Bioengineering tools to speed up the discovery and preclinical testing of vaccines for SARS-CoV-2 and therapeutic agents for COVID-19

Abstract

This review provides an update for the international research community on the cell modeling tools that could accelerate the understanding of SARS-CoV-2 infection mechanisms and could thus speed up the development of vaccines and therapeutic agents against COVID-19. Many bioengineering groups are actively developing frontier tools that are capable of providing realistic three-dimensional (3D) models for biological research, including cell culture scaffolds, microfluidic chambers for the culture of tissue equivalents and organoids, and implantable windows for intravital imaging. Here, we review the most innovative study models based on these bioengineering tools in the context of virology and vaccinology. To make it easier for scientists working on SARS-CoV-2 to identify and apply specific tools, we discuss how they could accelerate the discovery and preclinical development of antiviral drugs and vaccines, compared to conventional models.

Keywords: Coronavirus; antiviral; bioengineering; preclinical testing; target; vaccine.

Figure 1

Drug targets specific to the SARS-CoV-2 virus. A) The SARS-CoV-2 whole genome consists of three main open reading frames (ORF): ORF1a, ORF1b and ORF2-10. The most frequent targets for therapies are reported in gray with their PDB codes. B) Some proteins encoded in ORF1a and ORF1b represent the primary I potential drug target. Their inhibition blocks the viral RNA synthesis and replication. Pp1ab encodes for 16 non-structural proteins (NSPs) including the papain-like protease (PLpro) in the Nsp3region, main protease or C-like protease (Mpro or 3CLpro), ADP ribose phosphatase (ADRP), RNA-dependent RNA polymerase (RdRp) and helicase. ORF2-10 encodes for accessory proteins (e.g. 7a) and the structural and accessory proteins, spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). C) The spike protein is the most studied secondary II potential drug target. The virus cannot bind the cell receptors if the spike protein is inhibited . The spike protein has been studied the most, has been solved by electron microscopy (EM) and consists of: N-ter domain (NTD); receptor binding domain (RBD) consisting of receptor binding motif (RBM), subdomain 1 (SD1) and subdomain 2 (SD2); fusion peptide (FP); heptad repeat 1 (HR1); heptad repeat 2 (HR2); transmembrane region (TM) and intracellular domain (IC). The most studied RBD domain is RBM (in red), the domain mainly involved in host-cell interaction . RBM is believed to bind mainly with the ACE2 human cell receptor and, therefore, most of the relevant PDB codes, of which 6M0J, includes the RBD+ACE2 complex. The complete amino acid sequence for RBD is shown at the bottom of the figure. Amino acids corresponding to RBM are in red, the beta-sheet and alpha-helix structures are inside the dotted boxes. The contact residues at the RBD/ACE2 interface are shown in bold squares. *https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/.

Figure 2

Micro-fabricated “Nichoid” scaffold for 3D cell culture. A) Nichoid architecture. From left to right: scanning electron microscopy (SEM) image of a Nichoid single-module; SEM image of up-scaled repetitive matrixes of Nichoids; picture of 50 mm² micro patterned glass coverslip with Nichoids. Nichoid culture 3D-substrates are produced via two-photon laser polymerization of SZ2080 negative photoresist with near-infrared exposure following a CAD geometry. B) Possible microscopy configurations to use on Nichoid culture substrates. Thanks to the device's versatility, this scaffold enables optical accessibility both in transmission and in reflection modality. C) Immunofluorescence images obtained via confocal fluorescence microscopy of GFAP (red), β-ACTIN (green) and DNA (blue), in human Adipose derived Stem Cells (hADSCs) after a 7-day expansion inside the Nichoid and in standard conditions (2D-Control). The images demonstrate that nuclear morphology and protein organization and localization (both β-ACTIN and GFAP) differed between the two culture systems. Cytoskeletal markers merge (yellow signal) into cellular protrusions inside Nichoids, while a poor signal appears in flat conditions. D) Real-time PCR analysis of specific gene targets shows a significant gene expression difference (up-regulated in green, and down-regulated in red) between Nichoid and Control conditions, both using a standard culture medium and also using a culture medium that induces adipogenic or neural differentiation.

Figure 3

Millifluidic optically accessible bioreactor (MOAB) for perfused culture of 3D cell constructs. A) The system is composed of, from left to right: culture medium reservoirs, microfluidic pumps, bioreactor chamber and reservoirs for medium collection. CAD model of the 3D cell construct or organoid, cultured in the culture chambers, used for numerical simulations. B) Results of the computational fluid dynamics (CFD) simulations for a single culture chamber of the system. Top: fluid velocity (modulus of the velocity vector) is mapped on the whole culture chamber. Bottom: wall shear stress (WSS) is computed and mapped at the cell-culture medium interface. C) Photo of the MOAB showing the three independent chambers connected to the perfusion chamber by oxygenator tubes; the device is placed on a confocal microscope connected to a CPU allowing real time imaging of a perfused lymph-node-on-a-chip model, for the development and testing of vaccines and agents for cancer immune-therapy. D) Fibroblast reticular cells (yellow) are seeded on the 3D fiber scaffold. Dendritic cells introduced in suspension during culture medium flow adhere to the fibroblast reticular cells, migrate to the scaffold, and are activated to express the adhesion receptor ICAM-1 (red). E) Antigen-specific T cells (cyan) introduced with culture medium flow tend to adhere to dendritic cells expressing ICAM-1 (red) while crawling on the scaffold.

Figure 4

Miniaturized “Microatlas” imaging window for intravital microscopy. A frontier imaging window device used for mini invasive quantitative analyses of living organisms, without any persistent percutaneous access. The device incorporates micro-scaffolds micro-fabricated by two-photon polymerization. The device was developed for animal examinations with optical fluorescence microscopy techniques. A) The device was validated by studying the reaction of the foreign body of the implant, and the animal model used was the chicken embryo. The widely employed Chorioallantoic Membrane (CAM) assay was used to quantify and characterize the amount of reaction occurring inside the imaging device. Here an image of a living embryo on the 8^th day of incubation: the device was implanted in the membrane (red dot-circle). B) Rendering representation of the implant set-up. The device lies on the membrane. No conditioning factors were administered. C) Zoomed rendered detail of the tissue infiltration inside the micro-scaffold device. The mechanical conditioning, alone, guides the tissue regeneration in situ, allowing a fast neovascularization in the structure porosity. D) Example of possible fluorescence acquisitions that quantify the foreign reaction. In the upper part is the window imaging, with respect to the control, which is reported into the lower part. Second Harmonic Generation (left), tissue autofluorescence (middle) and nuclear dyes (right) were used for the examination in real-time. E) Graphical quantitative representation of the reaction occurring in terms of collagen fibers orientation, differential neo-vascularization rate and differential cellular density in subsequent incremental time-points. Scale bars: 50 µm.