The potential use of bacteriophages as antibacterial agents against Klebsiella pneumoniae.

One of the most common bacteria that cause nosocomial infections is Klebsiella pneumonia (K. pneumoniae), especially in patients who are very sick and admitted to the intensive care unit (ICU). The frequency of multi-drug-resistant Klebsiella pneumoniae (MDRKP) has dramatically increased worldwide in recent decades, posing an urgent threat to public health. The Western world's bacteriophage (phage) studies have been revitalized due to the increasing reports of antimicrobial resistance and the restricted development and discovery of new antibiotics. These factors have also spurred innovation in other scientific domains. The primary agent in phage treatment is an obligately lytic organism (called bacteriophage) that kills the corresponding bacterial host while sparing human cells and lessening the broader effects of antibiotic usage on commensal bacteria. Phage treatment is developing quickly, leading to many clinical studies and instances of life-saving medicinal use. In addition, phage treatment has a few immunological adverse effects and consequences in addition to its usefulness. Since K. pneumoniae antibiotic resistance has made treating multidrug-resistant (MDR) infections challenging, phage therapy (PT) has emerged as a novel therapeutic strategy. The effectiveness of phages has also been investigated in K. pneumoniae biofilms and animal infection models. Compared with antibiotics, PT exhibits numerous advantages, including a particular lysis spectrum, co-evolution with bacteria to avoid the emergence of phage resistance, and a higher abundance and diversity of phage resources than found in antibiotics. Moreover, phages are eliminated in the absence of a host bacterium, which makes them the only therapeutic agent that self-regulates at the sites of infection. Therefore, it is essential to pay attention to the role of PT in treating these infections. This study summarizes the state of knowledge on Klebsiella spp. phages and provides an outlook on the development of phage-based treatments that target K. pneumoniae in clinical trials.

Evaluation of the in vitro and in vivo efficiency of in-feed bacteriophage cocktail application to control Salmonella Typhimurium and Salmonella Enteritidis infection in broiler chicks.

RESEARCH HIGHLIGHTS: Bacteriophage (BP) cocktail was partially resistant to different temperatures and pH values.The BP cocktail showed lytic effects on different Salmonella isolates.The BP cocktail reduced Salmonella colonization in the internal organs of broilers.

Design of a multi-epitope-based vaccine consisted of immunodominant epitopes of structural proteins of SARS-CoV-2 using immunoinformatics approach.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown rapid global spread and has resulted in a significant death toll worldwide. In this study, we aimed to design a multi-epitope vaccine against SARS-CoV-2 based on structural proteins S, M, N, and E. We identified B- and T-cell epitopes and then the antigenicity, toxicity, allergenicity, and similarity of predicted epitopes were analyzed. T-cell epitopes were docked with corresponding HLA alleles. Consequently, the selected T- and B-cell epitopes were included in the final construct. All selected epitopes were connected with different linkers and flagellin and pan-HLA DR binding epitopes (PADRE) as an adjuvant were used in the vaccine construct. Furthermore, molecular docking was used to evaluate the complex between the final vaccine construct and two alleles, HLA-A*02:01 and HLA-DRB1*01:01. Finally, codons were optimized for in silico cloning into pET28a(+) vector using SnapGene. The final vaccine construct comprised 11 CTL, HTL, and B-cell epitopes corresponding to 394 amino acid residues. In silico evaluation showed that the designed vaccine might potentially promote an immune response. Further in vivo preclinical and clinical testing is required to determine the safety and efficacy of the designed vaccine.

New role of bacteriophages in medical oncology.

Targeted treatment of cancer is one of the most paramount approaches in cancer treatment. Despite significant advances in cancer diagnosis and treatment methods, there are still significant limitations and disadvantages in the field, including high costs, toxicity, and unwanted damage to healthy cells. The phage display technique is an innovative method for designing carriers containing exogenic peptides with cancer diagnostic and therapeutic properties. Bacteriophages possess unique properties making them effective in cancer treatment. These characteristics include the small size enabling them to penetrate vessels; having no pathogenicity to mammals; easy manipulation of their genetic information and surface proteins to introduce vaccines and drugs to cancer tissues; lower cost of large-scale production; and greater stimulation of the immune system. Bacteriophages will certainly play a more effective role in the future of medical oncology; however, studies are in the early stages of conception and require more extensive research. We aimed in this review to provide some related examples and bring insights into the potential of phages as targeted vectors for use in cancer diagnosis and treatment, especially regarding their capability in gene and drug delivery to cancer target cells, determination of tumor markers, and vaccine design to stimulate anticancer immunity.

Exosomes derived from rapamycin-treated 4T1 breast cancer cells induced polarization of macrophages to M1 phenotype.

M2 macrophages are the most prevalent type in the tumor microenvironment and their polarization to M1 type can be used as a potential cancer immunotherapy. Here, we investigated the role of tumor microenvironment and particularly purified exosomes in M2 to M1 macrophage polarization. Rapamycin treatment on triple-negative breast cancer cells (TNBC) was performed. Tumor cells-derived exosomes (called texosomes) were isolated and characterized using scanning electron microscopy, transmission electron microscopy, dynamic light scattering, high-performance liquid chromatography, Fourier transform infrared, and Western blot assays. M2 mouse peritoneal macrophages were treated with rapamycin or rapamycin-texosome. Then, M1/M2 phenotype-specific marker genes and proteins were measured to assess the degree of M2 to M1 polarization. Finally, nitric oxide (NO) production, phagocytosis, and efferocytosis assays were assessed to verify the functionality of the polarized macrophages. Purified rapamycin-texosomes significantly increased the expression of the M1 markers (Irf5, Nos2, and CD86) and decreased M2 markers (Arg, Ym1, and CD206). In addition, the levels of M1-specific cytokines tumor necrosis factor alpha and interleukin 1ß (IL-1ß) were increased, whereas the levels of M2 specific cytokines IL-10 and transforming growth factor beta were declined. Furthermore, texosome treatment increased NO concentration and phagocytosis and decreased efferocytosis indicating M1 polarization. These findings suggest rapamycin-texosomes can induce M2 to M1 macrophages polarization as a potential immunotherapy for TNBC.

Overview of the pre-clinical and clinical studies about the use of CAR-T cell therapy of cancer combined with oncolytic viruses.

BACKGROUND: Cancer is one of the critical issues of the global health system with a high mortality rate even with the available therapies, so using novel therapeutic approaches to reduce the mortality rate and increase the quality of life is sensed more than ever. MAIN BODY: CAR-T cell therapy and oncolytic viruses are innovative cancer therapeutic approaches with fewer complications than common treatments such as chemotherapy and radiotherapy and significantly improve the quality of life. Oncolytic viruses can selectively proliferate in the cancer cells and destroy them. The specificity of oncolytic viruses potentially maintains the normal cells and tissues intact. T-cells are genetically manipulated and armed against the specific antigens of the tumor cells in CAR-T cell therapy. Eventually, they are returned to the body and act against the tumor cells. Nowadays, virology and oncology researchers intend to improve the efficacy of immunotherapy by utilizing CAR-T cells in combination with oncolytic viruses. CONCLUSION: Using CAR-T cells along with oncolytic viruses can enhance the efficacy of CAR-T cell therapy in destroying the solid tumors, increasing the permeability of the tumor cells for T-cells, reducing the disturbing effects of the immune system, and increasing the success chance in the treatment of this hazardous disease. In recent years, significant progress has been achieved in using oncolytic viruses alone and in combination with other therapeutic approaches such as CAR-T cell therapy in pre-clinical and clinical investigations. This principle necessitates a deeper consideration of these treatment strategies. This review intends to curtly investigate each of these therapeutic methods, lonely and in combination form. We will also point to the pre-clinical and clinical studies about the use of CAR-T cell therapy combined with oncolytic viruses.

Detection of COVID-19 in tears of ICU-admitted patients with SARS-CoV-2 infection.

PURPOSE: Upon the outbreak of 2019, novel coronavirus (COVID-19) pandemic confirmed the cases surpassed 20 million. Despite a few reports identified the association of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with ocular manifestations, it may assess the ocular symptoms of patients with the COVID-19 by ophthalmologists facilitate the diagnosis and prevent transmission. METHODS: A total of 60 patients with the COVID-19 admitted to Baghiatallah hospital from March 2020 to May 2020 were retrospectively reviewed and analyzed for the ocular manifestations, blood tests, and reverse transcriptase-polymerase chain reaction (RT-PCR) for SARS-CoV-2 using nasopharyngeal and conjunctival swabs. RESULTS: Among 60 included patients with clinically confirmed COVID-19, the median age 58.36 years (IQR: 30-88 years), 27 (45%) were male. Furthermore, 29 (48%) and 5 (8%) patients yielded positive for SARS-CoV-2 on RT-PCR from nasopharyngeal swabs and conjunctival specimens, respectively. Among 60 patients, 10 (16%) and 3 (5%), respectively, had the ocular manifestations and positive results for SARS-CoV-2 on RT-PCR from conjunctival and nasopharyngeal swabs. CONCLUSION: Although the positive rate of tear RT-PCR rate is not noticeable as nasopharyngeal swabs yet, COVID-19 transmission through the eyes is biologically plausible.

Droplet digital PCR of viral DNA/RNA, current progress, challenges, and future perspectives.

High-throughput droplet-based digital PCR (ddPCR) is a refinement of the conventional polymerase chain reaction (PCR) methods. In ddPCR, DNA/RNA is encapsulated stochastically inside the microdroplets as reaction chambers. A small percentage of the reaction chamber contains one or fewer copies of the DNA or RNA. After PCR amplification, concentrations are determined based on the proportion of nonfluorescent partitions through the Poisson distribution. Some of the main features of ddPCR include high sensitivity and specificity, absolute quantification without a standard curve, high reproducibility, good tolerance to PCR inhibitor, and high efficacy compared to conventional molecular methods. These advantages make ddPCR a valuable addition to the virologist's toolbox. The following review outlines the recent technological advances in ddPCR methods and their applications in viral identification.

Detection of West Nile virus by real-time PCR in crows in northern provinces of Iran.

BACKGROUND & OBJECTIVES: West Nile virus (WNV) is a positive-sense, single-stranded RNA virion, that belongs to the Flaviviridae family. This virus is preserved in a bird-mosquito cycle that is capable of inducing diseases as a dead-end or endpoint host in humans as well as horses. In 2016, a suspicious case of crow population death was reported by the Department of Environment, Ministry of Health, Iran. Considering the mass migration of birds together with the WNV-related symptoms, including uncoordinated walking, ataxia, inability to fly, lack of awareness, and abnormal body posture, it was necessary to further investigate the possible causes of this incident. The objective of this study was molecular detection of WNV in crows utilizing the real-time PCR method in the northern provinces of Iran. METHODS: A total of 12 crows (8 dead, 4 alive) with a possible WNV infection, were collected from the northern provinces of Iran (Golestan, Mazandaran, and Guilan). A tissue sample of the liver, kidney, or lung was collected from all the crows, and RNA was isolated using an RNA extraction kit. A one-step real-time PCR method using a TaqMan probe was used for virus detection. RESULTS: All the infected crows were positive for WNV. The 132-bp real-time PCR amplicon of the genome was detected in all the samples. Comparative phylogenetic analysis revealed that WNV isolated from Iran clustered with strains from the USA, Hungary, and Culex pipiens. INTERPRETATION & CONCLUSION: The WNV genome sequence was detected in all the infected crows. The results confirmed the connection of this isolation with clade1a strains. Hence, determining the epidemiologic and prevalence characteristics of the WNV for transmission control is of critical importance in Iran.

The molecular epidemiology of respiratory viruses in military trainees in Iran.

Background: Military populations are more prone to respiratory infections worldwide. There is a dearth of research about the role of viral pathogens in the etiology of respiratory infections in military trainees in Iran. Hence, we aimed to investigate the molecular epidemiology and clinical symptoms of respiratory viruses among this population. Methods: This cross-sectional study was performed on 400 military trainees with symptoms of respiratory infection, referred to the military medical clinic center in the basic military training camp of the General Staff of the Armed Forces of the Islamic Republic of Iran. Nucleic acid extraction from the throat or nasopharyngeal swab samples was performed by an automated extraction system. The extracts were then analyzed by the CLART® PneumoVir array system for the detection of respiratory viruses. Results: All military trainees were male, aged between 18 and 57 years (mean: 21.69 years). Sore throat (75.5%), rhinorrhea (63.2%), cough (59.2%), fever (59.2%), and nasal congestion (50.5%) were amongst the most common symptoms. Overall, viral pathogens were detected in a total count of 124 (31%). The most commonly detected viruses were rhinovirus (7.2%), respiratory syncytial virus A (7.2%) and influenza B virus (6%). Conclusion: This study was an important first step for understanding the etiological role of viral pathogens in respiratory infection among military trainees population in Iran. Our results indicated that rhinovirus, respiratory syncytial virus A and influenza B virus are important viral pathogens causing respiratory infection in military trainees, respectively. However, further multi-center studies with larger sample size are strongly recommended to confirm our findings.

Human endogenous retrovirus env genes: Potential blood biomarkers in lung cancer.

Lung cancer, the leading cause of cancer mortality, needs urgent development of newly qualified diagnostic and therapeutic biomarkers. Recently, Human Endogenous Retroviruses (HERVs) have been introduced for cancer diagnosis. In this case-control study, we have collected blood samples from 60 lung cancer patients and 20 healthy controls. Quantitative gene expression analysis of various HERV env genes, including HERV-R, HERV-H, HERV-K, and HERV-P was performed by real-time PCR. Results indicate that expression of all four HERV env mRNAs is significantly increased in the blood of lung cancer patients than healthy controls (P-values<0.01). Furthermore, we have observed a positive and significant pairwise correlation between the expressions of four HERV env genes. The level of HERV env transcript in the blood of adenocarcinoma patients was generally much higher than squamous cell carcinoma (SCC) and small-cell lung cancer (SCLC) patients. Also, the expression of three HERV P, HERV H, and HERV K in the blood of lung cancer patients could significantly differentiate between adenocarcinoma and other types of lung cancer. In conclusion, these four HERV families could be considered as promising non-invasive blood-based biomarkers for prognosis, early detection, and monitoring of lung cancer.

The c-Met receptor: Implication for targeted therapies in colorectal cancer.

c-Met (mesenchymal-epithelial transition factor) is a tyrosine kinase receptor activated by hepatocyte growth factor and regulates multiple biological processes, such as cell scattering, survival, and proliferation. Aberrant c-Met signaling has been implicated in a variety of cancer types, including colorectal cancer. c-Met is genetically altered through various mechanisms that is associated with colorectal cancer progression and metastasis. Especially, in colorectal cancer, preclinical evidence for the aberrant activation of the c-Met signaling exists. Accordingly, molecular targeting of c-Met receptor could be a promising strategy, in the treatment of colorectal cancer patients. Recently, it was also shown that crosstalk between c-Met and other cell surface receptors attributes to tumorigenesis and development of therapeutic resistance. Characterization of the molecular mechanisms through which c-Met crosstalks with other receptors in favor of tumor formation and progression remains to explore. This review will describe the mechanisms of aberrant c-Met signaling in colorectal cancer and discuss on additional roles for c-Met receptor through crosstalk with other tyrosine kinase receptors and cell surface proteins in colorectal cancer. Novel therapeutic approaches for c-Met pathway targeting will also be discussed.

Selection of single chain antibody fragments binding to the extracellular domain of 4-1BB receptor by phage display technology.

The 4-1BB is a surface glycoprotein that pertains to the tumor necrosis factor-receptor family. There is compelling evidence suggesting important roles for 4-1BB in the immune response, including cell activation and proliferation and also cytokine induction. Because of encouraging results of different agonistic monoclonal antibodies against 4-1BB in the treatment of cancer, infectious, and autoimmune diseases, 4-1BB has been suggested as an attractive target for immunotherapy. In this study, single chain variable fragment phage display libraries, Tomlinson I+J, were screened against specific synthetic oligopeptides (peptides I and II) designed from 4-1BB extracellular domain. Five rounds of panning led to selection of four 4-1BB specific single chain variable fragments (PI.12, PI.42, PII.16, and PII.29) which showed specific reaction to relevant peptides in phage enzyme-linked immunosorbent assay. The selected clones were successfully expressed in Escherichia coli Rosetta-gami 2, and their expression was confirmed by western blot analysis. Enzyme-linked immunosorbent assay experiments indicated that these antibodies were able to specifically recognize 4-1BB without any cross-reactivity with other antigens. Flow cytometry analysis demonstrated an acceptable specific binding of the single chain variable fragments to 4-1BB expressed on CCRF-CEM cells, while no binding was observed with an irrelevant antibody. Anti-4-1BB single chain variable fragments enhanced surface CD69 expression and interleukin-2 production in stimulated CCRF-CEM cells which confirmed the agonistic effect of the selected single chain variable fragments. The data from this study have provided a rationale for further experiments involving the biological functions of anti-4-1BB single chain variable fragments in future studies.

The Effects of Mesenchymal Stem Cells Loaded with Oncolytic Coxsackievirus A21 on Mouse Models of Colorectal Cancer.

BACKGROUND: Cancer is a major cause of death worldwide. Colorectal cancer is the second most common type. Additional treatments like chemotherapy and radiation therapy may be recommended. Developing new techniques is vital due to drug resistance and a lack of targeted therapies. OBJECTIVE: In this study, the effects of mesenchymal stem cells (MSCs) loaded with oncolytic Coxsackievirus A21 (CVA21) on a mouse model of CRC were investigated. METHODS: The therapeutic potency of MSCs loaded with oncolytic CVA21 were evaluated in an experimental mouse model of colorectal cancer which received an injection CT26 cells per mouse subcutaneously. Splenocyte proliferation index, lactate dehydrogenase (LDH) assay, nitric oxide (NO) production assessment, and cytokine assay (IFN-γ, IL-4, IL-10, and TGF-ß) in the splenocyte supernatant were all used to evaluate the impact of MSCs loaded with CVA21. RESULTS: The results of this study showed that the treatment of a mouse model of colorectal cancer with MSCs loaded with oncolytic CVA21 could significantly suppress the tumor growth, which was accompanied by stimulation of splenocytes proliferation index, an increase of NO and LDH. Also, MSCs loaded with oncolytic CVA21 increased the secretion of IFN-γ and decreased the secretion of IL-4, IL-10, and TGF-ß. CONCLUSION: The results of the current study suggest that MSCs loaded with oncolytic CVA21 therapy for the CRC mouse model may have some potential advantages. On the other hand, the results of the study showed that, in addition to activating the acquired immune system, the use of MSCs loaded with oncolytic CVA21 also stimulates the innate immune system by increasing level of nitric oxide.

MicroRNA-122 Is More Effective than Rapamycin in Inhibition of Epithelial-mesenchymal Transition and mTOR Signaling Pathway in Triple Negative Breast Cancer.

The fundamental mechanism responsible for the aggressiveness of metastatic cancers such as triple-negative breast cancer (TNBC) is the epithelial-mesenchymal transition (EMT). In cancer microenvironments, the Phosphoinositide 3-kinases (PI3K)-Akt- mammalian target of rapamycin (mTOR) signaling pathway plays a critical role in regulating the EMT mechanism. The current study focuses on the impacts of rapamycin, a newly retargeted chemotherapeutic agent against mTOR, and MicroRNA (miR)-122 on the aggressive behavior of TNBC.  The half-maximal inhibitory concentration (IC50) of rapamycin on 4T1 cells was determined using an MTT assay. Also, miR-122 was transiently transfected into 4T1 cells to study its effect on the pathway. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to assess the expression level of central mTOR and EMT-related cascade genes. Moreover, cell mobility and migration were evaluated using scratch and migration assays, respectively. Both rapamycin and miR-122 significantly decreased the expression levels of PI3K, AKT, and mTOR, as well as ZeB1 and Snail genes. However, no significant change was observed in Twist gene expression. Furthermore, scratch and migration assays revealed that the migration of 4T1 cells was markedly reduced, especially following miR-122 induction. Our experimental findings and gene enrichment studies indicated that miR-122 mainly operates on multiple metabolic pathways, as well as EMT and mTOR, while rapamycin has restricted targets in cancer cells.  Consequently, miR-122 can be considered a potential cancer microRNA therapy option, which can be validated in the future in animal studies to demonstrate its efficacy in cancer control.

Role of Long Non-coding RNA HSD17B3-AS1 in Trauma for COVID-19.

COVID-19, an acute respiratory syndrome caused by the SARS-CoV-2 virus, was first reported in late 2019 in Wuhan, China, and rapidly escalated into a global pandemic. The condition can lead to organ dysfunction and ultimately death through its onset of acute respiratory distress syndrome (ARDS). Disease severity has been linked to proinflammatory cytokines which activate the NF-κB and STAT transcription factors in infected cells. It has been proven that lncRNAs play a very important role in reducing or increasing inflammatory factors. This makes them potentially valuable in recognizing pathogenesis pathways and therapeutic targets in COVID-19. Nanocurcumin is known as an antioxidant, tumor suppressor and anti-inflammatory substance, and it can be effective to reduce inflammation caused by the disease of COVID-19. This study analyzed Sequence Read Archive data from COVID-19 patients with acute versus milder symptoms, identifying dysregulated genes and non-coding RNAs. To verify this correlation, the expression of the candidate gene was evaluated with quantitative polymerase chain reaction (qPCR) in mouse models, while immunoglobulin (Ig) G titer was measured using enzyme-linked immunosorbent assay (ELISA) in mouse serum samples. Here we introduced a novel lncRNA called HSD17B3-AS1, suggested as a therapeutic target in COVID-19 patients with acute symptoms. Furthermore, we revealed nanocurcumin is reducing the expression of HSD17B3-AS1 which leads to reduced inflammation in mice. These results suggest that HSD17B3-AS1 plays a significant regulatory role in managing COVID-19, and the downregulation of HSD17B3-AS1 by Nanocurcumin presents a promising treatment option for minimizing complications in COVID-19 patients.

Antiproliferative effects of mesenchymal stem cells carrying Newcastle disease virus and Lactobacillus Casei extract on CT26 Cell line: synergistic effects in cancer therapy.

BACKGROUND AND AIMS: Colorectal Cancer (CRC) is a frequent malignancy with a high mortality rate. Specific inherited and environmental influences can affect CRC. Oncolytic viruses and bacteria in treating CRC are one of the innovative therapeutic options. This study aims to determine whether mesenchymal stem cells (MSCs) infected with the Newcastle Disease Virus (NDV) in combination with Lactobacillus casei extract (L. casei) have a synergistic effects on CRC cell line growth. MATERIALS AND METHODS: MSCs taken from the bone marrow of BALB/c mice and were infected with the 20 MOI of NDV. Then, using the CT26 cell line in various groups as a single and combined treatment, the anticancer potential of MSCs containing the NDV and L. casei extract was examined. The evaluations considered the CT26 survival and the rate at which LDH, ROS, and levels of caspases eight and nine were produced following various treatments. RESULTS: NDV, MSCs-NDV, and L. casei in alone or combined treatment significantly increased apoptosis percent, LDH, and ROS production compared with the control group (P˂0.05). Also, NDV, in free or capsulated in MSCs, had anticancer effects, but in capsulated form, it had a delay compared with free NDV. The findings proved that L. casei primarily stimulates the extrinsic pathway, while NDV therapy promotes apoptosis through the activation of both intrinsic and extrinsic apoptosis pathways. CONCLUSIONS: The results suggest that MSCs carrying oncolytic NDV in combination with L. casei extract as a potentially effective strategy for cancer immunotherapy by promoting the generation of LDH, ROS, and apoptosis in the microenvironment of the CT26 cell line.

Recombinant expression of SARS-CoV-2 receptor binding domain (RBD) in Escherichia coli and its immunogenicity in mice.

Objectives: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), giving rise to the coronavirus disease 2019 (COVID-19), has become a danger to wellbeing worldwide. Thus, finding efficient and safe vaccines for COVID-19 is of great importance. As a basic step amid contamination, SARS-CoV-2 employs the receptor-binding domain (RBD) of the spike protein to lock in with the receptor angiotensin-converting enzyme 2 (ACE2) on host cells. SARS-CoV-2 receptor-binding domain (RBD) is the main human antibody target for developing vaccines and virus inhibitors, as well as neutralizing antibodies. A bacterial procedure was developed for the expression and purification of the SARS-CoV-2 spike protein receptor-binding domain. Materials and Methods: In this research study, RBD was expressed by Escherichia coli and purified with Ni-NTA chromatography. Then it was affirmed by the western blot test. The immunogenicity and protective efficacy of RBD recombinant protein were assessed on BALB/c mice. Additionally, RBD recombinant protein was tested by ELISA utilizing sera of COVID-19 healing patients contaminated with SARS-CoV-2 wild type and Delta variation. Results: Indirect ELISA was able to detect the protein RBD in serum of the immunized mouse expressed in E. coli. The inactive SARS-CoV2 was detected by antibodies within the serum of immunized mice. Serum antibodies from individuals recovered from Covid19 reacted to the expressed protein. Conclusion: Our findings showed that RBD is of great importance in vaccine design and it can be used to develop recombinant vaccines through induction of antibodies against RBD.