Self-assembling amyloid-like nanostructures from SARS-CoV-2 S1, S2, RBD and N recombinant proteins

The intrinsic property of proteins to form α-helices and β-sheets leads to various types of aggregates including NP and amyloid fibrils [1]. The protein saNP are spontaneously and ubiquitously produced in both bacterial and eukaryotic cells and are involved in many normal physiological processes. Virus-like particles (VLP) are protein NP consisting of the viral structural proteins without nucleic acids. Their stability, the internal empty spaces, and the symmetric shape make them useful tools as antigens per se or as antigen display platforms [2]. The protein NP allow to overcome the low immunogenicity of both recombinant and isolated purified protein antigens. Simultaneous exposure of multiple copies of target antigen to the immune system and affinity binding with B-cell receptors induce higher antibody titers after NP administration [3,14]. Currently, several VLP-based vaccines against human papilloma virus [5,6], hepatitis B virus [[7], [8], [9]] and influenza virus [10] are available. Meanwhile other saNP and chimeric VLP are at preclinical and clinical development stages of the vaccines against SARS-CoV-2 [11], respiratory syncytial virus [12], human immunodeficiency virus (HIV), and influenza virus [13,14] as well as against bacterial [[15], [16], [17]] and protozoan (malaria) [18] pathogens.

Coronavirus infectious disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains the public health concern because of substantial morbidity and mortality resulted from the rapidly evolving new variants of the RNA-containing quasispecies. At least one-third of COVID-19 patients have neurological symptoms (loss of smell, sensory confusion, memory loss, severe headaches, mood and anxiety disorders, cognitive impairment) [19], neurological syndromes (Guillain–Barre Syndrome, encephalitis, encephalopathy, acute disseminated encephalomyelitis, intracranial hypertension) [20] with ischemic or hemorrhagic strokes [21]. The long persistence of such neurological sequelae at the remission stages is called post-COVID-19 syndrome, or long COVID-19. SARS-CoV-2 can cross the blood–brain barrier and infect neuronal cells causing their death [19] as well as destruction of vascular and immune cells of central nervous system (CNS) [20 and references therein]. It remains unclear if COVID-19 neuropathology arises because of direct viral infection of CNS or due to accompanying immune response and resulting hypercoagulability. The coronavirus neuroinvasiveness can be mediated by either the full virions or separate structural proteins being found in the CNS of laboratory mouse models and the post-mortem brain tissue of COVID-19 patients [19]. On the other hand neuroinflammation and microvascular injury are known to be responsible for amyloid-related neurodegenerative diseases such as Alzheimer’s (AD) and Parkinson’s [19,22].

COVID-19 is caused by the enveloped spherical β-coronavirus SARS-CoV-2 with a longest positive single-stranded RNA genome of up to 31 kb in length. The virions consist of structural proteins such as the spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins. Additionally, there is the hemagglutinin-esterase (HE) protein in some β-coronaviruses [23,24]. The S, M, and E proteins are embedded in the envelope and the N protein interacts with the viral RNA, forming the nucleocapsid [23]. S protein is necessary for β-coronaviruses to attach to the host cell and enter. The host protease furin cleaves the full-length precursor S glycoprotein into two associated polypeptides: S1 and S2 [25].

Currently there are more than 300 vaccine candidates in clinical and preclinical development, almost all of which target the surface spike (S) protein except for attenuated or inactivated whole virus. There are several vaccines licensed for emergency usage, which are mostly based on nanoparticle-formulated mRNA, inactivated whole virus, or adenovirus vectors. While all these vaccine platforms have certain advantages, some limitations have become evident. The main issue for mRNA vaccine is shortage of supply and the very low storage temperature. Both attenuated and inactivated vaccines are produced by conventional techniques, induce neutralizing antibodies with low titers and weak T-cellular response (if any). Adenoviruses-based vaccines against COVID-19 can be produced at large scale and stored at temperature 4 °C. However, pre-existing immunity against the widely spread human adenovirus vector backbone can significantly limit their efficiency. Combining different vaccines may overcome drawbacks of individual vaccines. Recently developed “mix and match” vaccines (such as ChAdOx1 nCoV-19 and BNT162b2) could elicit strong immune responses [26]. Despite multiple available vaccines against COVID-19 based on different platforms new approaches are being developed for prevention of further threats. Thus, VLP-based vaccine displaying the SARS-CoV-2 Wuhan variant spike (S1) protein receptor-binding domain (RBD) conjugated with the surface of preassembled bacteriophage AP205 VLP has been constructed [27].

Besides specific prophylactics antiviral nanomaterials with less specificity and therefore with a broader therapeutic capacity have been innovated and implemented. VivaGel, a well-known antiviral dendrimer gel, has been used for human immunodeficiency virus (HIV-1) and herpes simplex virus prevention by blocking the virus – cell interactions. Nanosilver display antiviral capacity through the interaction of the metallic atoms with subvirion components [28]. Gold NP, with a tunable surface chemistry design that mimics virus-adhesive heparan sulfate, have been applied to inhibit viral infections. An ultrathin nanosheets were fabricated from copper indium thiophosphate (CuInP2S 6, denoted CIPS) for anti-SARS-CoV-2 therapy, as a decontamination agent and surface-coating material to reduce SARS-CoV-2 infectivity. CIPS exhibits high and selective binding capacity (dissociation constant (K_D) < 1 pM) for the SARS-CoV-2 spike protein RBD domain, inhibiting virus entry and infection in angiotensin converting enzyme 2 (ACE2)-bearing cells, human airway epithelial organoids and human ACE2-transgenic mice. On association with CIPS, the virus is quickly phagocytosed and eliminated by macrophages, suggesting that CIPS could be successfully used to capture and facilitate virus elimination by the host [29].

Our research was aimed at study of nanostructures consisting of the recombinant SARS-CoV-2 S1, S2 and N proteins produced in E. coli and RBD gene fragment expressed in the human embryonic kidney cell line Expi293F as well as their penetration into various eukaryotic cells.