Observational Study of Receptor Binding Domain Spike Antibody Responses to 3 SARS-CoV-2 Vaccinations in Noninfected Subjects: Parallel Neutralizing Antibody and Cardiac Troponin I and T Observations.

BACKGROUND: Our goals were to demonstrate receptor binding domain spike 1 (RBD S1) protein antibody (Ab) kinetic responses to multiple vaccines over approximately 180 days, neutralizing Ab effectiveness, and high-sensitivity cardiac troponin I (hs-cTnI) and T (hs-cTnT) responses in postvaccinated, non-SARS-CoV-2-infected subjects. METHODS: Blood specimens were collected pre- and postvaccinations from seronegative subjects. RDB S1 Abs were measured by the novel Qorvo Biotechnologies Omnia platform. Neutralizing Abs and hs-cTnI and hs-cTnT were measured on the ET Healthcare Pylon 3D. RESULTS: Two-dose vaccines (Pfizer, Moderna) had peak RBD S1 Ab concentrations about 45 to 55 days after both doses and showed declines over the next 50 to 70 days. The Janssen vaccine showed lower RBD S1 Ab peak concentrations, continued to increase over time, and plateaued after 60 days. There was strong neutralizing Ab response post vaccinations, with only 3 specimens, shortly before and shortly after vaccination, not showing a response. Specimens showed no hs-cTnI (all < 3 ng/L) and hs-cTnT (all < 6 ng/L) increases or changes over time. CONCLUSIONS: We demonstrate in seronegative SARS-CoV-2 subjects that Pfizer and Moderna vaccinations provide strong, neutralizing RBD S1 Ab effectiveness, based on 2 different assays after 2 doses, with the Janssen single-dose vaccine showing a lower RBD S1 Ab response over 4 to 6 months. No myocardial injury was associated with the Pfizer postvaccination. The Qorvo Biotechnologies RBD S1 Ab assay measured on the Omnia platform has potential as a point-of-care platform.

Defining molecular and domain boundaries in the bacteriophage phi29 DNA packaging motor.

Cryo-electron microscopy (cryo-EM) studies of the bacteriophage phi29 DNA packaging motor have delineated the relative positions and molecular boundaries of the 12-fold symmetric head-tail connector, the 5-fold symmetric prohead RNA (pRNA), the ATPase that provides the energy for packaging, and the procapsid. Reconstructions, assuming 5-fold symmetry, were determined for proheads with 174-base, 120-base, and 71-base pRNA; proheads lacking pRNA; proheads with ATPase bound; and proheads in which the packaging motor was missing the connector. These structures are consistent with pRNA and ATPase forming a pentameric motor component around the unique vertex of proheads. They suggest an assembly pathway for the packaging motor and a mechanism for DNA translocation into empty proheads.

DNA packaging motor assembly intermediate of bacteriophage phi29.

Unraveling the structure and assembly of the DNA packaging ATPases of the tailed double-stranded DNA bacteriophages is integral to understanding the mechanism of DNA translocation. Here, the bacteriophage phi29 packaging ATPase gene product 16 (gp16) was overexpressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the phi29 precursor capsid (prohead) to produce a packaging motor intermediate that was fully active in in vitro DNA packaging. The formation of higher oligomers of the gp16 from monomers was concentration dependent and was characterized by analytical ultracentrifugation, gel filtration, and electron microscopy. The binding of multiple copies of gp16 to the prohead was dependent on the presence of an oligomer of 174- or 120-base prohead RNA (pRNA) fixed to the head-tail connector at the unique portal vertex of the prohead. The use of mutant pRNAs demonstrated that gp16 bound specifically to the A-helix of pRNA, and ribonuclease footprinting of gp16 on pRNA showed that gp16 protected the CC residues of the CCA bulge (residues 18-20) of the A-helix. The binding of gp16 to the prohead/pRNA to constitute the complete and active packaging motor was confirmed by cryo-electron microscopy three-dimensional reconstruction of the prohead/pRNA/gp16 complex. The complex was capable of supercoiling DNA-gp3 as observed previously for gp16 alone; therefore, the binding of gp16 to the prohead, rather than first to DNA-gp3, represents an alternative packaging motor assembly pathway.

Conservation of the capsid structure in tailed dsDNA bacteriophages: the pseudoatomic structure of phi29.

Bacteriophage phi29 is one of the smallest and simplest known dsDNA phages, making it amenable to structural investigations. The three-dimensional structure of a fiberless, isometric variant has been determined to 7.9 A resolution by cryo-electron microscopy (cryo-EM), allowing the identification of alpha helices and beta sheets. Their arrangement indicates that the folds of the phi29 and bacteriophage HK97 capsid proteins are similar except for an additional immunoglobulin-like domain of the phi29 protein. An atomic model that incorporates these two domains fits well into the cryo-EM density of the T = 3, fiberless isometric phi29 particle, and cryo-EM structures of fibered isometric and fiberless prolate prohead phi29 particles at resolutions of 8.7 A and 12.7 A, respectively. Thus, phi29 joins the growing number of phages that utilize the HK97 capsid structure, suggesting that this protein fold may be as prevalent in capsids of dsDNA phages as the jelly roll fold is in eukaryotic viruses.

Bacteriophage phi29 scaffolding protein gp7 before and after prohead assembly.

Three-dimensional structures of the double-stranded DNA bacteriophage phi29 scaffolding protein (gp7) before and after prohead assembly have been determined at resolutions of 2.2 and 2.8 A, respectively. Both structures are dimers that resemble arrows, with a four-helix bundle composing the arrowhead and a coiled coil forming the tail. The structural resemblance of gp7 to the yeast transcription factor GCN4 suggests a DNA-binding function that was confirmed by native gel electrophoresis. DNA binding to gp7 may have a role in mediating the structural transition from prohead to mature virus and scaffold release. A cryo-EM analysis indicates that gp7 is arranged inside the capsid as a series of concentric shells. The position of the higher density features in these shells correlates with the positions of hexamers in the equatorial region of the capsid, suggesting that gp7 may regulate formation of the prolate head through interactions with these hexamers.