Mucosal administration of a live attenuated recombinant COVID-19 vaccine protects nonhuman primates from SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 global pandemic. SARS-CoV-2 is an enveloped RNA virus that relies on its trimeric surface glycoprotein spike for entry into host cells. Here we describe the COVID-19 vaccine candidate MV-014-212, a live, attenuated, recombinant human respiratory syncytial virus expressing a chimeric SARS-CoV-2 spike as the only viral envelope protein. MV-014-212 was attenuated and immunogenic in African green monkeys (AGMs). One mucosal administration of MV-014-212 in AGMs protected against SARS-CoV-2 challenge, reducing by more than 200-fold the peak shedding of SARS-CoV-2 in the nose. MV-014-212 elicited mucosal immunoglobulin A in the nose and neutralizing antibodies in serum that exhibited cross-neutralization against virus variants of concern Alpha, Beta, and Delta. Intranasally delivered, live attenuated vaccines such as MV-014-212 entail low-cost manufacturing suitable for global deployment. MV-014-212 is currently in Phase 1 clinical trials as an intranasal COVID-19 vaccine.

Identification of a novel alkaline serine protease from gazami crab (Portunus trituberculatus) hepatopancreas and its hydrolysis of myofibrillar protein.

Serine proteases are thought to play a key role in the muscle softening of gazami crab (Portunus trituberculatus) during storage. A serine protease, Pt-sp2, was purified from the hepatopancreas of gazami crab using ammonium sulfate precipitation, anion-exchange and gel filtration chromatography, and was analyzed by mass spectrometry, transcriptome and bioinformatics. It revealed that Pt-sp2 was trypsin-like, with no 100% identical proteins in the NCBI database. The molecular weight of Pt-sp2 was approximately 37.2 kDa. Its optimum pH and temperature were 9.0 and 50 °C, respectively, using t-Butyloxy­carbonyl-Phe-Ser-Arg-4-methyl-coumaryl-7-amide as a substrate. Pt-sp2 was activated in the presence of Ca2+. Both soybean trypsin inhibitor and Nα-Tosyl-l-lysine chloromethyl ketone hydrochloride completely suppressed Pt-sp2 activity, while it was only partially inhibited by phenylmethylsulfonyl fluoride and EDTA. However, PMSF, Pepstatin A and cystatin inhibitor E-64 showed no inhibition on Pt-sp2 protease activity. The Km value of Pt-sp2 was 0.82 µM, and Pt-sp2 effectively hydrolyzed myofibrillar protein at 37 °C.

Long-Term Measurements of Human Inflammatory Cytokines Reveal Complex Baseline Variations between Individuals.

Comprehensive characterization of the healthy human proteome baseline is essential for personalized medicine. Baseline data are necessary to understand the variation between individuals, as well as longitudinal variation within individuals. Many important protein biomarkers, such as cytokines, exist at extremely low or undetectable levels in the healthy state. This paper describes results from a 14-week study of healthy human subjects using ultrasensitive single-molecule array (Simoa) assays to measure both intra and intersubject variation of 15 cytokines. The results show a wide variation in the ranges of some cytokines between individuals and demonstrate that individual baseline values will be essential for predicting disease presence and progression. Although all of the studied cytokines demonstrated high temporal stability (or low intrasubject variation) over the entire study period, there were two distinct groups of cytokines that demonstrated either high (IL-8, IFN-γ, IL-2, IL-6, and IL-1ß) or low (IL-15, TNF-α, IL-12 p70, IL-17A, GM-CSF, IL-12 p40, IL-10, IL-7, IL-1α, and IL-5) subject-to-subject variation. This work demonstrates that ultrasensitive assays are essential for characterizing human cytokines in healthy subjects. The results show that some cytokines vary by more than two orders of magnitude between individuals, making it an imperative to obtain individual baseline measurements if they are to play a role in health and disease diagnosis.

Incorporation of Slow Off-Rate Modified Aptamers Reagents in Single Molecule Array Assays for Cytokine Detection with Ultrahigh Sensitivity.

Slow off-rate modified aptamers (SOMAmers) are attractive protein recognition reagents due to their high binding affinities, stable chemical structures, easy production, and established selection process. Here, biotinylated SOMAmer reagents were incorporated into single molecule array (Simoa)-based assays in place of traditional detection antibodies for six cytokine targets. Optimization and validation were conducted for TNF-α as a demonstration using a capture antibody/detection-SOMAmer detection scheme to highlight the performance of this approach. The optimized assay has a broad dynamic range (>4 log10 units) and an ultralow detection limit of 0.67 fM (0.012 pg/mL). These results show comparable sensitivity to our antibody pair-based Simoa assays, and tens to thousands-fold enhancement in sensitivity compared with conventional ELISAs. High recovery percentages were observed in a spike-recovery test using human sera, demonstrating the feasibility of this novel Simoa assay in detecting TNF-α in clinically relevant samples. Detection SOMAmers were also used to detect other cytokines, such as IFN-γ, IL-1ß, IL-2, IL-6, and IL-10, in human samples. Although not yet demonstrated, in principle it should be possible to eventually replace both the capture and detector antibodies with corresponding SOMAmer pairs in sandwich immunoassays. The combination of the ultrasensitive Simoa platform with the higher reliability of SOMAmer binding reagents will greatly benefit both biomarker discovery and disease diagnostic fields.

Single molecule array (Simoa) assay with optimal antibody pairs for cytokine detection in human serum samples.

Concentrations of cytokines in bodily fluids reflect the physiological or pathological state of the patient and can be used for prognosis, disease diagnosis or for monitoring therapeutic efficacy. However, in the bodily fluids of healthy or sub-healthy individuals, many cytokines are present at concentrations that are near or below the detection limits of current methods. Here we selected antibody pairs to be employed in the single molecule array (Simoa) assay for ten cytokines including GM-CSF, TNF-α, IFN-γ, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-7, and IL-10. The limits of detection (LODs) obtained were as low as 90 aM-6 fM. These assays allow detection of cytokines in healthy human serum samples at levels significantly below the detection limits of conventional ELISA assays. We provide detailed antibody pair information as well as the concentration profiles of ten cytokines in healthy human serum to serve as reference data for further ultrasensitive immunoassay development and future clinical applications.

Protein induced aggregation of conjugated polyelectrolytes probed with fluorescence correlation spectroscopy: application to protein identification.

The interaction of a series of water-soluble conjugated polyelectrolytes with varying backbone structure, charge type (cationic and anionic), and charge density with a set of seven different proteins is explored by using fluorescence correlation spectroscopy (FCS). The FCS method affords the diffusion time for a particular CPE/protein pair, and this diffusion time is a reflection of the aggregation state of the polymer/protein in the solution. The diffusion time is larger for oppositely charged CPE/protein combinations, reflecting the tendency toward the formation of CPE/protein aggregates in these systems. However, by careful analysis of the data, other factors emerge, including possible effects of hydrophobic interaction in specific CPE/protein systems. The final diffusion time for each CPE/protein mixture varies and the diffusion time response pattern created by the six-CPE array for a typical protein is unique, and this effect was leveraged to develop a sensor array for protein identification by using linear-discriminant analysis (LDA) methods. By application of multimode linear discrimination analysis, the unknown protein samples have been successfully identified with a total accuracy of 93%.

Ion-induced aggregation of conjugated polyelectrolytes studied by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is applied to investigate aggregation behavior of an anionic conjugated polyelectrolyte (CPE) featuring branched carboxylate groups (PPE-(d)CO2) upon the addition of different metal ions. FCS results reveal that monovalent metal ions (Na(+), K(+)) bind to the CPE chain but do not induce aggregation, and divalent metal ions (Ca(2+), Cu(2+)) bind and induce the formation of small aggregates. Iron cations (Fe(2+) and Fe(3+)) bind strongly and induce the formation of large CPE aggregates. This is believed to be because of the propensity of these metals to bind to six ligands and thus bridge two of the triacid units on adjacent PPE-(d)CO2 chains. A comparison between the Stern-Volmer (SV) fluorescence quenching and the relative diffusion time change from FCS demonstrates that the most efficient SV quenching is observed for the ions that give rise to large increases in FCS diffusion time, underscoring the importance of ion-induced aggregation in the amplified quenching effect.

Helical Conjugated Polyelectrolyte Aggregation Induced by Biotin-Avidin Interaction.

Fluorescence correlation spectroscopy (FCS) is applied to demonstrate avidin-induced cross-linking in a system consisting of a helical anionic conjugated polyelectrolyte (P1) and a biotin-tetramethylrhodamine (TMR) conjugate (2). In a previous study, we used fluorescence spectroscopy to demonstrate that 2 binds to P1 via intercalation of the TMR chromophore into the P1 helix. Addition of avidin to the P1/2 complex induces little change in the fluorescence of the system; however, FCS reveals a remarkable increase in the diffusion time of the P1/2 complex in the presence of avidin. This change is attributed to supramolecular polymer aggregates produced by cross-link formation between the biotin unit of intercalated 2 and avidin. Atomic force microscopy imaging provides evidence supporting the existence of these aggregates. The highly sensitive FCS method is used to develop a novel sensor for the biotin-avidin interaction, with a detection limit of <100 pM for avidin.

Intercalation-FRET biosensor with a helical conjugated polyelectrolyte.

A biotin-tetramethylrhodamine (biotin-TMR) quencher-ligand interacts with a (phenylene-ethynylene) based helical conjugated polyelectrolyte (poly-1) via intercalation of the TMR unit into the helix. The interaction is signaled by efficient fluorescence resonance energy transfer (FRET) from the polymer to the TMR chromophore. Avidin addition to the poly-1/biotin-TMR intercalation complex does not interrupt FRET, instead resulting in the formation of avidin-biotin "cross-links". Mixing of biotin-TMR with avidin prior to addition of the polymer efficiently disrupts the FRET signal, giving rise to a sensor with a detection limit of 100 pM for avidin. Study of the FRET response as a function of biotin-TMR and avidin concentration affords insight into the interaction of the protein with the poly-1/biotin-TMR intercalation complex.