Intranasal SARS-CoV-2 RBD decorated nanoparticle vaccine enhances viral clearance in the Syrian hamster model.

Multiple vaccines have been developed and licensed for SARS-CoV-2. While these vaccines reduce disease severity, they do not prevent infection, and SARS-CoV-2 continues to spread and evolve. To prevent infection and limit transmission, vaccines must be developed that induce immunity in the respiratory tract. Therefore, we performed proof-of-principle vaccination studies with an intranasal nanoparticle vaccine against SARS-CoV-2. The vaccine candidate consisted of the self-assembling 60-subunit I3-01 protein scaffold covalently decorated with the SARS-CoV-2 receptor binding domain (RBD) using the SpyCatcher-SpyTag system. We verified the intended antigen display features by reconstructing the I3-01 scaffold to 3.4A using cryo-EM, and then demonstrated that the scaffold was highly saturated when grafted with RBD. Using this RBD-grafted SpyCage scaffold (RBD+SpyCage), we performed two unadjuvanted intranasal vaccination studies in the "gold-standard" preclinical Syrian hamster model. Hamsters received two vaccinations 28 days apart, and were then challenged 28 days post-boost with SARS-CoV-2. The initial study focused on assessing the immunogenicity of RBD+SpyCage, which indicated that vaccination of hamsters induced a non-neutralizing antibody response that enhanced viral clearance but did not prevent infection. In an expanded study, we demonstrated that covalent bonding of RBD to the scaffold was required to induce an antibody response. Consistent with the initial study, animals vaccinated with RBD+SpyCage more rapidly cleared SARS-CoV-2 from both the upper and lower respiratory tract. These findings demonstrate the intranasal SpyCage vaccine platform can induce protection against SARS-CoV-2 and, with additional modifications to improve immunogenicity, is a versatile platform for the development of intranasal vaccines targeting respiratory pathogens.

Interaction of coxsackievirus B3 with the full length coxsackievirus-adenovirus receptor.

Group B coxsackieviruses (CVB) utilize the coxsackievirus-adenovirus receptor (CAR) to recognize host cells. CAR is a membrane protein with two Ig-like extracellular domains (D1 and D2), a transmembrane domain and a cytoplasmic domain. The three-dimensional structure of coxsackievirus B3 (CVB3) in complex with full length human CAR and also with the D1D2 fragment of CAR were determined to approximately 22 A resolution using cryo-electron microscopy (cryo-EM). Pairs of transmembrane domains of CAR associate with each other in a detergent cloud that mimics a cellular plasma membrane. This is the first view of a virus-receptor interaction at this resolution that includes the transmembrane and cytoplasmic portion of the receptor. CAR binds with the distal end of domain D1 in the canyon of CVB3, similar to how other receptor molecules bind to entero- and rhinoviruses. The previously described interface of CAR with the adenovirus knob protein utilizes a side surface of D1.

Hot water immersion to eliminate Escherichia coli O157:H7 on the surface of whole apples: thermal effects and efficacy.

The effect of hot water immersion on both the reduction of Escherichia coli O157:H7 on the apple surface and internal temperatures of the apple was assessed in this study. Microbial reductions were measured experimentally, whereas internal temperatures were calculated through a mathematical analysis of experimental heat transfer data obtained from the apples. A method was developed to provide a purely surface-based inoculation of E. coli O157:H7. Rinsing produced no reduction, and treatments at 80 and 95 degrees C produced reductions of more than 5 logs in 15 s or less. The heat transfer analysis based on experimental data was used to calculate surface heat transfer coefficients and predict temperatures throughout the apple. The analysis indicated a low heat transfer rate. Although it reduces thermal degradation, a low heat transfer rate precludes thermal-based reduction of any internalized microorganisms.

Interaction of coxsackievirus A21 with its cellular receptor, ICAM-1.

Coxsackievirus A21 (CAV21), like human rhinoviruses (HRVs), is a causative agent of the common cold. It uses the same cellular receptor, intercellular adhesion molecule 1 (ICAM-1), as does the major group of HRVs; unlike HRVs, however, it is stable at acid pH. The cryoelectron microscopy (cryoEM) image reconstruction of CAV21 is consistent with the highly homologous crystal structure of poliovirus 1; like other enteroviruses and HRVs, CAV21 has a canyon-like depression around each of the 12 fivefold vertices. A cryoEM reconstruction of CAV21 complexed with ICAM-1 shows all five domains of the extracellular component of ICAM-1. The known atomic structure of the ICAM-1 amino-terminal domains D1 and D2 has been fitted into the cryoEM density of the complex. The site of ICAM-1 binding within the canyon of CAV21 overlaps the site of receptor recognition utilized by rhinoviruses and polioviruses. Interactions within this common region may be essential for triggering viral destabilization after attachment to susceptible cells.

Interaction of the poliovirus receptor with poliovirus.

The structure of the extracellular, three-domain poliovirus receptor (CD155) complexed with poliovirus (serotype 1) has been determined to 22-A resolution by means of cryo-electron microscopy and three-dimensional image-reconstruction techniques. Density corresponding to the receptor was isolated in a difference electron density map and fitted with known structures, homologous to those of the three individual CD155 Ig-like domains. The fit was confirmed by the location of carbohydrate moieties in the CD155 glycoprotein, the conserved properties of elbow angles in the structures of cell surface molecules with Ig-like folds, and the concordance with prior results of CD155 and poliovirus mutagenesis. CD155 binds in the poliovirus "canyon" and has a footprint similar to that of the intercellular adhesion molecule-1 receptor on human rhinoviruses. However, the orientation of the long, slender CD155 molecule relative to the poliovirus surface is quite different from the orientation of intercellular adhesion molecule-1 on rhinoviruses. In addition, the residues that provide specificity of recognition differ for the two receptors. The principal feature of receptor binding common to these two picornaviruses is the site in the canyon at which binding occurs. This site may be a trigger for initiation of the subsequent uncoating step required for viral infection.

Structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor.

Two human rhinovirus serotypes complexed with two- and five-domain soluble fragments of the cellular receptor, intercellular adhesion molecule-1, have been investigated by X-ray crystallographic analyses of the individual components and by cryo-electron microscopy of the complexes. The three-dimensional image reconstructions provide a molecular envelope within which the crystal structures of the viruses and the receptor fragments can be positioned with accuracy. The N-terminal domain of the receptor binds to the rhinovirus 'canyon' surrounding the icosahedral 5-fold axes. Fitting of molecular models into the image reconstruction density identified the residues on the virus that interact with those on the receptor surface, demonstrating complementarity of the electrostatic patterns for the tip of the N-terminal receptor domain and the floor of the canyon. The complexes seen in the image reconstructions probably represent the first stage of a multistep binding process. A mechanism is proposed for the subsequent viral uncoating process.