Recombinant highly antigenic truncated fusion-based protein as a diagnostic antigen for anti-SARS-CoV-2 nucleocapsid antibody ELISA.

Among the main structural protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), nucleocapsid phosphoprotein (NP) exhibits high immunogenicity and is the most abundant viral protein produced and shed during infection. Detection of antibodies against NP may help assess the number of individuals exposed to SARS-COV-2 or vaccinated against it. Based on these findings and other structural and antigenic evaluations, we designed a recombinant truncated fusion NP-based protein for application in an immunoassay for detecting immunoglobulins in patients who have recovered from COVID-19. In this research, we aligned the NPs from SARS-CoV and SARS-CoV-2 and selected highly antigenic parts of the SARS-CoV-2 sequences based on in-silico studies. The protein was expressed under optimum conditions in the bacterial host BL21 and purified by nickel immobilized metal affinity chromatography. Moreover, the purity level was assessed by SDS-PAGE and Western blotting whereas the folding of the protein was evaluated by circular dichroism. Ultimately, we used the purified recombinant protein in ELISA development in which 42 samples from convalescent patients were compared with 20 samples of the past 2019 patients who had attended laboratories for various clinical check-ups. The sensitivity and specificity were determined as 71% and 90%, respectively, in the optimum cut-off point measured by the receiver operating characteristic curve.

Molecular mechanisms and therapeutic effects of different vitamins and minerals in COVID-19 patients.

COVID-19 is a rapidly spreading disease, which has caught the world by surprise. Millions of people suffer from illness, and the mortality rates are dramatically high. Currently, there is no specific and immediate treatment for this disease. Remedies are limited to supportive regiments and few antiviral and anti-inflammatory drugs. The lack of a definite cure for COVID-19 is the reason behind its high mortality and global prevalence. COVID-19 can lead to a critical illness with severe respiratory distress and cytokine release. Increased oxidative stress and excessive production of inflammatory cytokines are vital components of severe COVID-19. Micronutrients, metalloids, and vitamins such as iron, manganese, selenium, Zinc, Copper, vitamin A, B family, and C are among the essential and trace elements that play a pivotal role in human nutrition and health. They participate in metabolic processes that lead to energy production. In addition, they support immune functions and act as antioxidants. Therefore, maintaining an optimal level of micronutrients intake, particularly those with antioxidant activities, is essential to fight against oxidative stress, modulate inflammation, and boost the immune system. Therefore, these factors could play a crucial role in COVID-19 prevention and treatment. In this review, we aimed to summarize antiviral properties of different vitamins and minerals. Moreover, we will investigate the correlation between them and their effects in COVID-19 patients.

A unique antigen against SARS-CoV-2, Acinetobacter baumannii, and Pseudomonas aeruginosa.

The recent outbreak of COVID-19 has increased hospital admissions, which could elevate the risk of nosocomial infections, such as A. baumannii and P. aeruginosa infections. Although effective vaccines have been developed against SARS-CoV-2, no approved treatment option is still available against antimicrobial-resistant strains of A. baumannii and P. aeruginosa. In the current study, an all-in-one antigen was designed based on an innovative, state-of-the-art strategy. In this regard, experimentally validated linear epitopes of spike protein (SARS-CoV-2), OmpA (A. baumannii), and OprF (P. aeruginosa) were selected to be harbored by mature OmpA as a scaffold. The selected epitopes were used to replace the loops and turns of the barrel domain in OmpA; OprF311-341 replaced the most similar sequence within the OmpA, and three validated epitopes of OmpA were retained intact. The obtained antigen encompasses five antigenic peptides of spike protein, which are involved in SARS-CoV-2 pathogenicity. One of these epitopes, viz. QTQTNSPRRARSV could trigger antibodies preventing super-antigenic characteristics of spike and alleviating probable autoimmune responses. The designed antigen could raise antibodies neutralizing emerging variants of SARS-CoV-2 since at least two epitopes are consensus. In conclusion, the designed antigen is expected to raise protective antibodies against SARS-CoV-2, A. baumannii, and P. aeruginosa.

Recombinant subunits of SARS‐CoV‐2 spike protein as vaccine candidates to elicit neutralizing antibodies

ObjectivesThe spike protein has been reported as one of the most critical targets for vaccine design strategies against the SARS‐CoV‐2 infection. Hence, we have designed, produced, and evaluated the potential use of three truncated recombinant proteins derived from spike protein as vaccine candidates capable of neutralizing SARS‐CoV‐2 virus.MethodsIn silico tools were used to design spike‐based subunit recombinant proteins (RBD (P1), fusion peptide (P2), and S1/S2 cleavage site (P3)). These proteins were checked for their ability to be identified by the anti‐SARS‐CoV‐2 antibodies by exposing them to COVID‐19 serum samples. The proteins were also injected into mice and rabbit, and the antibody titers were measured for 390 days to assess their neutralization efficiency.ResultsThe antibodies that existed in the serum of COVID‐19 patients were identified by designed proteins. The anti‐spike antibody titer was increased in the animals injected with recombinant proteins. The VNT results revealed that the produced antibodies could neutralize the cultured live virus.ConclusionTruncated subunit vaccines could also be considered as robust tools for effective vaccination against COVID‐19. Using a combination of in silico, in vitro, and in vivo experiments, it was shown that the injection of spike‐based truncated recombinant proteins could stimulate long‐lasting and neutralizing antibody responses.

Pierce into Structural Changes of Interactions Between Mutated Spike Glycoproteins and ACE2 to Evaluate Its Potential Biological and Therapeutic Consequences.

The structural consequences of ongoing mutations on the SARS-CoV-2 spike-protein remains to be fully elucidated. These mutations could change the binding affinity between the virus and its target cell. Moreover, obtaining new mutations would also change the therapeutic efficacy of the designed drug candidates. To evaluate these consequences, 3D structure of a mutant spike protein was predicted and checked for stability, cavity sites, and residue depth. The docking analyses were performed between the 3D model of the mutated spike protein and the ACE2 protein and an engineered therapeutic ACE2 against COVID-19. The obtained results revealed that the N501Y substitution has altered the interaction orientation, augmented the number of interface bonds, and increased the affinity against the ACE2. On the other hand, the P681H mutation contributed to the increased cavity size and relatively higher residue depth. The binding affinity between the engineered therapeutic ACE2 and the mutant spike was significantly higher with a distinguished binding orientation. It could be concluded that the mutant spike protein increased the affinity, preserved the location, changed the orientation, and altered the interface amino acids of its interaction with both the ACE2 and its therapeutic engineered version. The obtained results corroborate the more aggressive nature of mutated SARS-CoV-2 due to their higher binding affinity. Moreover, designed ACe2-baased therapeutics would be still highly effective against covid-19, which could be the result of conserved nature of cellular ACE2. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10989-021-10346-1.

Hotspots for mutations in the SARS-CoV-2 spike glycoprotein: a correspondence analysis.

Spike glycoprotein (Sgp) is liable for binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the host receptors. Since Sgp is the main target for vaccine and drug designing, elucidating its mutation pattern could help in this regard. This study is aimed at investigating the correspondence of specific residues to the SgpSARS-CoV-2 functionality by explorative interpretation of sequence alignments. Centrality analysis of the Sgp dissects the importance of these residues in the interaction network of the RBD-ACE2 (receptor-binding domain) complex and furin cleavage site. Correspondence of RBD to threonine500 and asparagine501 and furin cleavage site to glutamine675, glutamine677, threonine678, and alanine684 was observed; all residues are exactly located at the interaction interfaces. The harmonious location of residues dictates the RBD binding property and the flexibility, hydrophobicity, and accessibility of the furin cleavage site. These species-specific residues can be assumed as real targets of evolution, while other substitutions tend to support them. Moreover, all these residues are parts of experimentally identified epitopes. Therefore, their substitution may affect vaccine efficacy. Higher rate of RBD maintenance than furin cleavage site was predicted. The accumulation of substitutions reinforces the probability of the multi-host circulation of the virus and emphasizes the enduring evolutionary events.

SARS-CoV-2 Proteome Harbors Peptides Which Are Able to Trigger Autoimmunity Responses: Implications for Infection, Vaccination, and Population Coverage.

Autoimmune diseases (ADs) could occur due to infectious diseases and vaccination programs. Since millions of people are expected to be infected with SARS-CoV-2 and vaccinated against it, autoimmune consequences seem inevitable. Therefore, we have investigated the whole proteome of the SARS-CoV-2 for its ability to trigger ADs. In this regard, the entire proteome of the SARS-CoV-2 was chopped into more than 48000 peptides. The produced peptides were searched against the entire human proteome to find shared peptides with similar experimentally confirmed T-cell and B-cell epitopes. The obtained peptides were checked for their ability to bind to HLA molecules. The possible population coverage was calculated for the most potent peptides. The obtained results indicated that the SARS-CoV-2 and human proteomes share 23 peptides originated from ORF1ab polyprotein, nonstructural protein NS7a, Surface glycoprotein, and Envelope protein of SARS-CoV-2. Among these peptides, 21 peptides had experimentally confirmed equivalent epitopes. Amongst, only nine peptides were predicted to bind to HLAs with known global allele frequency data, and three peptides were able to bind to experimentally confirmed HLAs of equivalent epitopes. Given the HLAs which have already been reported to be associated with ADs, the ESGLKTIL, RYPANSIV, NVAITRAK, and RRARSVAS were determined to be the most harmful peptides of the SARS-CoV-2 proteome. It would be expected that the COVID-19 pandemic and the vaccination against this pathogen could significantly increase the ADs incidences, especially in populations harboring HLA-B*08:01, HLA-A*024:02, HLA-A*11:01 and HLA-B*27:05. The Southeast Asia, East Asia, and Oceania are at higher risk of AD development.

The immunology of SARS-CoV-2 infection, the potential antibody based treatments and vaccination strategies.

Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a potentially fatal agent for a new emerging viral disease (COVID-19) is of great global public health emergency. Herein, we represented potential antibody-based treatments especially monoclonal antibodies (mAbs) that may exert a potential role in treatment as well as developing vaccination strategies against COVID-19.Areas covered: We used PubMed, Google Scholar, and clinicaltrials.gov search strategies for relevant papers. We demonstrated some agents with potentially favorable efficacy as well as favorable safety. Several therapies are under assessment to evaluate their efficacy and safety for COVID19. However, the development of different strategies such as SARS-CoV-2-based vaccines and antibody therapy are urgently required beside other effective therapies such as plasma, anticoagulants, and immune as well as antiviral therapies. We encourage giving more attention to antibody-based treatments as an immediate strategy. Although there has not been any approved specific vaccine until now, developing vaccination strategies may have a protective effect against COVID-19.Expert opinion: An antiviral mAbs could be a safe and high-quality therapeutic intervention which is greatly recommended for COVID-19. Additionally, the high sequence homology between the SARS-CoV-2 and SARS/MERS viruses could shed light on developing to design a vaccine against SARS-CoV-2.

Design of an engineered ACE2 as a novel therapeutics against COVID-19.

The interaction between the angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a pivotal role in virus entry into the host cells. Since recombinant ACE2 protein has been suggested as an anti-SARS-CoV-2 therapeutic agent, this study was conducted to design an ACE2 protein with more desirable properties. In this regard, the amino acids with central roles in enzymatic activity of the ACE2 were substituted. Moreover, saturation mutagenesis at the interaction interface between the ACE2 and RBD was performed to increase their interaction affinity. The best mutations to increase the structural and thermal stability of the ACE2 were also selected based on B factors and mutation effects. The obtained resulted revealed that the Arg273Gln and Thr445Gly mutation have drastically reduced the binding affinity of the angiotensin-II into the active site of ACE2. The Thr27Arg mutation was determined to be the most potent mutation to increase the binding affinity. The Asp427Arg mutation was done to decrease the flexibility of the region with high B factor. The Pro451Met mutation along with the Gly448Trp mutation was predicted to increase the thermodynamic stability and thermostability of the ACE2. The designed therapeutic ACE2 would have no enzymatic activity while it could bear stronger interaction with Spike glycoprotein of the SARS-CoV-2. Moreover, decreased in vivo enzymatic degradation would be anticipated due to increased thermostability. This engineered ACE2 could be exploited as a novel therapeutic agent against COVID-19 after necessary evaluations.