Preclinical assessment of a recombinant RBD-Fc fusion protein as SARS-CoV-2 candidate vaccine.

Background: Waning immunity and emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlight the need for further research in vaccine development. Methods: A recombinant fusion protein containing the receptor-binding domain (RBD) fused to the human IgG1 Fc (RBD-Fc) was produced in CHO-K1 cells. RBD-Fc was emulsified with four adjuvants to evaluate its immunogenicity. The RBD-specific humoral and cellular immune responses were assessed by ELISA. The virus neutralizing potency of the vaccine was investigated using four neutralization methods. Safety was studied in mice and rabbits, and Antibody-Dependent Enhancement (ADE) effects were investigated by flow cytometry. Results: RBD-Fc emulsified in Alum induced a high titer of anti-RBD antibodies with remarkable efficacy in neutralizing both pseudotyped and live SARS-CoV-2 Delta variant. The neutralization potency dropped significantly in response to the Omicron variant. RBD-Fc induced both TH2 and particularly TH1 immune responses. Histopathologic examinations demonstrated no substantial pathologic changes in different organs. No changes in serum biochemical and hematologic parameters were observed. ADE effect was not observed following immunization with RBD-Fc. Conclusion: RBD-Fc elicits highly robust neutralizing antibodies and cellular immune responses, with no adverse effects. Therefore, it could be considered a promising and safe subunit vaccine against SARS-CoV-2.

Placenta: an old organ with new functions.

The transition from oviparity to viviparity and the establishment of feto-maternal communications introduced the placenta as the major anatomical site to provide nutrients, gases, and hormones to the developing fetus. The placenta has endocrine functions, orchestrates maternal adaptations to pregnancy at different periods of pregnancy, and acts as a selective barrier to minimize exposure of developing fetus to xenobiotics, pathogens, and parasites. Despite the fact that this ancient organ is central for establishment of a normal pregnancy in eutherians, the placenta remains one of the least studied organs. The first step of pregnancy, embryo implantation, is finely regulated by the trophoectoderm, the precursor of all trophoblast cells. There is a bidirectional communication between placenta and endometrium leading to decidualization, a critical step for maintenance of pregnancy. There are three-direction interactions between the placenta, maternal immune cells, and the endometrium for adaptation of endometrial immune system to the allogeneic fetus. While 65% of all systemically expressed human proteins have been found in the placenta tissues, it expresses numerous placenta-specific proteins, whose expression are dramatically changed in gestational diseases and could serve as biomarkers for early detection of gestational diseases. Surprisingly, placentation and carcinogenesis exhibit numerous shared features in metabolism and cell behavior, proteins and molecular signatures, signaling pathways, and tissue microenvironment, which proposes the concept of "cancer as ectopic trophoblastic cells". By extensive researches in this novel field, a handful of cancer biomarkers has been discovered. This review paper, which has been inspired in part by our extensive experiences during the past couple of years, highlights new aspects of placental functions with emphasis on its immunomodulatory role in establishment of a successful pregnancy and on a potential link between placentation and carcinogenesis.

Comparative Immunogenicity and Neutralization Potency of Four Approved COVID-19 Vaccines in BALB/c Mice.

Background: Since the outbreak of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), several vaccine candidates have been developed within a short period of time. Although the potency of these vaccines was evaluated individually, their comparative potency was not comprehensively evaluated. Objective: To compare the immunogenicity and neutralization efficacy of four approved COVID-19 vaccines in Iran, including: PastoCovac Plus, Sinopharm, SpikoGen, and Noora in BALB/c mice. Methods: Different groups of female BALB/c mice were vaccinated with three doses of each vaccine. The serum levels of antibodies against the viral receptor binding domain (anti-RBD) and spike (anti-spike) protein as well as the vaccine formulation (anti-vaccine) were evaluated using enzyme-linked immunosorbent assay (ELISA). The neutralization efficacy of these four vaccines was assessed through four neutralization assays: conventional virus neutralization test (cVNT), pseudotype virus neutralization test (pVNT), surrogate virus neutralization test (sVNT), and inhibition flow cytometry. Results: All four vaccines induced seroconversion in vaccinated animals. All vaccines successfully induced high levels of anti-vaccine antibody; however, PastoCovac Plus and Sinopharm vaccines induced significantly higher levels of anti-RBD antibody titer compared to Noora and SpikoGen. Moreover, the results of the antibody response were corroborated by the virus neutralization tests, which revealed very weak neutralization potency by Noora and SpikoGen in all tests. Conclusion: Our results indicate significant immunogenicity and neutralization efficacy induced by PastoCovac Plus and Sinopharm, but not by Noora and SpikoGen. This suggests the need for additional comparative assessment of the potency and efficacy of these four vaccines in vaccinated subjects.

Clinico-Pathological and Prognostic Significance of a Combination of Tumor Biomarkers in Iranian Patients With Breast Cancer.

PURPOSE: Breast cancer is one of the most common cancers in the world. It is a multifaceted malignancy with different histopathological and biological features. Molecular biomarkers play an essential role in accurate diagnosis, classification, prognosis, prediction of treatment response, and cancer surveillance. This study investigated the clinico-pathological and prognostic significance of HER3 and ROR1 in breast cancer samples. METHODS: Tissue microarrays (TMA) were constructed using tissue blocks of 444 Iranian breast cancer patients diagnosed with breast cancer. Overall survival (OS) and disease-free survival (DFS) were assessed after 5 years follow-up. TMA slides were stained with monoclonal antibodies against ROR1, HER3, ER, PR, Ki67, P53, HER2 and CK5/6 using IHC and correlation between the investigated tumor markers and the clinico-pathological parameters of patients were analyzed. RESULTS: Our results showed a significant correlation between ROR1 and ER, PR, HER3, and CK5/6 expression. Additionally, there was a significant correlation between HER3 and ER, PR, HER2, and Ki67 expression. Ki67 was also correlated with HER2 and P53 expression. HER3 expression was significantly correlated with tumor stage, lymph node metastasis, perineural invasion, and multifocal tumors. Furthermore, ROR1 expression was significantly associated with tumor metastasis, lympho-vascular invasion, and perineural invasion. While HER2-HER3 coexpression was significantly associated with poor OS, HER3-ROR1 coexpression was associated with lymph node invasion, lymph node metastasis, and distant metastasis. CONCLUSION: ROR1 and HER3 displayed significant association with different clinic-pathological features and in addition to the other tumor biomarkers could be considered as diagnostic and prognostic biomarkers in breast cancer patients.

Development and characterization of phage display-derived anti-toxin antibodies neutralizing TcdA and TcdB of Clostridioides difficile.

TcdA and TcdB are known as the major virulence attributes of Clostridioides difficile. Hence, neutralizing the TcdA and TcdB activities can be considered as an efficient therapeutic approach against C. difficile infection (CDI). In this work, we utilized phage display technique to select single-chain fragment variable (scFv) fragments as recombinant antibodies displayed on the surface of phages, which specifically target native TcdA, or TcdB (nTcdA and nTcdB), and their recombinant C-terminal combined repetitive oligopeptide (CROP) domains (rTcdA and rTcdB). After three rounds of biopanning, abundance of phage clones displaying high reactivity with TcdA or TcdB was quantified through enzyme-linked immunosorbent assay (ELISA). Furthermore, selected scFvs were characterized by cell viability and neutralization assays. The gene expression of immunological markers, IL-8 and TNF-α, was examined in treated Caco-2 cells by RT-qPCR. The epitopes of neutralizing scFvs were also identified by molecular docking. Totally, 18 scFv antibodies (seven for TcdA and 11 for TcdB) were identified by ELISA. Among selected scFvs, two clones for TcdA (rA-C2, A-C9) and three clones for TcdB (rB-B4, B-F5, B-F11) exhibited the highest neutralizing activity in Caco-2 and Vero cells. Moreover, the cocktail of anti-TcdA and anti-TcdB antibodies notably decreased the mRNA expression of TNF-α and IL-8 in Caco-2 cells. Molecular docking revealed that the interaction between scFv and toxin was mostly restricted to CROP domain of TcdA or TcdB. Our results collectively provided more insights for the development of neutralizing scFvs against C. difficile toxins using phage display. Further research is needed to meticulously evaluate the potential of scFvs as an alternative treatment for CDI using animal models and clinical trials.IMPORTANCETargeting the major toxins of Clostridioides difficile by neutralizing antibodies is a novel therapeutic approach for CDI. Here, we report a panel of new anti-TcdA (rA-C2, A-C9) and anti-TcdB (rB-B4, B-F5, and B-F11) recombinant antibody fragments (scFvs) isolated from Tomlinson I and J libraries using phage display technique. These scFv antibodies were capable of neutralizing their respective toxin and showed promise as potential therapeutics against TcdA and TcdB of C. difficile in different in vitro models. In addition, in silico analysis showed that at least two neutralization mechanisms, including inhibiting cell surface binding of toxins and inhibiting toxin internalization can be proposed for the isolated scFvs in this work. These findings provide more insights for the applicability of specific scFvs toward C. difficile toxins at in vitro level. However, further research is required to evaluate the potential application of these scFvs as therapeutic agents for CDI treatment in clinical setting.

Discovery of a novel marker for human granulocytes and tissue macrophages: RTL1 revisited.

Here, retrotransposon-like 1 (RTL1) is introduced as a marker for circulating and tissue neutrophils, tissue macrophages, and tumor-associated macrophages (TAM) and neutrophils (TAN). Anti-RTL1 polyclonal and monoclonal antibodies were produced, and their reactivity was examined by Western blotting (WB), ELISA, and immunostaining of human normal and cancer tissues. The reactivity of the anti-RTL1 antibodies with peripheral blood leukocytes and a panel of hematopoietic cell lines was examined. The generated antibodies specifically detected RTL1 in the WB of the placenta and U937 cells. The polyclonal antibody showed excellent reactivity with tissue-resident macrophages, Hofbauer cells, alveolar and splenic macrophages, Kupffer cells, and inflammatory cells in the tonsil, appendix, and gallbladder. In vitro GM-CSF-differentiated macrophages also showed a high level of intracellular RTL1 expression. TAM and TAN also showed excellent reactivity with this antibody. Almost all circulating granulocytes but not lymphocytes or monocytes expressed RTL1 at their surface. Serial sections of the appendix stained with CD15 and RTL1 and placenta stained with CD68 and RTL1 showed a considerable overlap in RTL1 expression in CD15+ granulocytes and CD68+ macrophages. A small percentage of myelomonocytic cell lines was positive for surface RTL1, while promyelocytic, monocytic, megaloblastic, and lymphoblastic cell lines were negative. Endothelial cells of normal and cancer tissues highly expressed RTL1. RTL1 could be considered a new marker for different normal tissue macrophages, TAM, circulating and tissue neutrophils, and TAN.

Investigation of Expression Profile of Placenta-specific 1 (PLAC1) in Acute Myeloid and Lymphoid Leukemias.

Background: Placenta-specific 1 (PLAC1) is one of the cancer-testis-placenta antigens that has no expression in normal tissue except placenta trophoblast and testicular germ cells, but is overexpressed in a variety of solid tumors. There is a lack of studies on the expression of PLAC1 in leukemia. We investigated expression of PLAC1 in Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL). Methods: In this study, we investigated expression pattern of PLAC1 gene in peripheral blood and bone marrow mononuclear cells of newly-diagnosed patients with AML (n=31) and ALL (n=31) using quantitative real-time PCR. Normal subjects (n=17) were considered as control. The PLAC1 protein expression in the samples were also detected using western blotting. Results: Our data demonstrated that PLAC1 transcripts had 2.7 and 2.9 fold-change increase in AML and ALL, respectively, compared to normal samples. PLAC1 transcript expression was totally negative in all studied normal subjects. Level of PLAC1 mRNA expression in ALL statistically increased compared to normal samples (p=0.038). However, relative mRNA expression of PLAC1 in AML was not significant in comparison to normal subjects (p=0.848). Furthermore, relative mRNA expression of PLAC1 in AML subtypes was not statistically significant (p=0.756). PLAC1 gene expression showed no difference in demographical clinical and para-clinical parameters. Western blotting confirmed expression of PLAC1 in the ALL and AML samples. Conclusion: Considering PLAC1 expression profile in acute leukemia, PLAC1 could be a potential marker in leukemia which needs complementary studies in the future.

Novel neutralizing mouse-human chimeric monoclonal antibodies against the SARS-CoV-2 receptor binding domain.

Introduction. Neutralizing antibodies have been widely used for the prophylaxis and treatment of COVID-19.Hypothesis. The major target for these neutralizing antibodies is the receptor-binding domain (RBD) of the viral spike protein.Aim. In the present study, we developed and characterized three neutralizing chimeric mouse-human mAbs for potential therapeutic purposes.Methodology. Light and heavy chain variable region genes of three mouse mAbs (m4E8, m3B6, and m1D1) were amplified and ligated to human Cγ1 and Cκ constant region genes by PCR. After cloning into a dual promoter mammalian expression vector, the final constructs were transiently expressed in DG-44 cells and the purified chimeric antibodies were characterized by ELISA and Western blotting. The neutralizing potency of the chimeric mAbs was determined by three different virus neutralization tests including sVNT, pVNT, and cVNT.Results. All three recombinant chimeric mAbs display human constant regions and are able to specifically bind to the RBD of SARS-CoV-2 with affinities comparable to the parental mAbs. Western blot analysis showed similar epitope specificity profiles for both the chimeric and the parental mouse mAbs. The results of virus neutralization tests (sVNT, pVNT, and cVNT) indicate that c4E8 had the most potent neutralizing activity with IC50 values of 1.772, 0.009, and 0.01 µg ml-1, respectively. All chimeric and mouse mAbs displayed a similar pattern of reactivity with the spike protein of the SARS-CoV-2 variants of concern (VOC) tested, including alpha, delta, and wild-type.Conclusion. The chimeric mAbs displayed neutralizing potency similar to the parental mouse mAbs and are potentially valuable tools for disease control.

Novel Monoclonal Antibodies Specific for Human Ki67 and P53 Tumor Markers in Breast Cancer Tissue Samples.

Background: Ki67 and P53 are important diagnostic and prognostic biomarkers expressed in several cancers. The current standard method for evaluating Ki67 and P53 in cancer tissues is immunohistochemistry (IHC), and having highly sensitive monoclonal antibodies against these biomarkers is necessary for an accurate diagnosis in the IHC test. Objective: To generate and characterize novel monoclonal antibodies (mAbs) against human Ki67 and P53 antigens for IHC purposes. Methods: Ki67 and P53-specific mAbs were produced by the hybridoma method and screened by enzyme-linked immunosorbent assay (ELISA) and IHC techniques. Selected mAbs were characterized using Western blot and flow cytometry, and their affinities and isotypes were determined by ELISA. Moreover, using the IHC technique in 200 breast cancer tissue samples, we assessed the specificity, sensitivity, and accuracy of the produced mAbs. Results: Two anti-Ki67 (2C2 and 2H1) and three anti-P53 mAbs (2A6, 2G4, and 1G10) showed strong reactivity to their target antigens in IHC. The selected mAbs were also able to recognize their targets by flow cytometry as well as Western blotting using human tumor cell lines expressing these antigens. The specificity, sensitivity, and accuracy calculated for clone 2H1 were 94.2%, 99.0%, and 96.6%, and for clone 2A6 were 97.3%, 98.1%, and 97.5%, respectively. Using these two monoclonal antibodies, we found a significant correlation between Ki67 and P53 overexpression and lymph node metastasis in patients with breast cancer. Conclusion: The present study showed that the novel anti-Ki67 and anti-P53 mAbs could recognize their respective antigens with high specificity and sensitivity and therefore can be used in prognostic studies.

Cancer Is Associated with the Emergence of Placenta-Reactive Autoantibodies.

Placenta-specific antigens are minimally expressed or unexpressed in normal adult tissues, while they are widely expressed in cancer. In the course of carcinogenesis, a vast array of autoantibodies (AAbs) is produced. Here, we used a quantitative approach to determine the reactivity of AAbs in the sera of patients with breast (BrC: N = 100, 100% female, median age: 51 years), gastric (GC: N = 30, 46.6% female, median age: 57 years), bladder (BC: N = 29, 34.4% female, median age: 57 years), and colorectal (CRC: N = 34, 41.1% female, median age: 51 years) cancers against first-trimester (FTP) and full-term placental proteome (TP) in comparison with age- and sex-matched non-cancer individuals. Human-on-human immunohistochemistry was used to determine reactive target cells in FTP. The effect of pregnancy on the emergence of placenta-reactive autoantibodies was tested using sera from pregnant women at different trimesters of pregnancy. Except for BC, patients with BrC (p < 0.0284), GC (p < 0.0002), and CRC (p < 0.0007) had significantly higher levels of placenta-reactive AAbs. BrC (p < 0.0001) and BC (p < 0.0409) in the early stages triggered higher autoantibody reactivity against FTP. The reactivities of BrC sera with FTP did not show an association with ER, PR, or HER2 expression. Pregnancy in the third trimester was associated with the induction of TP- and not FTP-reactive autoantibodies (=0.018). The reactivity of BrC sera with placental proteins was found to be independent of gravidity or abortion. BrC sera showed a very strong and specific pattern of reactivity with scattered cells beneath the syncytiotrophoblast layer. Our results reinforce the concept of the coevolution of placentation and cancer and shed light on the future clinical application of the placental proteome for the non-invasive early detection and treatment of cancer.

Diagnostic performance of a novel antigen-capture ELISA for the detection of SARS-CoV-2.

BACKGROUND AND AIMS: The coronavirus disease 2019 (COVID-19) pandemic is a serious health problem worldwide. Early virus detection is essential for disease control and management. Viral antigen detection by ELISA is a cost-effective, rapid, and accurate antigen diagnostic assay which could facilitate early viral detection. METHOD: An antigen-capture sandwich ELISA was developed using novel nucleocapsid (NP)-specific mouse monoclonal antibodies (MAbs). The clinical performance of the assay was assessed using 403 positive and 150 negative respiratory samples collected during different SARS-CoV-2 variants outbreaks in Iran. RESULTS: The limit of detection of our ELISA assay was found to be 43.3 pg/ml for recombinant NP. The overall sensitivity and specificity of this assay were 70.72% (95% CI: 66.01-75.12) and 100% (95% CI: 97.57-100), respectively, regardless of Ct values and SARS-CoV-2 variants. There was no significant difference in our assay sensitivity for the detection of Omicron subvariants compared to Delta variant. Assay sensitivity for the BA.5 Omicron subvariant was calculated as 91.89% (95% CI: 85.17-96.23) for samples with Ct values < 25 and 82.70% (95% CI: 75.19-88.71) for samples with Ct values < 30. CONCLUSION: Our newly developed ELISA method is reasonably sensitive and highly specific for detection of SARS-CoV-2 regardless of the variants and subvariants of the virus.

Histological Evaluation of Products of Conception, Who Benefits from It?

Background: Histopathological evaluation of the first trimester pregnancy loss has always been controversial. Although it is recommended, it is not a part of guidelines.Methods: Six hundred eighty-six samples in a referral infertility clinic were evaluated microscopically and categorized. Two hundred ninety-five cases were evaluated by genetic methods (Multiplex Ligation-dependent Probe Amplification).Results: From 569 samples with chorionic villi, 361 cases had history of three or more abortions. 18.3% of this group showed chronic intervillous of unknown etiology (CIUE) and 8.3% revealed intervilli fibrin deposition, both pathologies with a high risk of recurrence. History of a live child was significantly higher in CIUE group. 29% of genetically evaluated cases had a chromosomal abnormality.Conclusion: Histological evaluation of recurrent pregnancy loss could illuminate the cause of abortion in relatively acceptable percentage of cases, especially in mothers with higher number of previous abortion, mothers with a history of live child and in referral centers.

Human amniotic epithelial cells exert anti-cancer effects through secretion of immunomodulatory small extracellular vesicles (sEV).

We identified here mechanism by which hAECs exert their anti-cancer effects. We showed that vaccination with live hAEC conferred effective protection against murine colon cancer and melanoma but not against breast cancer in an orthotopic cancer cell inoculation model. hAEC induced strong cross-reactive antibody response to CT26 cells, but not against B16F10 and 4T1 cells. Neither heterotopic injection of tumor cells in AEC-vaccinated mice nor vaccination with hAEC lysate conferred protection against melanoma or colon cancer. Nano-sized AEC-derived small-extracellular vesicles (sEV) (AD-sEV) induced apoptosis in CT26 cells and inhibited their proliferation. Co-administration of AD-sEV with tumor cells substantially inhibited tumor development and increased CTL responses in vaccinated mice. AD-sEV triggered the Warburg's effect leading to Arginine consumption and cancer cell apoptosis. Our results clearly showed that it is AD-sEV but not the cross-reactive immune responses against tumor cells that mediate inhibitory effects of hAEC on cancer development. Our results highlight the potential anti-cancer effects of extracellular vesicles derived from hAEC.

Immunization with placenta-specific 1 (plac1) induces potent anti-tumor responses and prolongs survival in a mouse model of melanoma.

PURPOSE: Melanoma is a malignant and metastatic form of skin cancer, which is not diagnosed in early stages of the disease. Nowadays, immunotherapy is changing the treatment landscape for metastatic melanoma. Placenta-specific1 (PLAC1) is a cancer-testis-placenta (CTP) antigen with differential expression in melanoma tissues. Here, we evaluated the potential of plac1 to induce anti-cancer immune responses as well as to prevent cancer development in a mouse model of melanoma. METHODS: Two proteins containing full extracellular domain (ED) of mouse plac1+KDEL3 and full ED of mouse plac1+ tetanus toxin P2 and P30+ pan DR epitope (PADRE) â€‹+ â€‹KDEL3 were produced and injected in mice to evaluate their capacity to induce anti-cancer immune responses as well as their potential to prevent melanoma development. Induction of plac1-specific humoral and cellular responses as well as tumor-associated parameters were tested in a series of 36 mice. RESULTS: Sera of mice immunized with ED â€‹+ â€‹P2P30+PADRE â€‹+ â€‹KDEL3 contained antibodies able to react with surface plac1 in B16F10 â€‹cells. Both proteins induced proliferative cellular immune responses against B16F10 â€‹cells and plac1-specific cytotoxic T cells (CTL) and CD107a â€‹+ â€‹CTL responses, which was higher in mice immunized with ED â€‹+ â€‹P2P30+PADRE â€‹+ â€‹KDEL3. Splenocytes of mice vaccinated with ED â€‹+ â€‹P2P30+PADRE â€‹+ â€‹KDEL3 exerted a significant cytotoxicity against B16F10 â€‹cells. Vaccination with ED â€‹+ â€‹P2P30+PADRE â€‹+ â€‹KDEL3 significantly delayed B16F10-induced tumor onset, reduced tumor growth, and increased survival. Tumors induced by B16F10 expressed plac1 in vivo. CONCLUSION: Our results pave the way for development of effective melanoma preventive vaccine in humans, although further studies are needed.

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.

Generation and Characterization of Novel Diagnostic Mouse Monoclonal Antibodies Against Human Estrogen Receptor Alpha and Progesterone Receptor.

BACKGROUND: Estrogen and progesterone regulate the growth and development of several human cells and tissues. Their corresponding receptors (ER and PR) are important diagnostic and prognostic indicators for cancers of the breast and reproductive organs. Immunohistochemical analysis of ER and PR is the current standard method for evaluating the expression of these receptors in different cancers. Nonetheless, there is a significant lack of reproducibility of IHC results in laboratories worldwide, necessitating to develop more sensitive and specific antibodies for ER and PR IHC staining. METHODS: ER and PR-specific monoclonal antibodies (MAbs) were generated by immunizing mice with synthetic peptides from ERα and PR. The isotypes and affinity constants of the selected MAbs were determined, and their specificities were assessed by peptide-specific ELISA, IHC, Western-blot analysis, and flow cytometry. In addition, the reactivity of generated MAbs was compared with that of the commercially-available anti-ER and anti-PR antibodies in IHC using normal and cancerous tissue sections. Moreover, 200 breast cancer tissue samples were stained using the newly generated MAbs along with commercial antibodies by IHC, and the sensitivity, specificity and accuracy of our MAbs were evaluated. RESULTS: Among different MAbs generated in this study, two anti-ER and one anti-PR MAbs specifically detected the target antigens in normal and cancerous tissues in IHC. Further analyses confirmed the specificity of the MAbs in Western blotting and flow cytometry using a panel of ER and PR positive cell lines. The sensitivity, specificity and accuracy calculated for clone 1B9 (anti-ER) were 92.3%, 94.8% and 93%, and for clone 3D6 (anti-PR) were 93.0%, 94.3% and 93.5%, respectively. CONCLUSION: Our novel anti-ER and PR MAbs could be considered as suitable tools for diagnostic and research purposes.

Optimization of Expression and Purification of Recombinant Mouse plac1.

Background: Placenta-specific 1 (PLAC1) is one of the recently-discovered Cancer-Testis-Placenta (CTP) antigen with restricted normal tissue and ectopic expression in a wide range of cancer cells from different histological origins. The production of recombinant human PLAC1 has already been optimized; however, no study has been reported so far on the production and purification of mouse plac1. In this study, mouse plac1 expression and purification was optimized in a prokaryotic system and the effects of the generated proteins on inducing humoral responses in mice were investigated. Methods: A fusion protein containing full extracellular domain of mouse plac1, immunostimulatory peptides, tetanus toxin P2P30 and PADRE and KDEL3 signal (main plac1), and the same fragment without immunostimulatory peptides (control plac1) was produced. To optimize production and purification steps, different parameters including bacterial strain, cultivation temperature, cultivation time, IPTG concentration, culture medium, and also different buffers for purification of the recombinant proteins were tested. After confirming the identity of recombinant plac1 proteins with Western Blotting (WB) and ELISA assays, these proteins were subcutaneously injected in mice with Freund's adjuvant and the anti-plac1 antibody response was detected by ELISA. Results: The optimal expression level of main and control plac1 was obtained in BL21 (DE3) and TB culture medium in the presence of 0.25 mM IPTG after 24 hr of induction at 15°C. The buffer containing 2% sarkosyl produced higher yield and purity. Our results showed specific reactivity of anti-human recombinant plac1 polyclonal antibody with both main and control plac1 recombinant proteins in WB and ELISA analysis. Both proteins induced humoral responses in mice; however, anti-plac1 antibody titer was significantly higher in sera of mice immunized with main compared to control plac1. Conclusion: In this study, an optimized protocol for production and purification of mouse plac1 was reported and it was shown that insertion of immunostimulatory peptides in gene construct could efficiently enhance humoral immune responses against mouse plac1, which could potentially augment cellular immune responses against plac1 leading to more effective anti-cancer responses.

Introduction of an efficient method for placenta decellularization with high potential to preserve ultrastructure and support cell attachment.

The placenta, as a large discarded tissue and rich in extracellular matrix (ECM), is an excellent candidate for biological scaffolds in reconstructive medicine. Considering the importance of ECM structure in cell fate, the aim of this study was to achieve human placenta decellularization protocol that preserve the structure of scaffolds. Thus, human placenta was decellularized by four protocols and decellularization efficacy was compared by hematoxylin and eosin (H&E), 4',6-diamidino-2-phenylindole (DAPI) staining, and DNA measurement. Decellularized placenta structure preservation was assessed by Masson's trichrome staining, scanning electron microscopy (SEM), and immunofluorescence (IF) for collagen I, IV, and fibronectin. Finally, liquid displacement measured scaffolds' porosity. After culturing menstrual blood-derived stem cells (MenSCs) on placenta scaffolds, cell adhesion was investigated by SEM imaging, and cell viability and proliferation were assessed by MTT assay. According to H&E and DAPI staining, only protocols 1 and 3 could completely remove cells from the scaffolds. DNA measurements confirmed a significant reduction in the genetic material of decellularized scaffolds compared to native placenta. According to Masson's trichrome, IF, and SEM imaging, scaffold structure is better preserved in P3 than P1 protocol. Liquid displacement showed higher porosity of P3 scaffold than P1. SEM imaging confirmed cells adhesion to the decellularized placenta, and the attached cells showed good viability and maintained their proliferative capacity, indicating the suitability of the scaffolds for cell growth. Results introduced an optimized protocol for placenta decellularization that preserves the scaffold structure and supports cell adhesion and proliferation.

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.

2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin potential impacts on peripheral blood mononuclear cells of endometriosis women.

Endometriosis happens following the implantation of endometrial-derived tissues outside the uterine cavity. It has been suggested that 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD) is involved in endometriosis development. Furthermore, aryl hydrocarbon receptor (AHR), as a TCDD receptor, has been demonstrated to regulate immune responses. Nonetheless, data regarding the mechanisms, through which TCDD influences the immune system in endometriosis, are still inconclusive. Therefore, frequency of regulatory T cells (Tregs) and the expression of FOXP3, AHR and indoleamine 2, 3-dioxygenase 1 (IDO1) from endometriosis and non-endometriosis individuals were investigated in the absence and presence of TCDD; also, the concentration of IL-6 and kynurenine in the supernatant of cultures was assessed. The impact of TCDD-treated PBMCs on the migration capacity of menstrual blood-derived stromal stem cells (MenSCs) and monocyte chemoattractant protein-1 (MCP-1) and IL-6 production was determined. Here, we found that AHR and IDO1 expression levels were lower in endometriosis PBMCs; however, TCDD treatment increased AHR, FOXP3, IDO1, IL-6, and Treg levels in the endometriosis group (P ≤ 0.05-0.0001). TCDD-treated PBMCs increased the migration capacity of MenSCs and up-regulated MCP-1 and IL-6 levels in the PBMCs/MenSCs co-culture (P ≤ 0.01-0.0001). In conclusion, these results shed light on the probable mechanisms, through which AHR activation by chemical toxicants can impact inflammatory immune mediators involved in the development of endometriosis; also, these data support the idea that TCDD could promote endometriosis progression.