In silico and in vitro evaluation of a PE38 and Nb-based recombinant immunotoxin targeting the GRP78 receptor in cancer cells.

Cancer is a global health problem despite the most developed therapeutic modalities. The delivery of specific therapeutic agents to a target increases the effectiveness of cancer treatment by reducing side effects and post-treatment issues. Our aim in this study was to design a recombinant protein consisting of nanobody molecules and exotoxin that targets the surface GRP78 receptor on tumor cells. Bioinformatics methods make drug design and recombinant protein evaluation much easier before the laboratory steps. Two constructs were designed from a single-variable domain on heavy chain nanobody domains and PE toxin domains II, Ib, and III. The physicochemical properties, secondary structure, and solubility of the chimeric protein were analyzed using different software. Prostate cancer DU-145 and breast cancer MDA-MB-468 cell lines were used as GRP78-positive and negative controls, respectively. Accordingly, the cytotoxicity, binding affinity, cell internalization, and apoptosis were evaluated using MTT, enzyme-linked immunosorbent assay, and western blot. The results showed that in the DU-145 cell line, the cytotoxicity of two recombinant immunotoxins is dose and time-dependent. In MDA-MB-468 and HEK-293 cells, such an event does not occur. It is possible that two constructs designed for immunotoxins can attach to GRP78-positive cancer cells and then eradicate cancer cells by internalization and apoptosis. As our in vitro results were in line with in silico data confirming the Bioinformatics predictions, it can be concluded that the designed recombinant immunotoxins may exhibit therapeutic potential against GRP78-positive tumor cells.

The Assessment of Anti-SARS-CoV-2 Antibodies in Different Vaccine Platforms: A Systematic Review and Meta-Analysis of COVID-19 Vaccine Clinical Trial Studies.

BACKGROUND AND OBJECTIVE: The COVID-19 pandemic spread rapidly throughout the world and caused millions of deaths globally. Several vaccines have been developed to control the COVID-19 pandemic and reduce the burden it placed on public health. This study aimed to assess the efficacy of different vaccine platforms in inducing potent antibody responses. Moreover, the seroconversion rate and common side effects of vaccine platforms were evaluated. METHODS: This meta-analysis included clinical trials of COVID-19 vaccines that met the eligibility criteria. Electronic databases (including PubMed, Scopus, and Web of Science) and Google Scholar search engine were searched for eligible studies. Regarding the methodological heterogeneity between the included studies, we selected a random-effects model. The geometric mean ratio (GMR) was chosen as the effect size for this meta-analysis. RESULTS: Of the 1838 records identified through screening and after removing duplicate records, the full texts of 1076 records were assessed for eligibility. After the full-text assessment, 56 records were eligible and included in the study. Overall, vaccinated participants had a 150.8-fold increased rate of anti-spike IgG titres compared with the placebo group (GMR = 150.8; 95% CI, 95.9-237.1; I2 = 100%). Moreover, vaccinated participants had a 37.3-fold increased rate of neutralising antibody titres compared with the placebo group (GMR = 37.3; 95% CI, 28.5-48.7; I2 = 99%). The mRNA platform showed a higher rate of anti-spike IgG (GMR = 1263.5; 95% CI, 431.1-3702.8; I2 = 99%), while neutralising antibody titres were higher in the subunit platform (GMR = 53.4; 95% CI, 32.8-87.1; I2 = 99%) than in other platforms. Different vaccine platforms showed different rates of both anti-spike IgG and neutralising antibody titres with interesting results. The seroconversion rate of anti-spike IgG and neutralising antibody titres was more than 98% in the vaccinated participants. CONCLUSION: Inactivated and subunit vaccines produced a high percentage of neutralising antibodies and had a low common adverse reaction rate compared to other platforms. In this regard, subunit and inactivated vaccines can still be used as the main vaccine platforms for effectively controlling infections with high transmission rates.

In vitro analysis of single chain variable fragment-based immunotoxins against Erythropoietin-producing hepatocellular A2 receptor overexpressed in breast cancer cells.

Breast cancer is one of the leading causes of cancer deaths worldwide. Thereafter, designing new treatments with higher specificity and efficacy is urgently required. In this regard, targeted immunotherapy using immunotoxins has shown great promise in treating cancer. To target a breast cancer cell, the authors used the antibody fragment against a receptor tyrosine kinase, EphA2, which is overexpressed in many cancers. This fragment was conjugated to a plant toxin, subunit A of ricin, in two different orientations from N to C-terminal (EphA2- C-Ricin and EphA2- N-Ricin). Then, these two immunotoxins were characterized using in vitro cell-based assays. Three different cell lines were treated, MDA-MB-231 (breast cancer) which has a high level of EphA2 expression, MCF-7 (breast cancer) which has a low level of EphA2 expression, and HEK293 (human embryonic kidney) which has a very low level of EphA2 expression. Moreover, binding ability, cytotoxicity, internalization, and apoptosis capacity of these two newly developed immunotoxins were investigated. The flow cytometry using Annexin V- Propidium iodide (PI) method indicated significant induction of apoptosis only in the MDA-MB-231 cells at different concentrations. It was also found that construct I, EphA2- C-Ricin immunotoxin, could bind, internalize, and induce apoptosis better than the EphA2- N-Ricin immunotoxin. In addition, the obtained data suggested that the N or C-terminal orientation conformation is of significant importance.

Signal-On Fluorescence Biosensor for Detection of miRNA-21 Based on ROX labeled Specific Stem-Loop Probe.

Background: The abnormal expression of microRNA (miRNA) influences RNA transcription and protein translation, leading to tumor progression and metastasis. Today, reliably identifying aberrant miRNA expression remains challenging, especially when employing quick, simple, and portable detection methods. Objectives: This study aimed to diagnose and detect the miR-21 biomarker with high sensitivity and specificity. Methods: Our detection approach involves immobilizing ROX dye-labeled single-stranded DNA probes (ROX-labeled ssDNA) onto MWCNTs to detect target miRNA-21. Initially, adsorbing ROX-labeled ssDNA onto MWCNTs causes fluorescence quenching of ROX. Subsequently, introducing its complementary DNA (cDNA) forms double-stranded DNA (dsDNA), which results in the desorption and release from MWCNTs, thus restoring ROX fluorescence. Results: The study examined changes in fluorescence intensities before and after hybridization with miRNA-21. The fluorescence emission intensities responded linearly to increases in miR-21 concentration from 10-9 to 3.2 × 10-6 M. The developed fluorescence sensor exhibited a detection limit of 1.12 × 10-9 M. Conclusions: This work demonstrates that using a nano-biosensor based on carbon nanotubes offers a highly sensitive method for the early detection of colorectal cancer (CRC), supplementing existing techniques.

Screening and identification of DNA nucleic acid aptamers against F1 protein of Yersinia pestis using SELEX method.

BACKGROUND: Yersinia pestis is a bacterium that causes the disease plague. It has caused the deaths of many people throughout history. The bacterium possesses several virulence factors (pPla, pFra, and PYV). PFra plasmid encodes fraction 1 (F1) capsular antigen. F1 protein protects the bacterium against host immune cells through phagocytosis process. This protein is specific for Y. pestis. Many diagnostic techniques are based on molecular and serological detection and quantification of F1 protein in different food and clinical samples. Aptamers are small nucleic acid sequences that can act as specific ligands for many targets.This study, aimed to isolate the high-affinity ssDNA aptamers against F1 protein. METHODS AND RESULTS: In this study, SELEX was used as the main strategy in screening aptamers. Moreover, enzyme-linked aptamer sorbent assay (ELASA) and surface plasmon resonance (SPR) were used to determine the affinity and specificity of obtained aptamers to F1 protein. The analysis showed that among the obtained aptamers, the three aptamers of Yer 21, Yer 24, and Yer 25 were selected with a KD value of 1.344E - 7, 2.004E - 8, and 1.68E - 8 M, respectively. The limit of detection (LoD) was found to be 0.05, 0.076, and 0.033 µg/ml for Yer 21, Yer 24, and Yer 25, respectively. CONCLUSION: This study demonstrated that the synthesized aptamers could serve as effective tools for detecting and analyzing the F1 protein, indicating their potential value in future diagnostic applications.

Immunogenicity of different nanoparticle adjuvants containing recombinant RBD coronavirus antigen in animal model.

Ongoing mutations of SARS-CoV-2 present challenges for vaccine development, promising renewed global efforts to create more effective vaccines against coronavirus disease (COVID-19). One approach is to target highly immunogenic viral proteins, such as the spike receptor binding domain (RBD), which can stimulate the production of potent neutralizing antibodies. This study aimed to design and test a subunit vaccine candidate based on the RBD. Bioinformatics analysis identified antigenic regions of the RBD for recombinant protein design. In silico analysis identified the RBD region as a feasible target for designing a recombinant vaccine. Bioinformatics tools predicted the stability and antigenicity of epitopes, and a 3D model of the RBD-angiotensin-converting enzyme 2 complex was constructed using molecular docking and codon optimization. The resulting construct was cloned into the pET-28a (+) vector and successfully expressed in Escherichia coli BL21DE3. As evidenced by sodium dodecyl-polyacrylamide gel electrophoresis and Western blotting analyses, the affinity purification of RBD antigens produced high-quality products. Mice were immunized with the RBD antigen alone or combined with aluminum hydroxide (AlOH), calcium phosphate (CaP), or zinc oxide (ZnO) nanoparticles (NPs) as adjuvants. Enzyme-linked immunosorbent assay assays were used to evaluate immune responses in mice. In-silico analysis confirmed the stability and antigenicity of the designed protein structure. RBD with CaP NPs generated the highest immunoglobulin G titer compared to AlOH and ZnO after three doses, indicating its effectiveness as a vaccine platform. In conclusion, the recombinant RBD antigen administered with CaP adjuvant NPs induces potent humoral immunity in mice, supporting further vaccine development. These results contribute to ongoing efforts to develop more effective COVID-19 vaccines.

Unveiling promising immunogenic targets in Coxiella burnetii through in silico analysis: paving the way for novel vaccine strategies.

BACKGROUND: Coxiella burnetii, an intracellular pathogen, serves as the causative agent of zoonotic Q fever. This pathogen presents a significant threat due to its potential for airborne transmission, environmental persistence, and pathogenicity. The current whole-cell vaccine (WCV) utilized in Australia to combat Q fever exhibits notable limitations, including severe adverse reactions and limited regulatory approval for human use. This research employed the reverse vaccinology (RV) approach to uncover antigenic proteins and epitopes of C. burnetii, facilitating the development of more potent vaccine candidates. METHODS: The potential immunogenic proteins derived from C. burnetii RSA493/Nine Mile phase I (NMI) were extracted through manual, automated RV, and virulence factor database (VFDB) methods. Web tools and bioinformatics were used to evaluate physiochemical attributes, subcellular localization, antigenicity, allergenicity, human homology, B-cell epitopes, MHC I and II binding ratios, functional class scores, adhesion probabilities, protein-protein interactions, and molecular docking. RESULTS: Out of the 1850 proteins encoded by RSA493/NMI, a subset of 178 demonstrated the potential for surface or membrane localization. Following a series of analytical iterations, 14 putative immunogenic proteins emerged. This collection included nine proteins (57.1%) intricately involved in cell wall/membrane/envelope biogenesis processes (CBU_0197 (Q83EW1), CBU_0311 (Q83EK8), CBU_0489 (Q83E43), CBU_0939 (Q83D08), CBU_1190 (P39917), CBU_1829 (Q83AQ2), CBU_1412 (Q83BU0), CBU_1414 (Q83BT8), and CBU_1600 (Q83BB2)). The CBU_1627 (Q83B86 ) (7.1%) implicated in intracellular trafficking, secretion, and vesicular transport, and CBU_0092 (Q83F57) (7.1%) contributing to cell division. Additionally, three proteins (21.4%) displayed uncharacterized functions (CBU_0736 (Q83DJ4), CBU_1095 (Q83CL9), and CBU_2079 (Q83A32)). The congruent results obtained from molecular docking and immune response stimulation lend support to the inclusion of all 14 putative proteins as potential vaccine candidates. Notably, seven proteins with well-defined functions stand out among these candidates. CONCLUSIONS: The outcomes of this study introduce promising proteins and epitopes for the forthcoming formulation of subunit vaccines against Q fever, with a primary emphasis on cellular processes and the virulence factors of C. burnetii.

Designing and Expression of Recombinant Chimeric Spike Protein from SARS-CoV-2 in Escherichia coli and Its Immunogenicity Assessment.

Since December 2019, the world has been grappling with an ongoing global COVID-19 pandemic. Various virus variants have emerged over the past two years, each posing a greater threat than its predecessors. The recent appearance of the omicron variant (B.1.1.529) has raised significant alarm within the field of epidemiology due to its highly contagious nature and rapid transmission rate. The omicron variant possessed mutations in the key receptor-binding domain (RBD) region, the S region, and these modifications have shown a notable impact on the strain's susceptibility to neutralizing antibodies. Developing safe and efficient vaccines to prevent a future severe acute respiratory outbreak of coronavirus syndrome 2 (SARS-CoV-2) is significant. Viral surface spike proteins are ideal targets for vaccines. This study aimed to find a multi-subunit chimeric vaccine. After conducting bioinformatics analysis, the recombinant spike (RS) protein of SARS-CoV-2 was deliberately designed and subsequently produced using E. coli expression systems. The immunogenicity of RS and neutralizing antibody responses were evaluated on immunized BALB/c mice. There was a significant difference in antibody titers between RS-immunized mice and control groups. The endpoint of the serum antibody titer of mice immunized with our chimeric protein was 2.5 times higher than that of the negative control. The chimeric construct could present multiple antigens simultaneously, influentially affecting immunization. Sera from mice vaccinated by RS could recognize the SARS-CoV-2 virus and neutralize antibodies. Our chimeric peptide could bind to antibodies in the serum of patients infected with different serotypes of the SARS-CoV-2 virus, such as alpha, delta, and omicron variants. The results indicated that the RS protein would be a potential novel antigenic candidate for subunit vaccine development and could be used as a useful alternative to generate diagnostic serological tests for SARS-CoV-2 infection.

Anti-EGFR bioengineered bacterial outer membrane vesicles as targeted immunotherapy candidate in triple-negative breast tumor murine model.

Cancer immunotherapy employing checkpoint inhibitors holds great promise across diverse cancers; nonetheless, a substantial proportion of patients (ranging from 55 to 87%) remain unresponsive to this treatment. To amplify therapeutic efficiency, we propose a synergistic therapeutic strategy that entails the deployment of targeted nano-sized particles carrying Toll-like receptor (TLR) agonists to the tumor site. This innovative approach seeks to activate intratumoral antigen-presenting cells using bioengineered outer membrane vesicles (OMVs) derived from gram-negative bacteria. These OMVs possess inherent attributes of surface-exposed immune stimulators and TLR-activating components, rendering them intriguing candidates for investigation. These OMVs were meticulously designed to selectively target cancer cells exhibiting an overexpression of epidermal growth factor receptor (EGFR). To gauge the precision of this targeting, the conducted affinity-based assays aimed at determining the equilibrium dissociation constant of the single-chain variable fragment employed for this purpose. In vitro experiments confirmed the OMVs' proficiency in adhering to EGFR-overexpressed cancer cells. Moreover, the evaluation extended to an in vivo context, where the therapeutic effect of nanovesicles was appraised within the tumor microenvironment of the triple-negative breast cancer mouse model. Notably, both intraperitoneal and intratumoral administrations of nanovesicles exhibited the ability to activate natural killer cells and skew M2 macrophage towards an M1 phenotype. The combined scrutiny of in vitro and in vivo findings underscores the potential efficiency of OMVs as a promising strategy for future anti-tumor endeavors.

Protective immunization against Enterohemorrhagic Escherichia coli and Shigella dysenteriae Type 1 by chitosan nanoparticle loaded with recombinant chimeric antigens comprising EIT and STX1B-IpaD.

Increasing evidence demonstrated that Enterohemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae type 1 (S. dysenteriae1) are considered pathogens, that are connected with diarrhea and are still the greatest cause of death in children under the age of five years, worldwide. EHEC and S. dysenteriae 1 infections can be prevented and managed using a vaccination strategy against pathogen attachment stages. In this study, the chitosan nanostructures were loaded with recombinant EIT and STX1B-IpaD polypeptides. The immunogenic properties of this nano-vaccine candidate were investigated. The EIT and STX1B-IpaD recombinant proteins were heterologous expressed, purified, and confirmed by western blotting. The chitosan nanoparticles, were used to encapsulate the purified proteins. The immunogenicity of recombinant nano vaccine candidate, was examined in three groups of BalB/c mice by injection, oral delivery, and combination of oral-injection. ELISA and antibody titer, evaluated the humoral immune response. Finally, all three mice groups were challenged by two pathogens to test the ability of the nano-vaccine candidate to protect against bacterial infection. The Sereny test in guinea pigs was used to confirm the neutralizing effect of immune sera in controlling S. dysenteriae 1, infections. SDS-PAGE and western blotting, confirmed the presence and specificity of 63 and 27 kDa recombinant EIT and STX1B-IpaD, respectively. The results show that the nanoparticles containing recombinant proteins could stimulate the systemic and mucosal immune systems by producing IgG and IgA, respectively. The challenge test showed that, the candidate nano-vaccine could protect the animal model from bacterial infection. The combination of multiple recombinant proteins, carrying several epitopes and natural nanoparticles could evocate remarkable humoral and mucosal responses and improve the protection properties of synthetic antigens. Furthermore, compared with other available antigen delivery methods, using oral delivery as immune priming and injection as a booster method, could act as combinatorial methods to achieve a higher level of immunity. This approach could present an appropriate vaccine candidate against both EHEC and S. dysenteriae 1.

A novel EGFR-specific recombinant ricin-panitumumab (scFv) immunotoxin against breast and colorectal cancer cell lines; in silico and in vitro analyses.

The Epidermal Growth Factor Receptor (EGFR) has been of high importance as it is over expressed in a wide diversity of epithelial cancers, promoting cell proliferation and survival pathways. Recombinant immunotoxins (ITs) have emerged as a promising targeted therapy for cancer treatment. In this study, we aimed to investigate the antitumor activity of a novel recombinant immunotoxin designed against EGFR. Using an in silico approach, we confirmed the stability of the RTA-scFv fusion protein. The immunotoxin was successfully cloned and expressed in the pET32a vector, and the purified protein was analyzed by electrophoresis and western blotting. In vitro evaluations were conducted to assess the biological activities of the recombinant proteins (RTA-scFv, RTA, scFv). The novel immunotoxin demonstrated significant anti-proliferative and pro-apoptotic effects against cancer cell lines. The MTT cytotoxicity assay revealed a decrease in cell viability in the treated cancer cell lines. Additionally, Annexin V/Propidium iodide staining followed by flow cytometry analysis showed a significant induction of apoptosis in the cancer cell lines, with half maximal inhibitory concentration (IC50) values of 81.71 nM for MDA-MB-468 and 145.2 nM for HCT116 cells (P < 0.05). Furthermore, the EGFR-specific immunotoxin exhibited non-allergenic properties. The recombinant protein demonstrated high affinity binding to EGFR. Overall, this study presents a promising strategy for the development of recombinant immunotoxins as potential candidates for the treatment of EGFR-expressing cancers.

Cytotoxic and apoptotic effects of chemically synthesized silver nanoparticles loaded with recombinant Staphylococcus LukS-PV toxin.

Chronic myeloid leukemia (CML) accounts for approximately 15% of leukemias. LukS-PV, a Panton-Valentine leucocidin (PVL) component, is secreted by Staphylococcus aureus. Silver nanoparticles have increasingly been used for different purposes, most notably for drug delivery and anticancer agents. In this work, the cytotoxicity effect of recombinant LukS-PV protein, chemically synthesized AgNPs, and recombinant LukS-PV protein-loaded silver nanoparticles was investigated on human Chronic myeloid leukemia K562 cells and human normal embryonic kidney HEK293 cells. Cell apoptosis was investigated by staining with Annexin V/propidium iodide. The recombinant LukS-PV protein-loaded silver nanoparticles exhibited dose-dependent cytotoxicity and induced apoptosis in the K562 cells but had little effect on normal HEK293 cells. After 24 h of exposure to recombinant LukS-PV protein-loaded silver nanoparticles (IC50 concentration), flow cytometry showed that 31.17% of K562 cells were apoptotic. These results indicate that recombinant LukS-PV protein-loaded silver nanoparticles maybe are a potential chemotherapeutic agent candidate against K562 cells. Hence, silver nanoparticles could be used as drug carriers for toxin release to cancer cells.

The anticancer effect of recombinant LukS-PV protein and silver nanoparticles loaded with this protein.

LukS-PV is a component of Panton-Valentine leucocidin (PVL) and is secreted by Staphylococcus aureus. Silver nanoparticles exhibit considerable potential as anticancer agents and drug delivery systems. Drug delivery is a way to deliver medicinal combinations to achieve a beneficial therapeutic effect. In the current study, recombinant LukS-PV protein-loaded silver nanoparticles were prepared and their cytotoxicity effect was analyzed on human breast cancer cells and human normal embryonic kidneys cells by MTT assay. Apoptosis was investigated by staining with Annexin V/propidium iodide. The recombinant LukS-PV protein-loaded silver nanoparticles showed dose-dependent cytotoxicity and induced apoptosis in the MCF7 cells and had a lesser effect on HEK293 cells. After 24 h exposure to the recombinant LukS-PV protein-loaded silver nanoparticles (IC50), Annexin V-FITC/PI FCM revealed that 33.2% of MCF7 cells were apoptotic. In conclusion, recombinant LukS-PV protein-loaded silver nanoparticles probably cannot be a better alternative for the targeted healing approaches to cancer therapies. Hence, it is suggested that silver nanoparticles could be utilized as a delivery system for releasing toxins into cancer cells.

Immune response induced by recombinant pres2/S-protein and a pres2-S-protein fused with a core 18-27 antigen fragment of hepatitis B virus compared to conventional HBV vaccine.

Although comprehensive vaccination is the cornerstone of public health programs to control hepatitis B virus (HBV) infections, 5% of people who receive the existing vaccine do not develop proper immunity against HBV. To overcome this challenge, researchers have tried using various protein fragments encoded by the virus genome to achieve better immunization rates. An important antigenic component of HBsAg called the preS2/S or M protein has also received much attention in this area. The gene sequences of preS2/S and Core18-27 peptide were extracted from the GenBank (NCBI). Final gene synthesis was conducted with pET28. Groups of BALB/c mice were immunized with 10 µg/ml of recombinant proteins and 1 µg/ml CPG7909 adjuvant. Serum levels of IF-γ, TNF-α, IL-2, IL-4, and IL-10 were measured by ELISA assay method on spleen cell cultures on day 45, and IgG1, IgG2a, and total IgG titers obtained from mice serum were quantified on days 14 and 45. Statistical analysis did not show any significant difference between the groups regarding IF-γ level. There were, however, significant differences in terms of IL-2 and IL-4 levels between the groups receiving preS2/S-C18-27 with and without adjuvant and the groups receiving both preS2/S and preS2/S-C18-27 (Plus Recomb-Plus Recomb: the group of mice that received both preS2/S and preS2/S-C18-27 simultaneously). The strongest total antibody production was induced by immunization with both recombinant proteins without CPG adjuvant. The groups that received both preS2/S and preS2/S-C18-27, whether with or without adjuvant, were significantly different from those that received the conventional vaccine considering most abundant interleukins. This difference suggested that higher levels of efficacy can be achieved by the use of multiple virus antigen fragments rather than using a single fragment.

Antigenicity and immunogenicity of SARS-CoV-2 surface glycoprotein fragment in CHO cells.

Background and Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein that projects from the virus surface is highly immunogenic. It is considered to be the target of many neutralizing antibodies as well as a target in vaccine design efforts. Evaluation the immunogenicity of a recombinant fragment of the spike protein (rfsp) that is comprised of Receptor Binding Domain (RBD), S1/S2 cleavage site, and fusion peptide (FP) as immunogenic proteins of SARS-COV-2, in BALB/c mice and evaluation of the efficacy of epitopes rfsp as a multi-subunit chimeric vaccine. Materials and Methods: The present study made use of CHO-K1 (Chinese hamster ovary K1) cells to create a cell line for constant expression rfsp. The rfsp was purified with Ni-NTA chromatography and confirmed by Western blotting. The immunogenicity and neutralizing antibody efficacy of rfsp were evaluated in BALB/c mice. ELISA was employed to test rfsp via sera of COVID-19 convalescent patients infected with SARS-CoV-2 alpha and delta variants. Results: Our results showed significant differences in antibody titers in immunized mice compared to the control groups and neutralizing antibodies were positive, sera from mice immunized are capable of bound SARS-CoV-2 virus, chimer peptide is capable bound antibodies patients infected with SARS-CoV-2 and patients infected with delta variant SARS-CoV-2. Conclusion: Overall, these results indicate that rfsp protein would be a novel potential antigen candidate for the development of a subunit SARS CoV-2 vaccine and rfsp has the potential to be a useful option for the development of the assays for serodiagnosis of SARS-CoV-2 infection.

Investigate the immunogenic and protective effect of trivalent chimeric protein containing IpaD-StxB-TolC antigens as a vaccine candidate against S. dysenteri and S. flexneri.

BACKGROUNDS: Shigella spp. causes bloody diarrhea and leads to death, especially in children. Chimeric proteins containing virulence factors can prevent Shigella infection. The purpose of this study is to investigate the immunogenic and protective effect of trivalent chimeric protein containing IpaD-StxB-TolC antigens against shiga toxin, S. dysenteri and S. flexneri in vitro and in vivo conditions. METHODS: Recombinant vector was transferred to E. coli BL21. The expression of the chimeric protein was confirmed by SDS PAGE and purified using the Ni-NTA column. Mice were immunized with recombinant protein and antibody titer was evaluated by ELISA. 10, 25 and 50 LD50 of Shiga toxin neutralization was evaluated in vitro (Vero cell line) and in vivo conditions. Also, the challenge of immunized mice with 10, 25 and 50 LD50 of S. dysentery and S. flexneri was done. RESULTS: The expression and purification of the recombinant protein with 60.6 kDa was done. ELISA showed increased antibody titer against the chimeric protein. MTT assay indicated that 1/8000 dilution of the sera had a 51% of cell viability against the toxin in Vero cell line. The challenge of mice immunized with toxin showed that the mice had complete protection against 10 and 25 LD50 of toxin and had 40% survival against 50 LD50. Mice receiving 10 and 25 LD50 of S. dysenteri and S. flexneri had 100% protection and in 50 LD50 the survival rate was 60 and 50%, respectively. Organ burden showed that the amount of bacterial colonization in immunized mice was 1 × 104 CFU/mL, which was significantly different from the control group. CONCLUSION: This study showed that chimeric proteins can create favorable immunogenicity in the host as vaccine candidates.

Preparation of chitosan nanoparticle containing recombinant CD44v antigen and evaluation of its immunization capacity against breast cancer in BALB/c mice.

OBJECTIVE(S): Breast tumors show heterogeneity containing cancer stem cells as a small subpopulation of a tumor mass. CD44 as a cancer stem cells antigen is abnormally expressed by carcinomas of epithelial origin. Also, overexpression of CD44 variable isoforms (CD44v) is associated with malignancy in breast cancer. In the present research, our objective was to evaluate the immunogenicity of prepared nanoparticles containing a novel recombinant CD44v (rCD44v) protein in the mouse model. MATERIALS AND METHODS: CD44 gene was expressed in E. coli BL21 DE3 using the pET28a-CD44 vector. The expressed rCD44v protein was purified, encapsulated into the chitosan nanoparticles, and administered to BALB/c mice. ELISA was used to evaluate the immunoglobulin levels of immunized animals. For challenge experiment, 2 × 106 4T1-CD44 tumor cells were injected subcutaneously in mice, and tumor size, necrosis, and metastases were measured. Finally, cell proliferation assay, cytokines assay, and neutralization assay of the mouse anti-rCD44v on the human breast cancer cell line were examined. RESULTS: The measured size of chitosan-rCD44v nanoparticles was 146.5 nm. Recombinant CD44v encapsulated by chitosan nanoparticles increases immunological responses via the adjuvant nature of chitosan nanoparticles. In the immunized mice, IgG and IgA titers were significantly increased. Tumor growth in injection and nano-injection test groups compared with the mice control groups displayed a significant reduction (P < 0.05). A high amount of splenocytes secreting IFNγ and IL-17 was seen in immunized mice with rCD44v (P < 0.05). Furthermore, a smaller size of lung metastases compared to the control mice groups was detected. CONCLUSION: The encapsulated rCD44v within the chitosan nanoparticles induced a significant immune response in mice and can establish significant protection against breast cancer. Therefore, it can be considered a vaccine candidate for breast cancer therapeutic modalities.

Immunosenescence and inflamm-ageing in COVID-19.

The destructive effects of coronavirus disease 2019 (COVID-19) on the elderly and people with cardiovascular disease have been proven. New findings shed light on the role of aging pathways on life span and health age. New therapies that focus on aging-related pathways may positively impact the treatment of this acute respiratory infection. Using new therapies that boost the level of the immune system can support the elderly with co-morbidities against the acute form of COVID-19. This article discusses the effect of the aging immune system against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the pathways affecting this severity of infection.

In silico designing and expression of novel recombinant construct containing the variable part of CD44 extracellular domain for prediagnostic breast cancer.

BACKGROUND: CD44, as a tumor-associated marker, can be used to detect stem cells in breast cancer. While CD44 is expressed in normal epithelial cells, carcinoma cells overexpress CD44. AIMS: In the current study, we designed a recombinant protein that included the variable component of the CD44 (CD44v) extracellular domain to apply in clinical diagnosis of breast cancer. METHODS: A total of 100 CD44v amino-acid residues were determined, and the structure was examined using bioinformatics tools. The construct was inserted into the PET28a vector and transformed in E. coli BL21(DE3). A nearly 12 kDa fusion protein was obtained by Ni-NTA affinity metal chromatography. Recombinant CD44v was examined by Western blotting, ELISA, and immunohistochemistry (IHC) assays. RESULTS: The findings revealed that the structure of rCD44v was stable, and its antigenic domain was exposed. The recombinant CD44v was confirmed by western blotting, and the presence of antibodies against recombinant CD44v protein in the patient's serum was detected by the ELISA. Our data demonstrated a link between CD44v serum levels and the prevalence of breast cancer. CONCLUSION: Assessments of antiCD44v antibodies with rCD44v could be a useful tool for identifying breast cancer in its early stages, which can lead to better outcomes.