FAM72A antagonizes UNG2 to promote mutagenic repair during antibody maturation.

Activation-induced cytidine deaminase (AID) catalyses the deamination of deoxycytidines to deoxyuracils within immunoglobulin genes to induce somatic hypermutation and class-switch recombination1,2. AID-generated deoxyuracils are recognized and processed by subverted base-excision and mismatch repair pathways that ensure a mutagenic outcome in B cells3-6. However, why these DNA repair pathways do not accurately repair AID-induced lesions remains unknown. Here, using a genome-wide CRISPR screen, we show that FAM72A is a major determinant for the error-prone processing of deoxyuracils. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a-/- mice exhibit reduced class-switch recombination and somatic hypermutation frequencies at immunoglobulin and Bcl6 genes, and reduced genome-wide deoxyuracils. The somatic hypermutation spectrum in B cells from Fam72a-/- mice is opposite to that observed in mice deficient in uracil DNA glycosylase 2 (UNG2)7, which suggests that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2, resulting in reduced levels of UNG2 protein in the G1 phase of the cell cycle, coinciding with peak AID activity. FAM72A therefore causes U·G mispairs to persist into S phase, leading to error-prone processing by mismatch repair. By disabling the DNA repair pathways that normally efficiently remove deoxyuracils from DNA, FAM72A enables AID to exert its full effects on antibody maturation. This work has implications in cancer, as the overexpression of FAM72A that is observed in many cancers8 could promote mutagenesis.

Revisiting of Properties and Modified Polyethylenimine-Based Cancer Gene Delivery Systems.

A new era of medical technology in cancer treatment is a directly specific modification of gene expression in tumor cells by nucleic acid delivery. Currently, the main challenge to achieving this goal is to find a non-toxic, safe, and effective strategy for gene transfer to cancer cells. Synthetic composites based on cationic polymers have historically been favored in bioengineering due to their ability to mimic bimolecular structures. Among them, polyethylenimines (PEIs) with superior properties such as a wide range of molecular weight and a flexible structure may propel the development of functional combinations in the biomedical and biomaterial fields. Here, in this review, we will focus on the recent progressions in the formulation optimization of PEI-based polyplex in gene delivery to treat cancer. Also, the effect of PEI's intrinsic characteristics such as structure, molecular weight, and positive charges which influence the gene delivery efficiency will be discussed.

Overexpression of bHLH domain of HIF-1 failed to inhibit the HIF-1 transcriptional activity in hypoxia.

BACKGROUND: Hypoxia inducible factor-1 (HIF-1) is considered as the most activated transcriptional factor in response to low oxygen level or hypoxia. HIF-1 binds the hypoxia response element (HRE) sequence in the promoter of different genes, mainly through the bHLH domain and activates the transcription of genes, especially those involved in angiogenesis and EMT. Considering the critical role of bHLH in binding HIF-1 to the HRE sequence, we hypothesized that bHLH could be a promising candidate to be targeted in hypoxia condition. METHODS: We inserted an inhibitory bHLH (ibHLH) domain in a pIRES2-EGFP vector and transfected HEK293T cells with either the control vector or the designed construct. The ibHLH domain consisted of bHLH domains of both HIF-1a and Arnt, capable of competing with HIF-1 in binding to HRE sequences. The transfected cells were then treated with 200 µM of cobalt chloride (CoCl2) for 48 h to induce hypoxia. Real-time PCR and western blot were performed to evaluate the effect of ibHLH on the genes and proteins involved in angiogenesis and EMT. RESULTS: Hypoxia was successfully induced in the HEK293T cell line as the gene expression of VEGF, vimentin, and ß-catenin were significantly increased after treatment of untransfected HEK293T cells with 200 µM CoCl2. The gene expression of VEGF, vimentin, and ß-catenin and protein level of ß-catenin were significantly decreased in the cells transfected with either control or ibHLH vectors in hypoxia. However, ibHLH failed to be effective on these genes and the protein level of ß-catenin, when compared to the control vector. We also observed that overexpression of ibHLH had more inhibitory effect on gene and protein expression of N-cadherin compared to the control vector. However, it was not statistically significant. CONCLUSION: bHLH has been reported to be an important domain involved in the DNA binding activity of HIF. However, we found that targeting this domain is not sufficient to inhibit the endogenous HIF-1 transcriptional activity. Further studies about the function of critical domains of HIF-1 are necessary for developing a specific HIF-1 inhibitor.

Exhaustion of T lymphocytes in the tumor microenvironment: Significance and effective mechanisms.

T lymphocytes play crucial roles in adaptive immune responses to tumors. However, due to different tolerance mechanisms and inhibitory effects of the tumor microenvironment (TME) on T cells, responses to tumors are insufficient. In fact, cellular and molecular suppressive mechanisms repress T cell responses in the TME, resulting in senescent, anergic and exhausted lymphocytes. Exhaustion is a poor responsive status of T cells, with up-regulated expression of inhibitory receptors, decreased production of effective cytokines, and reduced cytotoxic activity. Low immunogenicity of tumor antigens and inadequate presentation of tumor-specific antigens results in inappropriate activation of naive T lymphocytes against tumor antigens. Moreover, when effector cytotoxic T cells enter TME, they encounter a complicated network of cells and cytokines that suppress their effectiveness and turn them into exhausted T cells. Thus, the mechanism of T cell exhaustion in cancer is different from that in chronic infections. In this review we will discuss the main components such as inhibitory receptors, inflammatory cells, stromal cells, cytokine milieu as well as environmental and metabolic conditions in TME which play role in development of exhaustion. Furthermore, recent therapeutic methods available to overcome exhaustion will be discussed.

The Effect of Licorice Protein Fractions on Proliferation and Apoptosis of Gastrointestinal Cancer Cell Lines.

BACKGROUND: Several methods for the treatment of colon cancer have been introduced, but none of them are safe and effective. We planned to evaluate the inhibitory effect of protein extract of licorice root on HT-29 and CT26 cell lines proliferation and apoptosis. METHODS: Protein extract of licorice root was prepared in phosphate-buffered solution, and SDS-PAGE was used to isolate its fractions. HT-29, CT-26, and HEK293 cell lines were treated with various concentrations of the fractions and full extract of licorice. Cytotoxicity of licorice at various concentrations was assessed using MTT assay. Flow cytometry analysis was applied to evaluate the apoptosis. RESULTS: Our results demonstrated that the concentrations of 5 µg/mL from 25 to 33 kDa fraction and concentration of 8 µg/mL from 62 kDa fraction had a significant inhibitory effect on both cancerous cell lines (P < 0.05), with no significant effect on the noncancerous cell line. The concentrations of 50 and 100 µg/mL from full extracts significantly increased apoptosis in CT26 cells [35.52 ± 7.5 (P = 0.048*) and 47.72 ± 8 (P 0.026*), respectively], but not in HT29 and noncancerous cell lines. CONCLUSIONS: Protein compounds of licorice showed anticancer properties and were able to induce apoptosis in both human colon cancer and mouse colon carcinoma cell lines.

Localization of immunodominant epitopes within the "a" determinant of hepatitis B surface antigen using monoclonal antibodies.

The common "a" determinant is the major immunodominant region of hepatitis B surface antigen (HBsAg) shared by all serotypes and genotypes of hepatitis B virus (HBV). Antibodies against this region are thought to confer protection against HBV and are essential for viral clearance. Mutations within the "a" determinant may lead to conformational changes in this region, which can affect the binding of neutralizing antibodies. There is an increasing concern about identification and control of mutant viruses which is possible by comprehensive structural investigation of the epitopes located within this region. Anti-HBs monoclonal antibodies (mAbs) against different epitopes of HBsAg are a promising tool to meet this goal. In the present study, 19 anti-HBs mAbs were employed to map epitopes localized within the "a" determinant, using a panel of recombinant mutant HBsAgs. The topology of the epitopes was analyzed by competitive enzyme-linked immunosorbent assay (ELISA). Our results indicate that all of the mAbs seem to recognize epitopes within or in the vicinity of the "a" determinant of HBsAg. Different patterns of binding with mutant forms were observed with different mAbs. Amino acid substitutions at positions 123, 126, 129, 144, and 145 dramatically reduced the reactivity of antibodies with HBsAg. The T123N mutation had the largest impact on antibody binding to HBsAg. The reactivity pattern of our panel of mAbs with mutant forms of HBsAg could have important clinical implications for immunoscreening, diagnosis of HBV infection, design of a new generation of recombinant HB vaccines, and immunoprophylaxis of HBV infection as an alternative to therapy with hepatitis B immune globulin (HBIG).

Biomolecular markers in assessment and treatment of asthma.

Asthma is a chronic and heterogeneous inflammatory disorder with several different phenotypes. Whereas clinical features of asthma are non-specific and pulmonary function tests are often insensitive, further development is needed for efficient treatment or even early diagnosis. Recently, several airway inflammatory biomarkers have emerged as valuable tools in diagnosis and management of asthma. The analysis of molecular markers of airways inflammation has provided promising and non-invasive techniques that facilitate the detection of disease phenotypes as well as measurement of therapeutic efficacy. Although conventional treatments remain the preferred therapy, they do not adequately control some severe cases of asthma. Novel therapeutic agents have been developed to target various biomarkers involved in the inflammatory responses and have been investigated in patients with asthma. In this article, we summarized the most studied asthma biomarkers, derived from a variety of biological sources including exhaled gases, induced sputum, serum and urine. Likewise, the effects of current anti-inflammatory asthma treatments on inflammatory biomarkers and some promising biomarkers for developing new targeted therapies are also discussed.

An Updated Review of Interleukin-2 Therapy in Cancer and Autoimmune Diseases.

Interleukin-2 (IL-2) is a cytokine that acts in dual and paradoxical ways in the immunotherapy of cancers and autoimmune diseases. Numerous clinical trial studies have shown that the use of different doses of this cytokine in various autoimmune diseases, transplantations, and cancers has resulted in therapeutic success. However, side effects of varying severity have been observed in patients. In recent years, to prevent these side effects, IL-2 has been engineered to bind more specifically to its receptors on the cell surface, decreasing IL-2 toxicities in patients. In this review article, we focus on some recent clinical trial studies and analyze them to determine the appropriate dose of IL-2 drug with the least toxicities. In addition, we discuss the engineering performed on IL-2, which shows that engineered IL-2 increases the specificity function of IL-2 and decreases its adverse effects.

The Evaluation of the N-cadherin Promoter's ability to Block EMT by Specific Expression of Diphtheria Toxin in EMT-induced A549 Cell Lines.

During epithelial to mesenchymal transition, the ability of cancer cells to transform and metastasize is primarily determined by N-cadherin-mediated migration and invasion. This study aimed to evaluate whether the N-cadherin promoter can induce diphtheria toxin expression as a suicide gene in epithelial to mesenchymal transition (EMT)-induced cancer cells and whether this can be used as potential gene therapy. To investigate the expression of diphtheria toxin under the N-cadherin promoter, the promoter was synthesized, and was cloned upstream of diphtheria toxin in a pGL3-Basic vector. The A-549 cells was transfected by electroporation. After induction of EMT by TGF-ß and hypoxia treatment, the relative expression of diphtheria toxin, mesenchymal genes such as N-cadherin and Vimentin, and epithelial genes such as E-cadherin and ß-catenin were measured by real-time PCR. MTT assay was also performed to measure cytotoxicity. Finally, cell motility was assessed by the Scratch test. After induction of EMT in transfected cells, the expression of mesenchymal markers such as Vimentin and N-cadherin significantly decreased, and the expression of ß-catenin increased. In addition, the MTT assay showed promising toxicity results after induction of EMT with TGF-ß in transfected cells, but toxicity was less effective in hypoxia. The scratch test results also showed that cell movement was successfully prevented in EMT-transfected cells and thus confirmed EMT occlusion. Our findings indicate that by using structures containing diphtheria toxin downstream of a specific EMT promoter such as the N-cadherin promoter, the introduced toxin can kill specifically and block EMT in cancer cells.

Competitive Effect of Overexpressed C-terminal of Snail-1 (CSnail) in Control of the Growth and Metastasis of Melanoma Cells.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) plays a role in the invasion and metastasis of cancer cells. During this phenomenon, Snail can promote tumor progression by upregulating mesenchymal factors and downregulating the expression of pro-apoptotic proteins. OBJECTIVE: Therefore, interventions on the expression rate of Snails may show beneficial therapeutic applications. METHODS: In this study, the C-terminal region of Snail1, capable of binding to E-box genomic sequences, was subcloned into the pAAV-IRES-EGFP backbone to make complete AAV-CSnail viral particles. B16F10 as a metastatic melanoma cell line, with a null expression of wild type TP53 was transduced by AAV-CSnail. Moreover, the transduced cells were analyzed for in vitro expression of apoptosis, migration, and EMT-related genes, and in vivo inhibition of metastasis. RESULTS: In more than 80% of the AAV-CSnail transduced cells, the CSnail gene expression competitively reduced the wild-type Snail functionality and consequently lowered the mRNA expression level of EMT-related genes. Furthermore, the transcription level of cell cycle inhibitory factor p21 and pro-apoptotic factors were promoted. The scratch test showed a decrease in the migration ability of AAV-CSnail transduced group compared to control. Finally, metastasis of cancer cells to lung tissue in the AAV-CSnail-treated B16F10 melanoma mouse model was significantly reduced, pointing out to prevention of EMT by the competitive inhibitory effect of CSnail on Snail1 and increased apoptosis of B16F10 cells. CONCLUSION: The capability of this successful competition in reducing the growth, invasion, and metastasis of melanoma cells indicates that gene therapy is a promising strategy for the control of the growth and metastasis of cancer cells.

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.

Allergen-specific immunotherapy by recombinant Der P1 allergen-derived peptide-based vaccine in an allergic mouse model.

AIM: Increased IgE levels have made house dust mite allergens one of the most frequent causes of allergies worldwide. Treatment reduces the IgE antibodies and types two cytokines, namely interleukin-4 (IL-4) and IL-13. Although existing treatments significantly reduce IgE or IL-4/IL-13, they are very costly. This study aimed to construct a recombinant protein derived from rDer p1 peptides in the form of an immunotherapy approach and to measure the response of IgE and IgG antibodies. METHODS: The proteins were isolated, purified, and evaluated using the SDS-PAGE and Bradford test and confirmed by using Western blot. To evaluate immunotherapy efficiency, 24 BALB/C mice were sensitized intraperitoneally with house dust mites (HDM) adsorbed to Aluminum hydroxide (Alum) and randomly divided into four groups of six: control sensitized, HDM extract, rDer p1, and DpTTDp vaccine. To immunization, four groups of random mice were each treated with phosphate-buffered saline, 100 µg of rDer p1 protein, DpTTDp, or HDM extract, every 3 days. Direct ELISA determined HDM-specific IgG and IgE subclasses. Data were analyzed in SPSS and Graph pad prism software. Values of p < .05 were considered significant. RESULTS: After immunization of mice, the rDer P1 and recombinant vaccine like HDM extract increased IgG antibody titer and decreased IgE-dependent reactivity in allergic mice to rDer P1. Also, the levels of inflammatory IL-4 and IL-13 cytokines as allergic stimulants decreased. CONCLUSION: The use of present available recombinant proteins is considered a viable, cost-effective, and long-term option for providing effective HDM allergy immunotherapy vaccines without side effects.

Reversing T Cell Exhaustion by Converting Membrane PD-1 to Its Soluble form in Jurkat Cells; Applying The CRISPR/Cas9 Exon Skipping Strategy.

OBJECTIVE: T-cells express two functional forms of the programmed cell death protein 1 (PD-1): membrane (mPD-1) and soluble (sPD-1). The binding of mPD-1 and its ligand (PD-L1) on tumor cells could lead activated lymphocytes toward exhaustion. Selective deletion of the transmembrane domain via alternative splicing of exon-3 in PD-1 mRNA could generate sPD-1. Overexpression of sPD-1 could disrupt the mPD-1/PD-L1 interaction in tumor-specific T cells. We investigated the effect of secreted sPD-1 from pooled engineered and non-engineered T cell supernatant on survival and proliferation of lymphocytes in the tumor microenvironment (TME). MATERIALS AND METHODS: In this experimental study, we designed two sgRNA sequences upstream and downstream of exon-3 in the PDCD1 gene. The lentiCRISPRv2 puro vector was used to clone the dual sgRNAs and produce lentiviral particles to transduce Jurkat T cells. Analysis assays were used to clarify the change in PD-1 expression pattern in the pooled (engineered and non-engineered) Jurkat cells. Co-culture conditions were established with PD-L1+ cancer cells and lymphocytes. RESULTS: CRISPR/Cas9 could delete exon-3 of the PDCD1 gene in the engineered cells based on the tracking of indels by decomposition (TIDE) and interference of CRISPR edit (ICE) sequencing analysis reports. Our results showed a 12% reduction in mPD-1 positive cell population after CRISPR manipulation and increment in sPD-1 concentration in the supernatant. The increased sPD-1 confirmed its positive effect on proliferation of lymphocytes co-cultured with PDL1+ cancer cells. The survival percent of lymphocytes co-cultured with the pooled cells supernatant was 12.5% more than the control. CONCLUSION: The CRISPR/Cas9 exon skipping approach could be used in adoptive cell immunotherapies to change PD-1 expression patterns and overcome exhaustion.

The immunostimulant effects of the rice ragged stunt virus genome on the growth and metastasis of breast cancer in mouse model.

There are multiple treatment strategies that have been reported for breast cancer, while new and effective therapies against it are still necessary. Stimulating the immune system and its components against cancer cells is one of the unique treatment strategies of immunotherapy and long dsRNAs are immunostimulant in this regard. Based on bioinformatics approaches, a fragment of the Rice ragged stunt RNA virus genome was selected and synthesized according to its immunogenicity. Based on the in vitro transcription technique, dsRNA was synthesized and its binding ability to the PEI/PEI-Ac Polyethylenimine (PEI) or Acetylated polyethylenimine (PEI-Ac) was verified by the gel retardation assay. Then, the PEI-Ac was synthesized by adding acetyl groups to the PEI, and the results of the 1H NMR method indicated its successful synthesis. After cancer induction by 4 T1 cells in Balb/C mice, intraperitoneal (IP) and intratumoral (IT) treatment by the PEI/PEI-Ac-dsRNA were performed and the tumor growth inhibition was evaluated. Results demonstrated that PEI/PEI-Ac-dsRNA can lead to a decrease in tumor weight and volume in both the IP and IT routes. Also, by using macro-metastatic nodule counting and hematoxylin and eosin (H&E) staining we showed that PEI/PEI-Ac-dsRNA can prevent micro and macro-metastasis in the lung. Therefore, the PEI/PEI-Ac-dsRNA acts as an effective inhibitor of growth and metastasis of the breast cancer models. We showed that viral dsRNA can exert its antitumor properties by stimulating TNF-α and IFN-γ. In general, our results revealed that dsRNA derived from the plant virus genome stimulates the intrinsic immune system and can be a potential immune stimulant drug for cancer treatment.

Cobalt Chloride-induced Hypoxia Can Lead SKBR3 and HEK293T Cell Lines toward Epithelial-mesenchymal Transition.

Hypoxia is a common characteristic of the tumor microenvironment. In response to hypoxia, expression of the hypoxia-inducible factor (HIF) can lead to activation of downstream molecular events such as epithelial-mesenchymal transition (EMT), invasion, and angiogenesis. In this study, CoCl2 was used to simulate hypoxia in SKBR3 and HEK293T cell lines to investigate whether this treatment can induce hypoxia-associated EMT and invasion in the studied cells. SKBR3 and HEK293T cells were treated with different concentrations of CoCl2 at different exposure times and their viability was analyzed. To confirm successful hypoxia induction, the expression levels of HIF1α and vascular endothelial growth factor A (VEGFA) mRNA were assessed. Additionally, the expression of EMT-associated markers including snail, E-cadherin, N-cadherin, and vimentin, as well as invasion-related genes including matrix metalloproteinase-2 (MMP2) and MMP9 was measured. We found that cell viability in CoCl2-treated cells was concentration-dependent and was not affected at low doses. While the expression of HIF and VEGFA genes was upregulated following hypoxia induction. E-cadherin expression was significantly downregulated in HEK293T cells; while, N-cadherin and snail were upregulated in both cell lines. Moreover, an increment of MMP expression was only observed in SKBR3 cells. Taken together, the findings indicated that CoCl2 can mimic hypoxia in both cell lines, but EMT was triggered in SKBR3 cells more effectively than in HEK293T cells, and invasion was only stimulated in SKBR3 cells. In conclusion, SKBR3 cancer cells can be used as an EMT model to better understand its control and manipulation mechanisms and to investigate new therapeutic targets for the suppression of tumor metastasis.

A Comprehensive Overview of Allergen-specific Immunotherapy Types, Recombinant and Natural Extract Allergens in the Diagnosis and Treatment of Allergies.

Allergen-specific immunotherapy (AIT) involves administering allergen extracts. It is used to desensitize allergic patients. Herbal allergen extracts that are optimum in efficacy and fewest in side effects are still challenging to produce. To overcome these limitations, oral immunotherapy, epicutaneous immunotherapy, intralymphatic immunotherapy, and artificial recombinant allergen preparations have been evaluated. Recombinant allergens have become more popular with the development of molecular diagnostics and therapeutics. Besides food and drug allergens, pollen, fungal spores, and other allergens have been studied. Based on related clinical studies, this comprehensive overview will present the latest perspectives on AIT methods and available allergenic products, as well as discuss the challenges and opportunities for treating allergic disorders.

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.

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).

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.