The Trend of CRISPR-Based Technologies in COVID-19 Disease: Beyond Genome Editing.

Biotechnological approaches have always sought to utilize novel and efficient methods in the prevention, diagnosis, and treatment of diseases. This science has consistently tried to revolutionize medical science by employing state-of-the-art technologies in genomic and proteomic engineering. CRISPR-Cas system is one of the emerging techniques in the field of biotechnology. To date, the CRISPR-Cas system has been extensively applied in gene editing, targeting genomic sequences for diagnosis, treatment of diseases through genomic manipulation, and in creating animal models for preclinical researches. With the emergence of the COVID-19 pandemic in 2019, there is need for the development and modification of novel tools such as the CRISPR-Cas system for use in diagnostic emergencies. This system can compete with other existing biotechnological methods in accuracy, precision, and wide performance that could guarantee its future in these conditions. In this article, we review the various platforms of the CRISPR-Cas system meant for SARS-CoV-2 diagnosis, anti-viral therapeutic procedures, producing animal models for preclinical studies, and genome-wide screening studies toward drug and vaccine development.

Epitope mapping of severe acute respiratory syndrome coronavirus 2 neutralizing receptor binding domain-specific monoclonal antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the outbreak led to the coronavirus disease 2019 (COVID-19) pandemic. Receptor binding domain (RBD) of spike (S) protein of SARS-CoV-2 is considered as a major target for immunotherapy and vaccine design. Here, we generated and characterized a panel of anti-RBD monoclonal antibodies (MAbs) isolated from eukaryotic recombinant RBD-immunized mice by hybridoma technology. Epitope mapping was performed using a panel of 20-mer overlapping peptides spanning the entire sequence of the RBD protein from wild-type (WT) Wuhan strain by enzyme-linked immunosorbent assay (ELISA). Several hybridomas showed reactivity toward restricted RBD peptide pools by Pepscan analysis, with more focus on peptides encompassing aa 76-110 and 136-155. However, our MAbs with potent neutralizing activity which block SARS-CoV-2 spike pseudovirus as well as the WT virus entry into angiotensin-converting enzyme-2 (ACE2) expressing HEK293T cells showed no reactivity against these peptides. These findings, largely supported by the Western blotting results suggest that the neutralizing MAbs recognize mainly conformational epitopes. Moreover, our neutralizing MAbs recognized the variants of concern (VOC) currently in circulation, including alpha, beta, gamma, and delta by ELISA, and neutralized alpha and omicron variants at different levels by conventional virus neutralization test (CVNT). While the neutralization of MAbs to the alpha variant showed no substantial difference as compared with the WT virus, their neutralizing activity was lower on omicron variant, suggesting the refractory effect of mutations in emerging variants against this group of neutralizing MAbs. Also, the binding reactivity of our MAbs to delta variant showed a modest decline by ELISA, implying that our MAbs are insensitive to the substitutions in the RBD of delta variant. Our data provide important information for understanding the immunogenicity of RBD, and the potential application of the novel neutralizing MAbs for passive immunotherapy of SARS-CoV-2 infection.

Does prior immunization with measles, mumps, and rubella vaccines contribute to the antibody response to COVID-19 antigens?

BACKGROUND: Incidence and severity of SARS-CoV2 infection are significantly lower in children and teenagers proposing that certain vaccines, routinely administered to neonates and children may provide cross-protection against this emerging infection. OBJECTIVE: To assess the cross-protection induced by prior measles, mumps and rubella (MMR) vaccinations against COVID-19. METHODS: The antibody responses to MMR and tetanus vaccines were determined in 53 patients affected with SARS-CoV2 infection and 52 age-matched healthy subjects. Serum levels of antibodies specific for NP and RBD of SARS-CoV2 were also determined in both groups of subjects with ELISA. RESULTS: Our results revealed significant differences in anti-NP (P<0.0001) and anti-RBD (P<0.0001) IgG levels between patients and healthy controls. While the levels of rubella- and mumps specific IgG were not different in the two groups of subjects, measles-specific IgG was significantly higher in patients (P<0.01). The serum titer of anti-tetanus antibody, however, was significantly lower in patients compared to healthy individuals (P<0.01). CONCLUSION: Our findings suggest that measles vaccination triggers those B cells cross-reactive with SARS-CoV2 antigens leading to the production of increased levels of measles-specific antibody.

Epitope mapping of severe acute respiratory syndrome coronavirus 2 neutralizing receptor binding domain-specific monoclonal antibodies

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the outbreak led to the coronavirus disease 2019 (COVID-19) pandemic. Receptor binding domain (RBD) of spike (S) protein of SARS-CoV-2 is considered as a major target for immunotherapy and vaccine design. Here, we generated and characterized a panel of anti-RBD monoclonal antibodies (MAbs) isolated from eukaryotic recombinant RBD-immunized mice by hybridoma technology. Epitope mapping was performed using a panel of 20-mer overlapping peptides spanning the entire sequence of the RBD protein from wild-type (WT) Wuhan strain by enzyme-linked immunosorbent assay (ELISA). Several hybridomas showed reactivity toward restricted RBD peptide pools by Pepscan analysis, with more focus on peptides encompassing aa 76–110 and 136–155. However, our MAbs with potent neutralizing activity which block SARS-CoV-2 spike pseudovirus as well as the WT virus entry into angiotensin-converting enzyme-2 (ACE2) expressing HEK293T cells showed no reactivity against these peptides. These findings, largely supported by the Western blotting results suggest that the neutralizing MAbs recognize mainly conformational epitopes. Moreover, our neutralizing MAbs recognized the variants of concern (VOC) currently in circulation, including alpha, beta, gamma, and delta by ELISA, and neutralized alpha and omicron variants at different levels by conventional virus neutralization test (CVNT). While the neutralization of MAbs to the alpha variant showed no substantial difference as compared with the WT virus, their neutralizing activity was lower on omicron variant, suggesting the refractory effect of mutations in emerging variants against this group of neutralizing MAbs. Also, the binding reactivity of our MAbs to delta variant showed a modest decline by ELISA, implying that our MAbs are insensitive to the substitutions in the RBD of delta variant. Our data provide important information for understanding the immunogenicity of RBD, and the potential application of the novel neutralizing MAbs for passive immunotherapy of SARS-CoV-2 infection.

COVID-19: A systematic review and update on prevention, diagnosis, and treatment.

Since the rapid onset of the COVID-19 or SARS-CoV-2 pandemic in the world in 2019, extensive studies have been conducted to unveil the behavior and emission pattern of the virus in order to determine the best ways to diagnosis of virus and thereof formulate effective drugs or vaccines to combat the disease. The emergence of novel diagnostic and therapeutic techniques considering the multiplicity of reports from one side and contradictions in assessments from the other side necessitates instantaneous updates on the progress of clinical investigations. There is also growing public anxiety from time to time mutation of COVID-19, as reflected in considerable mortality and transmission, respectively, from delta and Omicron variants. We comprehensively review and summarize different aspects of prevention, diagnosis, and treatment of COVID-19. First, biological characteristics of COVID-19 were explained from diagnosis standpoint. Thereafter, the preclinical animal models of COVID-19 were discussed to frame the symptoms and clinical effects of COVID-19 from patient to patient with treatment strategies and in-silico/computational biology. Finally, the opportunities and challenges of nanoscience/nanotechnology in identification, diagnosis, and treatment of COVID-19 were discussed. This review covers almost all SARS-CoV-2-related topics extensively to deepen the understanding of the latest achievements (last updated on January 11, 2022).

Identification of immunodominant epitopes on nucleocapsid and spike proteins of the SARS-CoV-2 in Iranian COVID-19 patients.

Given the emergence of SARS-CoV-2 virus as a life-threatening pandemic, identification of immunodominant epitopes of the viral structural proteins, particularly the nucleocapsid (NP) protein and receptor-binding domain (RBD) of spike protein, is important to determine targets for immunotherapy and diagnosis. In this study, epitope screening was performed using a panel of overlapping peptides spanning the entire sequences of the RBD and NP proteins of SARS-CoV-2 in the sera from 66 COVID-19 patients and 23 healthy subjects by enzyme-linked immunosorbent assay (ELISA). Our results showed that while reactivity of patients' sera with reduced recombinant RBD protein was significantly lower than the native form of RBD (P < 0.001), no significant differences were observed for reactivity of patients' sera with reduced and non-reduced NP protein. Pepscan analysis revealed weak to moderate reactivity towards different RBD peptide pools, which was more focused on peptides encompassing amino acids (aa) 181-223 of RBD. NP peptides, however, displayed strong reactivity with a single peptide covering aa 151-170. These findings were confirmed by peptide depletion experiments using both ELISA and western blotting. Altogether, our data suggest involvement of mostly conformational disulfide bond-dependent immunodominant epitopes in RBD-specific antibody response, while the IgG response to NP is dominated by linear epitopes. Identification of dominant immunogenic epitopes in NP and RBD of SARS-CoV-2 could provide important information for the development of passive and active immunotherapy as well as diagnostic tools for the control of COVID-19 infection.

Differential Antibody Response to SARS-CoV-2 Antigens in Recovered and Deceased Iranian COVID-19 Patients.

The coronavirus infectious disease 2019 (COVID-19), which is initiated by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has imposed critical challenges to global health. Understanding the kinetic of SARS-CoV-2-specific IgM and IgG responses in different subsets of COVID-19 patients is crucial to get insight into the humoral immune response elicited against the virus. We investigated IgM and IgG responses against SARS-CoV-2 nucleocapsid (N) and receptor-binding domain (RBD) of spike protein in two groups of recovered and deceased COVID-19 patients. The levels of IgM and IgG specific to N and RBD proteins were detected by ELISA. N- and RBD-specific IgM was higher in deceased patients in comparison with recovered patients, while there was no significant difference in N- and RBD-specific IgG between the two groups. A significant correlation was observed between IgG and IgM titers against RBD and N, in both groups of patients. These results argue against impaired antibody response in deceased COVID-19 patients.