An intranasal nanoparticle STING agonist has broad protective immunity against respiratory viruses and variants

Respiratory viral infections, especially Influenza (endemic) or SARS-CoV-2 (pandemic since 2020), cause morbidity and mortality worldwide. Despite remarkable progress in the development and deployment of vaccines, they are clearly impacted by the rapid emergence of viral variants. The development of an off-the-shelf, effective, safe, and low-cost drug for prophylaxis against respiratory viral infections is a major unmet medical need. Here, we developed NanoSTING, a liposomally encapsulated formulation of the endogenous STING agonist, 2-3 cGAMP, to function as an immunoantiviral. NanoSTING rapidly activates the bodys innate immune system to facilitate a broad-spectrum antiviral response against SARS-CoV-2 and influenza variants in hamsters and mice. We demonstrate that a single intranasal dose of NanoSTING can: (1) treat infections throughout the respiratory system and minimize clinical symptoms, (2) protect against highly pathogenic strains of SARS-CoV-2 (alpha and delta), (3) provide durable protection against reinfection from the same strains without the need for retreatment, (4) prevent transmission of the highly infectious SARS-CoV-2 Omicron strain, and (5) provide protection against both oseltamivir-sensitive and resistant strains of influenza. Mechanistically, administration of NanoSTING rapidly upregulated interferon-stimulated and antiviral pathways in both the nasal turbinates and lung. Our results support using NanoSTING as a thermostable, immunoantiviral with broad-spectrum antiviral properties making it appealing as a therapeutic for prophylactic or early post-exposure treatment.

Cytotoxic T lymphocytes targeting a conserved SARS-CoV-2 spike epitope are efficient serial killers

Understanding the cellular immune response to infections, cancers and vaccines lags behind the investigation of humoral responses. While neutralizing antibody responses wane over time, the ability of T cells to recognize viruses including SARS-CoV-2 is instrumental to providing long-term immunity. Although T-cell receptor (TCR) repertoire screening can provide insights into the skewing of a T-cell response elicited upon vaccination or infection, they unfortunately provide no assessment into the functional capacity of T cells or their ability to eliminate virally infected targets. We have used time-lapse imaging microscopy in nanowell grids (TIMING) to integrate the migration of individual T cells with analysis of effector functions including cytokine secretion and cytotoxicity. Machine learning is then applied to study thousands of videos of dynamic interactions as T cells with specificity for SARS-CoV-2 eliminate targets bearing spike protein as a surrogate for viral infection. Our data provide the first direct evidence that cytotoxic T lymphocytes from a convalescent patient targeting an epitope conserved across all known variants of concern (VoC) are serial killers capable of eliminating multiple infected targets. These data have implications for development of vaccines to provide broad and sustained cellular immunity and for the recovery and monitoring of individuals who have been exposed to SARS-CoV-2. Multidisciplinary abstractWe present an imaging platform that uses artificial intelligence (AI) to track thousands of individual cell-cell interactions within nanowell arrays. We apply this platform to quantify how the T cell component of adaptive immunity responds to infections. Our results show that T cells specific for a conserved epitope within the SARS-CoV-2 spike protein are serial killers that can rapidly eliminate virally infected targets. The ability to map the functional capacity of T cells and their ability to kill infected cells provides fundamental insights into the immunology of vaccines and recovery from infections. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/477535v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@3e3eaborg.highwire.dtl.DTLVardef@844cacorg.highwire.dtl.DTLVardef@1c5ecfdorg.highwire.dtl.DTLVardef@14a0b44_HPS_FORMAT_FIGEXP M_FIG C_FIG

Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2

A safe and durable vaccine is urgently needed to tackle the COVID19 pandemic that has infected >15 million people and caused >620,000 deaths worldwide. As with other respiratory pathogens, the nasal compartment is the first barrier that needs to be breached by the SARS-CoV-2 virus before dissemination to the lung. Despite progress at remarkable speed, current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity. We report the development of an intranasal subunit vaccine that contains the trimeric or monomeric spike protein and liposomal STING agonist as adjuvant. This vaccine induces systemic neutralizing antibodies, mucosal IgA responses in the lung and nasal compartments, and T-cell responses in the lung of mice. Single-cell RNA-sequencing confirmed the concomitant activation of T and B cell responses in a germinal center-like manner within the nasal-associated lymphoid tissues (NALT), confirming its role as an inductive site that can lead to long-lasting immunity. The ability to elicit immunity in the respiratory tract has can prevent the initial establishment of infection in individuals and prevent disease transmission across humans.

Molecular characterization and phylogenetic study of velogenic Newcastle disease virus isolates in Iran.

The pathogenicity and genetic characterizations of six Newcastle disease virus (NDV) isolates obtained from chicken farms in six different regions in Iran were carried out using conventional and molecular techniques. Based on the pathogenicity indices (MDT, ICPI, and IVPI), all of these isolates were found to be velogenic (highly virulent) strains. A sequence analysis of the full-length mRNA encoding the fusion glycoprotein precursor (F0) of the NDV's fusion proteins F1 and F2 in these six isolates showed the presence of point mutations in form of nucleic acid substitutions at positions 82((C→T)), 83((T→C)), 736((A→G)), and 1,633((G→A)). However, the nucleic acid residues at positions 330-347 of the precursor F0 gene, corresponding to the cleavage site of the F0 protein, were found to have remained conserved among the six NDV isolates. A phylogenetic comparison between the six Iranian isolates and the NDVs whose F0 gene sequences were previously deposited in GenBank Database showed that all of the newly characterized Iranian NDV isolates belonged to genotype VII.

Which form of collagen is suitable for nerve cell culture?

In this study, we investigated the effects of hydrolyzed and non-hydrolyzed collagen and two-dimensional and three-dimensional collagen matrices on cell survival, attachment and neurite outgrowth of primary cultured nerve cells using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and inverted microscopy. Hydrolyzed collagen facilitated nerve cell survival and neurite outgrowth, but it had no obvious influences on cell attachment. In contrast, non-hydrolyzed two-dimensional collagen matrix had no obvious effects on neurite outgrowth. These findings suggest that hydrolyzed collagen is an ideal nerve cell culture media.

Genetic polymorphism at the candidate gene in Iranian Sistani cattle (Bos indicus).

The genotypes for Leptin, Kappa-Casein, Calpastatin and BoLA-DRB3 loci were determined by polymerase chain reaction and restriction enzyme digestion method in native Iranian breed cattle, Sistani. Blood samples were collected from Sistani Breeding Station located in Zehak, Zabol in Iran. The extraction of genomic DNA was based on Guanidin Thiocyanate-Silica gel method. After PCR reaction, amplicons were digested with appropriate restriction enzymes. The Calpastatin locus had 3 genotypes with frequencies of 0.62, 0.29 and 0.09 for MM, MN and NN, respectively; kappa-Casein and Leptin had 3 genotypes with frequencies of 0.27, 0.57 and 0.16 for kappa-Casein, 0.77, 0.22 and 0.01 for Leptin for AA, AB and BB genotypes, respectively. For BoLA-DRB3 we identified 19 alleles, that DRB3. 2*8 had the highest allelic frequency (22.4%) and DRB3. 2*3, *29, *37 and *51 had the lowest allelic frequency (1%). One of the 19 alleles had a new pattern. Average heterozygosity values for all loci were low. Chi2-test did not confirm the Hardy-Weinberg equilibrium for Leptin and Calpastatin in this population. These data provide evidence that Iranian's Sistani breed have a good genetic variability, which opens interesting prospects for future selection programs, especially marker-assistant selection.

Effect of the water extracts of propolis on stimulation and inhibition of different cells.

The water extracts of propolis (WEP) could inhibit growth of different cell lines namely McCoy, HeLa, SP2/0, HEp-2, and BHK21 and stimulate growth of normal cell named human lymphocyte, rat kidney, rat liver, and rat spleen. In these experiments 1 and 2 mg of WEP were added to 1 ml RPMI media with 5% FCS. Cell counts and cell viability of propolis-treated and propolis-free cells were assessed by Trypan blue dye exclusion test and MTT assay. The results showed that in case of McCoy, HeLa, SP20, HEp-2, and BHK21 cell lines, the water extracts of propolis could inhibit cell growth as well as reduction on size of the cells. In contrast the same amount of WEP could stimulate growth of normal cells up to 60% with the same concentration used for cell lines. Thus our study indicates that although WEP consists only of the soluble part of propolis, it enables to inhibit different cell lines and increase growth of normal cells. This indicates also that WEP contains the specific compounds with bioactivity against cell lines. Although propolis contain different number of compounds it is clear that WEP has enough biological compounds useful for the treatment of some diseases, medical and related applications.

Potential application of protease isolated from Pseudomonas aeruginosa PD100

A protease isolated from Pseudomonas aeruginosa PD100 could act in the presence of SDS and Tween 80. This protease could be useful for degradation of protein in the presence of solvent, dehairing of cow skin and degradation of natural proteins. The immobilized protease showed 15-20% increases in temperature stability and the entrapped enzyme retained 83% of its initial activity after six cycles. With respect to properties of the enzyme and its capability for degradation of different protein sources, this protease finds potential application for waste treatment, used in detergents and leather industry.

Purification and characterization of an extracellular alpha-amylase from Bacillus subtilis AX20.

A Bacillus subtilis AX20 from soil with ability to produce extracellular alpha-amylases was isolated. The characterization of microorganism was performed by biochemical tests as well as 16S rDNA sequencing. Maximum amylase activity (38 U/ml) was obtained at stationery phase when the culture was grown at 37 degrees C. The enzyme was purified to homogeneity with an overall recovery of 24.2% and specific activity of 4133 U/mg. The native protein showed a molecular mass of 149 kDa composed of a homodimer of 78 kDa polypeptide by SDS-PAGE. The optimum pH and temperature of the amylase were 6 and 55 degrees C, respectively. The enzyme was inhibited by Hg(2+), Ag(2+), and Cu(2+) and it did not show an obligate requirement of metal ions. The enzyme was not inhibited by EDTA or EGTA, suggesting that this enzyme is not a metalloenzyme. The end products of corn starch and soluble starch were glucose (70-75%) and maltose (20-25%). Rapid reduction of blue value and the end products suggest an endo mode of action for the amylase. The purified amylase shows interesting properties useful for industrial applications.