SPIKENET: An Evidence-Based Therapy for Long COVID

Abstract

The COVID-19 pandemic has been one of the most impactful events in our lifetime, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Multiple SARS-CoV-2 variants were reported globally, and a wide range of symptoms existed. Individuals who contract COVID-19 continue to suffer for a long time, known as long COVID or post-acute sequelae of COVID-19 (PASC). While COVID-19 vaccines were widely deployed, both unvaccinated and vaccinated individuals experienced long-term complications. To date, there are no treatments to eradicate long COVID. We recently conceived a new approach to treat COVID in which a 15-amino-acid synthetic peptide (SPIKENET, SPK) is targeted to the ACE2 receptor binding domain of SARS-CoV-2, which prevents the virus from attaching to the host. We also found that SPK precludes the binding of spike glycoproteins with the receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) of a coronavirus, murine hepatitis virus-1 (MHV-1), and with all SARS-CoV-2 variants. Further, SPK reversed the development of severe inflammation, oxidative stress, tissue edema, and animal death post-MHV-1 infection in mice. SPK also protects against multiple organ damage in acute and long-term post-MHV-1 infection. Our findings collectively suggest a potential therapeutic benefit of SPK for treating COVID-19.

Keywords: COVID-19; SPIKENET; long COVID; molecular mechanism; multiorgan dysfunction; therapy.

Figure 1

SPIKENET structure and binding affinity to the ACE2 binding domain of the SARS-CoV-2 spike glycoprotein. Structure of SPIKENET, a 15-amino-acid synthetic peptide targeted to the ACE2 binding domain of the SARS-CoV-2 spike glycoprotein (top image). The computational protein docking approach shows particular SPIKENET binding affinity to the ACE2 and CEACAM1 binding domains of the SARS-CoV-2 and MHV-1 spike glycoproteins (S1), respectively (bottom images) [13].

Figure 2

Spectroscopic analysis shows highly specific SPIKENET (SPK) binding affinity to the ACE2 binding domain of the SARS-CoV-2 spike glycoprotein (S1)—Ab, absorbance; Wl, wavelength [13].

Figure 3

The LIGPLOT diagram of the protein–peptide interaction between the RBD and SPIKENET. The pink SPIKENET peptide residues are shown on the top, and the yellow amino acid residues of the RBD from SARS-CoV-2—two are shown at the bottom. The SPIKENET peptide consists of 15 amino acid residues, 14 of which are shown in non-bonded interactions with the RBD of SARS-CoV-2, proving that SPIKENET has a significant binding affinity with the spike glycoprotein-1. These findings strongly suggest that SPIKENET is a potent competitive inhibitor of S1 [13].

Figure 4

The protein docking approach shows SPIKENET binding affinity to the CEACAM1a binding domain of the MHV-1 spike protein [13].

Figure 5

SPIKENET binding affinity to the CEACAM1a (CCM) binding domain of the MHV-1-NTD with molecular dynamic studies. The RMSD of both complexes with their respective native proteins are shown in (a,b). The structures showed complete equilibration in the system when comparing the RMSD of both NTD and NTD + SPK (a) after 50 ns dynamic simulation. The SPK peptide was well stabilized and had a high affinity at the CCM binding location of NTD. However, the RMSD analysis of CCM and CCM+ SPK structures after the 50 ns dynamic simulation exhibited more flexibility at the NTD binding site of CCM than native CCM (b), suggesting the SPK peptide detachment and displacement over the CCM [13].

Figure 6

MHV-1-inoculated mice lost 20–40% of body weight over days 3–7. Treatment of MHV-1-inoculated mice with SPIKENET (3 doses, on days 2, 4, and 6 post-MHV-1 injection with 5 mg/kg) reversed such loss. These findings correlated well with the animal survival post-SPIKENET treatment in SARS-CoV-2-infected K18-hACE2 transgenic mice (n = 10). Error bars represent mean ± SEM [13].

Figure 7

SPIKENET treatment reduces the death rate in MHV-1-infected mice. Female A/J mice were inoculated with MHV-1 intranasally. SPIKENET (5 mg/kg b.wt.) was injected subcutaneously when mice showed sickness (i.e., 2 days after MHV-1 exposure). SPIKENET was injected 2 times every alternate day. SPIKENET diminished the animals’ deaths. ANOVA, n = 5 for control, n = 16 for virus alone, and n = 7 for MHV1 + SPK group. * = p < 0.05 versus control; † = p < 0.05 verses MHV-1 infected mice, Error bars represent mean ± SEM [13].

Figure 8

Elevated edema was observed in MHV-1-infected brain, lung, liver, kidney, and heart, as compared to control, which was similar to patients with COVID-19. Treatment of MHV-1-infected mice with SPK (5 mg/kg; 3 injections from 2 to 6 days) showed edema levels comparable to control levels on day 7. ANOVA, n = 5 for control, n = 16 for virus alone, and n = 7 for MHV1 + SPK group. * = p < 0.05 versus control; † = p < 0.05 verses MHV-1 infected mice, Error bars represent mean ± SEM [13].

Figure 9

Oxidative stress post-MHV-1 infection in mice. (A) Representative immunofluorescence images from four individual animals show increased levels of 4-hydroxynonenol (4-HNE) and malondialdehyde (MDA) in the lung, liver, kidney, brain, and heart. Treatment of MHV-1-inoculated mice with SPK (5 mg/kg) prevented such an increase. (B,C) Quantitation of 4-HNE and MDA levels with and without SPK post-MHV-1 infection. ANOVA, n = 4. * = p < 0.05 versus control; † = p < 0.05 verses MHV-1-infected mice. Scale bar = 25 μm. Error bars represent mean ± SEM [13].

Figure 10

Effect of SPIKENET (SPK) on hydrogen peroxide (H2O2)-induced oxidative stress (protein carbonyl formation) (12 h) (A) and LPS-induced LDH release (36 h) (B) in primary cultures of rat brain microglia, as well as H2O2-induced increase in cell volume (24 h) in primary cultures of rat brain astrocytes (C). SPK significantly diminished these effects in glial cells (30 min post-treatment). Exposure of primary microglia to LPS (24 h) showed an increase in DCF fluorescence (D), as well as an increase in IL-6 level (E) in cell culture medium, and such an increase was diminished and blocked by treatment of cells (30 min post-treatment) with SPIKENET. C, control; LPS, lipopolysaccharide. * = p < 0.05 vs. control. † = p < 0.05 vs. LPS. C, control; AIU, arbitrary intensity units; LPS, lipopolysaccharide. Error bars represent mean ± SEM [13].

Figure 11

Altered AQP levels were identified in various organs post-MHV-1 inoculation. While increased AQP1 was identified in multiple organs, AQP1 levels were decreased in lungs post-MHV-1. Further, treatment of MHV-1-inoculated mice with SPK (5 mg/kg) reversed these changes. Scale bar = 25 μm [13].

Figure 12

SPIKENET (SPK) diminishes MHV-1-induced pathological changes in the brain, lungs, heart, liver, kidney, and skin during long-term infection. Representative histological images of hematoxylin and eosin (H&E) stained brain, lung, heart, liver, kidney, and skin tissue sections of a standard mouse (A,D,G,J,M,P) and an infected mouse at 12 months (B,E,H,K,N,Q). MHV-1-inoculated mice treated with 5 mg/kg SPIKENET (SPK) eased all of these changes (C,F,I,L,O,R) (H&E original magnification is 400× for (A–O) and 66× for (P–R)) [8,13,21].

Figure 13

Altered mRNA expressions 12 months post-MHV-1 infection (chronic). (A) EGFR mRNA level does not significantly change in disease and treatment with SPK. (B) TGF-β1 mRNA level is significantly increased in the infected group, while its mRNA level is decreased substantially following SPK treatment. (C) FGF-23 mRNA level is significantly increased in the infected group, and therapy with SPK leads to substantial elevation. Values are the mean SD of three independent experiments. * = p < 0.05, ** = p < 0.01, and **** = p < 0.0001 are statistically significant. ns = nonsignificant [20].

Figure 14

The MHV-1 coronavirus-mediated signaling systems in various organs result in pathological and functional consequences. SPK ameliorated or prevented such defects in these organs.

Figure 15

MHV-1 coronavirus instigated signaling pathways across diverse organs, leading to pathological and functional repercussions. SPK mitigated or averted such abnormalities within these organs.