The orchestration of the recruitment process in response to the SARS-CoV-2 spike protein involves a unique role for macrophage-activation signaling.

In a recent work that was published in Scientific Reports, researchers created peptides from the SARS-CoV-2 spike (S) protein to analyze the inflammatory alterations linked to the coronavirus illness 2019 (COVID-19) using zebrafish models.

Despite vaccination and other mitigation measures, the COVID-19 pandemic nonetheless overwhelms healthcare institutions around the world and affects millions of people. The efficacy of current treatments has been jeopardized by the ongoing appearance of novel SARS-CoV-2 variants of concern (VOCs), which has necessitated the development of innovative medicines.

Enhancing our knowledge of the pathophysiology of COVID-19 may help us create more potent treatments that will raise the bar for medical care and lessen the COVID-19 burden worldwide. Since manipulating the complete virus necessitates high biosafety standards, alternative approaches, such as peptide synthesis from SARS-CoV-2 proteins, may offer a more practical and speedy answer.

Concerning the study
The goal of the current work was to see whether the pathophysiology of COVID-19 could be clarified using zebrafish models and the peptide synthesis technique.

Two peptides, PSPD2002 and PSPD2003, were created by the team using an in-silico study of the SARS-CoV-2 spike (S) protein, and they were verified both in vitro and in vivo. With the use of anti-S immunoglobulin G (IgG), the peptides were measured. To extract neutrophils and macrophages and evaluate their activation and inflammatory responses to the peptide challenge in vitro, fluorescence-activated cell sorting (FACS) was used.

After six days of fertilization, the peptides were then administered into transgenic zebrafish larvae to measure macrophage polarization, survival, and oxidative stress indicators. High-performance liquid chromatography (HPLC) was used to separate the peptides from the SARS-CoV-2 spike protein. To assess the impact of the peptides on ACE2 activity, human angiotensin-converting enzyme 2 (ACE2) from the Saccharomyces cerevisiae strain was produced in BY4742 yeast.

Additionally, simulations of molecular dynamics (MD) were carried out. The COVID-19-related inflammation was assessed using confocal microscopy (CFM), and zebrafish splenic, hepatic, intestinal, and muscular tissues were collected for histological analysis. Cytotoxicity tests and measurements of oxidative stress were carried out.

Nitric oxide (NO), thiobarbituric acid reactive species (TBARS), reactive oxygen species (ROS), and hydrogen peroxide (H2O2) were all indicators of oxidative stress. ELISAs, or enzyme-linked immunosorbent assays, were used to quantify protein levels. To evaluate membrane-bound L-selectin (CD62L)-based neutrophil activation using flow cytometry and cytotoxicity using murine alveolar macrophage AMJ2-C11 cells, respectively.

The peptides came from regions with active S-ACE2 interaction. The S protein-based peptide compounds interacted with adhesion molecules and other receptors from zebrafish and humans, including the T-cell receptor (TCR) and major histocompatibility complex (MHC), according to in-silico experiments and MD simulations. They were also shown to be stably bound to ACE-2 receptors.

In zebrafish and humans, PSPD2002 exhibited binding to ACE2 receptors with values of 7.4 and -8.0 kcal per mol, respectively. For PSPD2003, the comparable affinity values were -8.2 and -8.1 kcal per mol. Nitric oxide, C-X-C motif ligand-2 (CXCL-2), and tumor necrosis factor-alpha (TNF-) production in macrophages activated by peptides was found to be enhanced.

Similar to the changes seen in COVID-19 patients, peptide inoculation in zebrafish larvae triggered inflammatory processes defined by the recruitment of macrophages, histological abnormalities, and increased mortality. Both peptides were detectable by anti-S IgG and could be measured in vitro. They interacted with zebrafish and human immune receptors and showed substantial antigenic potential. The peptides may therefore be employed in the creation of COVID-19 vaccines and other treatments.

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Nitric oxide production by PSPD2003 was shown to be dose-dependent, and PSPD2003 also increased the synthesis of CXCL2 and TNF. PSPD2002 and PSPD2003, administered at concentrations of 10.0 and 100.0 g/ml, respectively, activated neutrophils; however, regardless of the presence or absence of lipopolysaccharide (LPS), the peptides were unable to alter CD62L activity on the cellular surface. The results showed that the peptides caused pro-inflammatory reactions that were mediated by macrophages.

Animal survival rates were decreased by PSPD2002 (10.0 g/ml) and PSPD2003 (1.0 and 10.0 g/ml), and PSPD2003 caused more intense inflammation than PSPD2002, indicating that it was more cytotoxic than PSPD2002. Higher peptide doses produced inflammation more quickly, reaching a peak two days after vaccination and then resolving more quickly. The peptide infusions reduced the levels of H2O2 and SOD but increased the formation of malondialdehyde (MDA). While PSPD2003 raised catalase (CAT) levels, PSPD2002 decreased nitrite levels.

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The redox profiles of the zebrafish larvae were associated with the degree of immune infiltration. Findings from the docking analysis demonstrated that all peptide-antioxidant interactions had affinity levels that were negatively above the 6.00 kcal per mol threshold. The scientists measured affinities of -6.70 and 7.20 kcal per mol for PSPD2022 interactions with CAT and SOD, respectively. For PSPD2003, the matching enzymes’ affinity values were -7.20 and 6.60 kcal per mol, respectively.

Overall, the study’s findings suggested that using peptide synthesis to assess SARS-CoV-2-associated inflammation in the host would be a useful strategy. Zebrafish may also be employed in animal experiments to mimic COVID-19-related inflammation in people.

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