal based bio-formulation of consortium without adding any chemical fertilizer was observed in field trials of Nohradhar of Sirmaur district. An alternative choice as a biocontrol agent as well as for PGP activities, the novel and most robust isolate Serratia marcescens IS-2 had revealed to have a variety of bioactive metabolic products with abilities against nematodes, bacteria, and fungi.

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.

Inhaled delivery of a lipid nanoparticle encapsulated messenger RNA encoding a ciliary protein for the treatment of primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a respiratory disease caused by dysfunction of the cilia with currently no approved treatments. This predominantly autosomal recessive disease is caused by mutations in any one of over 50 genes involved in cilia function; DNAI1 is one of the more frequently mutated genes, accounting for approximately 5-10% of diagnosed PCD cases. A codon-optimized mRNA encoding DNAI1 and encapsulated in a lipid nanoparticle (LNP) was administered to mice via aerosolized inhalation resulting in the expression human DNAI1 in the multiciliated cells of the pseudostratified columnar epithelia. The spatial localization of DNAI1 expression in the bronchioles indicate that delivery of the DNAI1 mRNA transpires the lower airways. In a PCD disease model, exposure to the LNP-encapsulated DNAI1 mRNA resulted in increased ciliary beat frequency using high speed videomicroscopy showing the potential for an mRNA therapeutic to correct cilia function in patients with PCD due to DNAI1 mutations.

Long Term Immune Response Produced by the SputnikV Vaccine.

SputnikV is a vaccine against SARS-CoV-2 developed by the Gamaleya National Research Centre for Epidemiology and Microbiology. The vaccine has been shown to induce both humoral and cellular immune responses, yet the mechanisms remain largely unknown. Forty SputnikV vaccinated individuals were included in this study which aimed to demonstrate the location of immunogenic domains of the SARS-CoV-2 S protein using an overlapping peptide library. Additionally, cytokines in the serum of vaccinated and convalescent COVID-19 patients were analyzed. We have found antibodies from both vaccinated and convalescent sera bind to immunogenic regions located in multiple domains of SARS-CoV-2 S protein, including Receptor Binding Domain (RBD), N-terminal Domain (NTD), Fusion Protein (FP) and Heptad Repeats (HRs). Interestingly, many peptides were recognized by immunized and convalescent serum antibodies and correspond to conserved regions in circulating variants of SARS-CoV-2. This breadth of reactivity was still evident 90 days after the first dose of the vaccine, showing that the vaccine has induced a prolonged response. As evidenced by the activation of T cells, cellular immunity strongly suggests the high potency of the SputnikV vaccine against SARS-CoV-2 infection.

"Smart" Nanoparticles Enhance the Cytoplasmic Delivery of Anti-RhoC Silencing RNA and Inhibit the Migration and Invasion of Aggressive Breast Cancer Cells.

Rho-GTPases are small GTP-binding proteins that contribute to the epithelial-to-mesenchymal transition by regulating several cellular processes including organization of the actin cytoskeleton, cell motility, transcription, and cell proliferation. Overexpression of RhoC-GTPases (RhoC) in breast cancer has been implicated in poor disease prognosis due to increased cancer cells invasion, migration, and motility, which warranted its consideration as a therapeutic target for inhibiting breast cancer metastasis. Using silencing RNA (siRNA) molecules to knockdown RhoC expression is a promising approach to inhibit breast cancer metastases. However, transforming anti-RhoC siRNA molecules into a viable therapy remains a challenge due to the lack of a biocompatible carrier that can selectively deliver the RNA cargo into breast cancer cells. We report the use of a degradable, pH-sensitive, ß-cyclodextrin (ßCD)-based polymeric carrier that condenses anti-RhoC siRNA forming "smart" particles. These smart anti-RhoC particles were efficiently internalized, successfully escaped the endosome, and delivered the RNA cargo into the cytoplasm of SUM149 and MDA-MB-231 breast cancer cells. Our results show that anti-RhoC particles used at a low N/P ratio of 2.5/1 suppressed RhoC protein levels by 100% and 90% in SUM149 and MDA-MB-231 cells, respectively. Further, anti-RhoC particles inhibited the invasion, motility, and migration of SUM149 and MDA-MB-231 cells by 40-47%, 57-60%, and 61.5-73%, respectively. Smart particles encapsulating the scrambled siRNA sequence did not affect RhoC protein expression or the invasion, motility, and migration of SUM149 and MDA-MB-231 cells, which indicate the biocompatibility of the polymeric carrier and selectivity of the observed RhoC knockdown. These results collectively indicate the therapeutic potential of smart anti-RhoC particles in arresting the metastatic spread of breast cancer cells.

A novel GO hoisted SnO2-BiOBr bifunctional catalyst for the remediation of organic dyes under illumination by visible light and electrocatalytic water splitting.

It is imperative to develop affordable multi-functional catalysts based on transition metals for various applications, such as dye degradation or the production of green energy. For the first time, we propose a simple chemical bath method to create a SnO2-BiOBr-rGO heterojunction with remarkable photocatalytic and electrocatalytic activities. After introducing graphene oxide (GO) into the SnO2-BiOBr nanocomposite, the charge separation, electron mobility, surface area, and electrochemical properties were significantly improved. The X-ray diffraction results show the successful integration of GO into the SnO2-BiOBr nanocomposite. Systematic material characterization by scanning and transmission electron microscopy showed that the photocatalysts are composed of uniformly distributed SnO2 nanoparticles (∼11 nm) on the regular nanosheets of BiOBr (∼94 nm) and rGO. The SnO2-BiOBr-rGO photocatalyst has outstanding photocatalytic activity when it comes to reducing a variety of organic dyes like rhodamine B (RhB) and methylene blue (MB). Within 90 minutes of visible light illumination, degradation of a maximum of 99% for MB and 99.8% for RhB was noted. The oxygen evolution reaction (OER) and hydrogen evolution reaction (HER) performance was also tested for the ternary nanocomposite, and significantly lower overpotential values of 0.34 and -0.11 V (vs. RHE) at 10 mA cm-2 were observed for the OER and HER, respectively. Furthermore, the Tafel slope values are 34 and 39 mV dec-1 for the OER and HER, respectively. The catalytic degradation of dyes with visible light and efficient OER and HER performance offer this work a broad spectrum of potential applications.

Mutational analysis of catalytic site domain of CCHFV L RNA segment.

INTRODUCTION: Crimean-Congo haemorrhagic fever virus (CCHFV) has tripartite RNA genome and is endemic in various countries of Asia, Africa and Europe. METHOD: The present study is focused on mutation profiling of CCHFV L segment and phylogenetic clustering of protein dataset into six CCHFV genotypes. RESULTS: Phylogenetic tree rooted with NCBI reference sequence (YP_325663.1) indicated less divergence from genotype III and the sequences belonging to same genotypes have shown less divergence among each other. Mutation frequency at 729 mutated positions was calculated and 563, 49, 33, 46 and 38 amino acid positions were found to be mutated at mutation frequency intervals of 0-0.2, 0.21-0.4, 0.41-0.6, 0.61-0.8 and 0.81-1.0 respectively. Thirty-eight highly frequent mutations (0.81-1.0 interval) were found in all genotypes and mapping in L segment (encoded for RdRp) revealed four mutations (V2074I, I2134T/A, V2148A and Q2695H/R) in catalytic site domain and no mutation in OTU domain. Molecular dynamic simulation and in silico analysis showed that catalytic site domain displayed large deviation and fluctuation upon introduction of these point mutations. CONCLUSION: Overall study provides strong evidence that OTU domain is highly conserved and less prone to mutation whereas point mutations recorded in catalytic domain have affected the stability of protein and were found to be persistent in the large population.

Solvent-Free Synthesis of S,N-Doped Carbon Dots for Extended Visible-Light-Induced Oxidase-Mimicking Activities and Antimicrobial Applications.

Photo-oxidase nanozymes are emerging enzyme-mimicking materials that produce reactive oxygen species (ROS) upon light illumination and subsequently catalyze the oxidation of the substrate. Carbon dots are promising photo-oxidase nanozymes due to their biocompatibility and straightforward synthesis. Carbon dot-based photo-oxidase nanozymes become active for ROS generation under UV or blue light illumination. In this work, sulfur and nitrogen doped carbon dots (S,N-CDs) were synthesized by solvent-free, microwave assisted technique. We demonstrated that sulfur, nitrogen doping of carbon dots (band gap of 2.11 eV) has enabled photo-oxidation of 3,3,5,5'-tetramethylbenzidine (TMB) with extended visible light (up to 525 nm) excitation at pH 4. The photo-oxidase activities by S,N-CDs produce Michaelis-Menten constant (Km ) of 1.18 mM and the maximum initial velocity (Vmax ) as 4.66×10-8  Ms-1 , under 525 nm illumination. Furthermore, visible light illumination can also induce bactericidal activities with growth inhibition of Escherichia coli (E. coli). These results demonstrate that S,N-CDs can increase intracellular ROS in the presence of LED light illumination.

Identification of conserved immunogenic peptides of SARS-CoV-2 nucleocapsid protein.

The emergence of the new SARS-CoV-2 variants has led to major concern regarding the efficacy of approved vaccines. Nucleocapsid is a conserved structural protein essential for replication of the virus. This study focuses on identifying conserved epitopes on the nucleocapsid (N) protein of SARS-CoV-2. Using 510 unique amino acid sequences of SARS-CoV-2 N protein, two peptides (193 and 215 aa) with 90% conservancy were selected for T cell epitope prediction. Three immunogenic peptides containing multiple T cell epitopes were identified which were devoid of autoimmune and allergic immune response. These peptides were also conserved (100%) in recent Omicron variants reported in Jan-August 2023. HLA analysis reveals that these peptides are predicted as binding to large number of HLA alleles and 71-90% population coverage in six continents. Identified peptides displayed good binding score with both HLA class I and HLA class II molecules in the docking study. Also, a vaccine construct docked with TLR-4 receptor displays strong interaction with 20 hydrogen bonds and molecular simulation analysis reveals that docked complex are stable. Additionally, the immunogenicity of these N protein peptides was confirmed using SARS-CoV-2 convalescent serum samples. We conclude that the identified N protein peptides contain highly conserved and antigenic epitopes which could be used as a target for the future vaccine development against SARS-CoV-2.Communicated by Ramaswamy H. Sarma.

Antibody and T Cell Immune Responses to SARS-CoV-2 Peptides in COVID-19 Convalescent Patients.

Identifying immunogenic targets of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is critical to advance diagnostic and disease control strategies. We analyzed humoral (ELISA) and T-cell (ELISpot) immune responses to spike (S) and nucleocapsid (N) SARS-CoV-2 proteins as well as to human endemic coronavirus (eCoV) peptides in serum from convalescent coronavirus disease 2019 (COVID-19) patients from Tatarstan, Russia. We identified multiple SARS-CoV-2 peptides that were reactive with serum antibodies and T cells from convalescent COVID-19. In addition, age and gender associated differences in the reactivity to S and N protein peptides were identified. Moreover, several SARS-CoV-2 peptides tested negatively correlated with disease severity and lung damage. Cross-reactivity to eCoV peptides was analyzed and found to be lower in COVID-19 compared to controls. In this study, we demonstrate the changing pattern of immunogenic peptide reactivity in COVID-19 serum based on age, gender and previous exposure to eCoVs. These data highlight how humoral immune responses and cytotoxic T cell responses to some of these peptides could contribute to SARS-CoV-2 pathogenesis.

Immunogenic SARS-CoV-2 S and N Protein Peptide and Cytokine Combinations as Biomarkers for Early Prediction of Fatal COVID-19.

Early indications of the likelihood of severe coronavirus disease 2019 COVID-19 can influence treatments and could improve clinical outcomes. However, knowledge on the prediction markers of COVID-19 fatality risks remains limited. Here, we analyzed and quantified the reactivity of serum samples from acute (non-fatal and fatal) and convalescent COVID-19 patients with the spike surface glycoprotein (S protein) and nucleocapsid phosphoprotein (N protein) SARS-CoV-2 peptide libraries. Cytokine activation was also analyzed. We demonstrated that IgM from fatal COVID-19 serum reacted with several N protein peptides. In contrast, IgM from non-fatal serum reacted more with S protein peptides. Further, higher levels of pro-inflammatory cytokines were found in fatal COVID-19 serum compared to non-fatal. Many of these cytokines were pro-inflammatory and chemokines. Differences in IgG reactivity from fatal and non-fatal COVID-19 sera were also demonstrated. Additionally, the longitudinal analysis of IgG reactivity with SARS-CoV-2 S and N protein identified peptides with the highest longevity in humoral immune response. Finally, using IgM antibody reactivity with S and N SARS-CoV-2 peptides and selected cytokines, we have identified a panel of biomarkers specific to patients with a higher risk of fatal COVID-19 compared with that of patients who survive. This panel could be used for the early prediction of COVID-19 fatality risk.

Mutational Frequencies of SARS-CoV-2 Genome during the Beginning Months of the Outbreak in USA.

SARS-CoV-2 has spread very quickly from its first reported case on 19 January 2020 in the United Stated of America, leading WHO to declare pandemic by 11 March 2020. RNA viruses accumulate mutations following replication and passage in human population, which prompted us to determine the rate and the regions (hotspots) of the viral genome with high rates of mutation. We analyzed the rate of mutation accumulation over a period of 11 weeks (submitted between 19th January to 15 April 2020) in USA SARS-CoV-2 genome. Our analysis identified that majority of the viral genes accumulated mutations, although with varying rates and these included NSP2, NSP3, RdRp, helicase, Spike, ORF3a, ORF8, and Nucleocapsid protein. Sixteen mutations accumulated in Spike protein in which four mutations are located in the receptor binding domain. Intriguingly, we identified a fair number of viral proteins (NSP7, NSP9, NSP10, NSP11, Envelop, ORF6, and ORF7b proteins), which did not accumulate any mutation. Limited changes in these proteins may suggest that they have conserved functions, which are essential for virus propagation. This provides a basis for a better understanding of the genetic variation in SARS-CoV-2 circulating in the US, which could help in identifying potential therapeutic targets for controlling COVID-19.

Effect of N-acetylgalactosamine ligand valency on targeting dendrimers to hepatic cancer cells.

The display of N-acetylgalactosamine (NAcGal) ligands has shown great potential in improving the targeting of various therapeutic molecules to hepatocellular carcinoma (HCC), a severe disease whose clinical treatment is severely hindered by limitations in delivery of therapeutic cargo. We previously used the display of NAcGal on generation 5 (G5) polyamidoamine (PAMAM) dendrimers connected through a poly(ethylene glycol) (PEG) brush (i.e. G5-cPEG-NAcGal; monoGal) to effectively target hepatic cancer cells and deliver a loaded therapeutic cargo. In this study, we were interested to see if tri-valent NAcGal ligands (i.e. NAcGal3) displayed on G5 dendrimers (i.e. G5-cPEG-NAcGal3; triGal) could improve their ability to target hepatic cancer cells compared to their monoGal counterparts. We therefore synthesized a library of triGal particles, with either 2, 4, 6, 8, 11, or 14 targeting branches (i.e. cPEG-NAcGal3) attached. Conventional flow cytometry studies showed that all particle formulations can label hepatic cancer cells in a concentration-dependent manner, reaching 90-100% of cells labeled at either 285 or 570 nM G5, but interestingly, monoGal labeled more cells at lower concentrations. To elucidate the difference in internalization of monoGal versus triGal conjugates, we turned to multi-spectral imaging flow cytometry and quantified the amount of internalized (I) versus surface-bound (I0) conjugates to determine the ratio of internalization (I/I0) in all treatment groups. Results show that regardless of NAcGal valency, or the density of targeting branches, all particles achieve full internalization and diffuse localization throughout the cell (I/I0 ∼ 3.0 for all particle compositions). This indicates that while tri-valent NAcGal is a promising technique for targeting nanoparticles to hepatic cancer cells, mono-valent NAcGal is more efficient, contrary to what is observed with small molecules.

Synergistic inhibition of aggressive breast cancer cell migration and invasion by cytoplasmic delivery of anti-RhoC silencing RNA and presentation of EPPT1 peptide on "smart" particles.

Overexpression of RhoC protein in breast cancer patients has been linked to increased cancer cell invasion, migration, and metastases. Suppressing RhoC expression in aggressive breast cancer cells using silencing RNA (siRNA) molecules is a viable strategy to inhibit the metastatic spread of breast cancer. In this report, we describe the synthesis of a series of asymmetric pH-sensitive, membrane-destabilizing polymers engineered to complex anti-RhoC siRNA molecules forming "smart" nanoparticles. Using ß-CD as the particle core, polyethylene glycol (PEG) chains were conjugated to the primary face via non-cleavable bonds and amphiphilic polymers incorporating hydrophobic and cationic monomers were grafted to the secondary face via acid-labile linkages. We investigated the effect of PEG molecular weight (2 & 5 kDa) on transfection capacity and serum stability of the formed particles. We evaluated the efficacy of EPPT1 peptides presented on the free tips of the PEG brush to function as a targeting ligand against underglycosylated MUC1 (uMUC1) receptors overexpressed on the surface of metastatic breast cancer cells. Results show that "smart" nanoparticles successfully delivered anti-RhoC siRNA into the cytoplasm of aggressive SUM149 and MDA-MB-231 breast cancer cells, which resulted in a dose-dependent inhibition of cell migration and invasion. Further, EPPT1-targeted nanoparticles demonstrate a synergistic inhibition of cell migration and invasion imparted via RhoC knockdown and EPPT1-mediated signaling via the uMUC1 receptor.