Decidualization, the process of endometrial stem/stromal cell (EnSCs) differentiation, is essential for embryo implantation and pregnancy maintenance. Menstrual blood-derived stem/stromal cells (MenSCs), although often considered as surrogates of EnSCs, represent a distinct population. Pharmacologic modulation of cellular senescence using senomorphics has emerged as a promising strategy in reproductive medicine. This study investigates the decidualization capacity of EnSCs and MenSCs and evaluates how senomorphic agents influence their senescence, metabolic profile, and inflammatory response. Primary EnSCs and MenSCs were isolated, characterized, and subjected to in vitro decidualization using standardized protocols. Cells were classified as well-decidualized (WD) or poorly-decidualized (PD) based on the extent of decidualization. Six senomorphic compounds were applied before and during decidualization. Senescence-associated β-galactosidase activity, IL-6 secretion, glycolytic intermediates, and global metabolomic changes were assessed before and after treatment with senomorphics. MenSCs exhibited accelerated but limited and prolonged decidualization capacity compared to EnSCs. Metabolic reprogramming in EnSCs at day 6 resembled that of MenSCs at day 3. Decidualization induced differential changes in glycolysis-related metabolites, senescence markers, and IL-6, especially in PD cells. Treatment with six senomorphics modulated these effects in a context-dependent manner. Exposure during decidualization increased senescence in both WD and PD sources, whereas pretreatment increased senescence in WD EnSCs but decreased it in PD EnSCs. Notably, senomorphics shifted the metabolomic profile of PD EnSCs toward a WD-like state. EnSCs and MenSCs differ in decidualization dynamics, metabolism, and response to senomorphic modulation. Senomorphics may be strategically employed to reduce senescence in patients with impaired endometrial decidualization, offering therapeutic potential in reproductive pharmacology.

BACKGROUND:Programmed death-ligand 1 (PD-L1) is one of the main immunoregulatory proteins. It binds to its receptor, PD-1, on the surface of T cells and suppresses their anti-tumor activity. Therefore, elevated levels of PD-L1 protein could predict a potential benefit from PD-L1/PD-1 checkpoint inhibitor immunotherapy. Immunohistochemistry (IHC) is the current standard technique for assessing PD-L1 expression in tumor tissues, and a sensitive and specific monoclonal antibody (mAb) to PD-L1 is a critical parameter for accurate results. Thus, in the current study, we aimed to produce new anti-PD-L1 mAbs with potential diagnostic applications in IHC. METHODS:By immunizing mice with human recombinant PD-L1 protein, specific mAbs were produced via the hybridoma method and then screened using enzyme-linked immunosorbent assay (ELISA) and IHC techniques in parallel. Furthermore, isotypes and affinity constants of the selected and purified mAbs were determined, and their specificities were evaluated in Western blot, flow cytometry, IHC, and immunocytochemistry (ICC) assays. Afterward, their reactivity was compared with commercially available anti-PD-L1 mAb in optimized IHC using human placenta tissue and 54 paraffin-embedded bladder cancer tissue samples, and the analytical sensitivity, specificity, and accuracy of homemade IHC were assessed. RESULTS:Among 30 clones that produce significant amounts of specific antibodies against PD-L1 in ELISA, three anti-PD-L1 mAbs (clones 1D6, 1D8, and 1F8) demonstrated high reactivity to their target antigen in human placental and cancerous tissues in IHC. Moreover, one of the selected mAbs, clone 1D6, was also able to specifically recognize its target by flow cytometry, western blotting, and ICC using a panel of human PD-L1 positive and negative tumor cell lines and placenta tissue. Compared to commercially available anti-PD-L1, the analytical sensitivity, specificity, and accuracy calculated for clone 1D6 were 94.87 %, 80 %, and 91.83 %, and for clone 1D8, they were 97.67 %, 81.81 %, and 94.4 %, respectively. CONCLUSION:This study's results demonstrated that the novel anti-PD-L1 mAbs could recognize the target antigen with high specificity and sensitivity. Therefore, they might be appropriate tools for research and diagnosis.

Each immunoglobulin molecule consists of two functional independent regions, including variable and constant regions. Toxin neutralization by antibody is mainly mediated by the variable regions. Here, we report that constant region chimerization of a mouse anti-tetanus toxin monoclonal antibody (MAb), 1F3E3, confers enhanced in vivo toxin neutralizing activity of the chimeric MAb compared to its parental murine counterpart. A mouse-human chimeric MAb, c-1F3E3, was generated by recombinant DNA technology and its structural and functional characteristics, including affinity constant, reactivity pattern to tetanus toxin fragments, inhibiting the toxin binding to GT1b ganglioside receptors as well as in vivo toxin neutralizing activity in mice were assessed and compared with those of the murine MAb. Both the murine and chimeric MAbs displayed similar reactivity patterns as well as similar toxin binding affinity constant. The target epitope of both MAbs was located in fragment C of the toxin and both were able to inhibit binding of the toxin to GT1b ganglioside at a comparable level. While 1F3E3 MAb failed to neutralize the toxin in vivo, the chimeric MAb (c-1F3E3) showed a strong and dose-dependent toxin neutralizing potency leading to improved protection and survival in mice. Mouse-human chimerization of an anti-tetanus toxin MAb improved its in vivo toxin neutralizing activity, implying contribution of the constant region of the MAb in toxin neutralization.

Pertussis is a highly contagious respiratory disease caused by the gram-negative bacterium Bordetella pertussis (Bp). The disease is most severe in infants and young children, whereas adolescents and adults typically experience milder symptoms but serve as important reservoirs for transmission. Despite widespread vaccination efforts, pertussis continues to pose a significant public health challenge. Historically, the first generation of pertussis vaccines, formulated as inactivated whole cell pertussis (wP) vaccines, were associated with notable side effects, prompting the development of safer acellular pertussis (aP) vaccines. The second generation of pertussis vaccines contains purified components of Bp and provides protection comparable to that of the older whole-cell vaccines. However, recent studies have reported a resurgence of pertussis, attributed to several factors, including improved diagnostic methods, waning immunity following vaccinations, and the emergence of antigenically divergent or vaccine-adapted strains. To address these challenges, researchers are developing next-generation pertussis vaccines using various approaches, such as transitioning from intramuscular to intranasal administration, formulating outer membrane vesicle (OMV)-based vaccines, designing live attenuated pertussis vaccines, and exploring nucleic acid-based vaccines and novel adjuvants aimed at inducing long-lasting mucosal and systemic immunity. This review primarily focuses on assessing the efficacy of the next-generation intranasally administered pertussis vaccines in both pre-clinical and clinical settings.

Despite global vaccination efforts, hepatitis B virus (HBV) infection remains a major health threat, causing over a million deaths annually. Hepatitis B e-antigen (HBeAg) is an indicator of HBV replication and high infectivity. HBeAg is an essential serological marker for monitoring response to treatment and/or determining the stage of chronic HBV infection. Here, we produced a panel of mouse hybridomas secreting monoclonal antibodies (MAbs) to HBeAg by fusing a mouse myeloma cell line with splenocytes from mice immunized with recombinant HBeAg. Anti-HBe MAbs were then characterized by competition ELISA and Western blotting. We designed and optimized an in-house sandwich ELISA using HBeAg-specific rabbit polyclonal and mouse monoclonal antibodies. The diagnostic performance of the assay was then compared to a commercial HBeAg detection ELISA kit using 176 HBeAg[-] and 44 HBeAg[+] serum samples, showing a significant positive correlation (r = 0.8250; P < 0.0001). The in-house ELISA showed reasonable sensitivity (97.56 %) and specificity (99.40 %), with a cut-off value and area under the curve of 0.193 and 0.9884, respectively. Additionally, the assay showed high repeatability, with intra- and inter-assay coefficients of variation of 2.46 % and 11.38 %, respectively. Our designed HBeAg-detecting sandwich ELISA has the potential for use in clinical diagnosis.

BACKGROUND:Pertussis is a pulmonary disease caused by the gram-negative bacteria Bordetella pertussis (BP) with a high fatality rate among newborns and young children. Pertussis toxin (PT) is essential for pertussis pathogenesis as well as production of acellular pertussis vaccines (aPV). Traditional PT purification procedures are laborious and yield low purity and recovery rates. Also, due to the low production levels of PT by BP and the difficulties of purification, an appropriate immunoassay is needed to monitor PT concentrations upstream and downstream of the production process. This study investigates production and application of monoclonal and polyclonal antibodies for efficient PT purification and quantification. METHODS:Rabbits and mice were immunized with native PT to produce polyclonal and monoclonal antibodies (MAbs). The MAbs were selected based on affinity, isotype and specificity, as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The native PT antigen was purified using an immunoaffinity column. The purity and recovery rates of native PT were analyzed by ELISA, SDS-PAGE, and immunoblotting. Additionally, monoclonal and polyclonal antibodies were used to establish an ELISA assay for measurement of PT concentration. RESULTS:A highly pure PT with recovery rates of around 74 ± 4.9 % was obtained following purification by immunoaffinity column, using polyclonal antibodies. Furthermore, the designed ELISA demonstrated suitable reactivity for measurement of the PT antigen. CONCLUSION:Our results indicate suitability of the produced monoclonal and polyclonal anti-PT antibodies for purification and monitoring of PT by immunoaffinity chromatography and ELISA, respectively. The immunoaffinity method offers an efficient replacement for PT purification in the context of developing aPV.

Placenta-Specific Protein 1 (PLAC1) is essential for normal placental and embryonic development. It is widely expressed in various types of cancer cells. We produced a panel of anti-mouse plac1 monoclonal antibodies (mAbs) with different applications. Two recombinant proteins were produced containing either the extracellular domain (ED) plus tetanus toxin P2, P30, pan-DR epitope (PADRE), and KDEL3 (main plac1) or ED plus KDEL3 (control plac1). Recombinant proteins were used for immunization and screening. Positive clones were selected by ELISA and flow cytometry. Purified mAbs were tested by ELISA, WB, flow cytometry, immunohistochemistry (IHC), and immunofluorescent (IF). A combination of bioinformatics tools was used to predict the target epitope (s) of the mAbs. Eight anti-mouse plac1 mAbs (all IgG1/kappa 1) were generated, all reacting with high affinity in ELISA. Seven clones recognized plac1 in both reduced and non-reduced Western blots, while one only recognized the non-reduced form. Cross-inhibition ELISA revealed that all mAbs recognized overlapping epitopes with a shared motif except for 5C9. Four clones reacted with the native antigen in flow cytometry, but none were functional in IF or IHC staining. The produced multifunctional mAbs can be used to investigate different aspects of PLAC1 biology in reproduction and cancer.

Neutralizing antibodies (NAbs) targeting receptor-binding domain (RBD) or spike of SARS-CoV-2 play an important role in blocking virus entry to the host cells and detecting their levels is critical for the assessment of humoral protective immune response following vaccination or recovery from SARS-CoV-2 infection. Here, we compared the performance of four virus neutralization tests to measure neutralizing antibodies in various sample types. We analyzed 25 serum samples obtained from mice or rabbits immunized with different vaccine platforms, and also 11 mouse anti-RBD monoclonal antibodies (MAbs) using surrogate virus neutralization test (SVNT), pseudovirus neutralization test (PVNT), conventional virus neutralization test (CVNT), and one-step or two-step inhibition flowcytometry virus neutralization test (IFVNT). All four VNTs showed significant correlations with each other, though PVNT and CVNT displayed significantly lower limit of detection (LoD) compared to the other two assays. In conclusion, our findings indicate that all four VNT assays give valid and accurate results and could be employed to determine the level of SARS-CoV-2 neutralizing monoclonal and polyclonal antibodies.

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.

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.

Filamentous hemagglutinin (FHA) is a critical adhesion molecule produced by Bordetella pertussis (BP), the causative agent of highly contagious respiratory infection known as whooping cough. FHA plays a pivotal role in the pathogenesis of whooping cough and is a key component of acellular pertussis vaccines (aPV). However, conventional purification methods for FHA often involve labor-intensive processes and result in low purity and recovery rates. Therefore, this study explores the use of monoclonal and polyclonal antibodies as specific tools to achieve highly pure and efficient FHA purification. To generate FHA-specific antibodies, polyclonal antibodies were produced by immunizing sheep and monoclonal antibodies (MAbs) were generated by immunizing mice with recombinant and native FHA. The MAbs were selected based on affinity, isotypes, and specificity, which were assessed through ELISA and Western blot assays. Two immunoaffinity columns, one monoclonal and one polyclonal, were prepared for FHA antigen purification. The purity and recovery rates of these purifications were determined using ELISA, SDS-PAGE, and immunoblotting. Furthermore, the MAbs were employed to develop an ELISA assay for FHA antigen concentration determination. The study's findings revealed that immunoaffinity column-based purification of FHA resulted in a highly pure antigen with recovery rates of approximately 57% ± 6.5% and 59% ± 7.9% for monoclonal and polyclonal columns, respectively. Additionally, the developed ELISA exhibited appropriate reactivity for determining FHA antigen concentration. This research demonstrates that affinity chromatography is a viable and advantageous method for purifying FHA, offering superior purity and recovery rates compared to traditional techniques. This approach provides a practical alternative for FHA purification in the context of aPV development.

Acellular pertussis vaccines (aPVs) have been developed as an alternative to whole-cell pertussis vaccines (wPVs) because of their similar efficacy but reduced reactogenicity. The aPV contains 3 or more immunogenic components of Bordetella pertussis. We aimed to evaluate the immunogenicity and protective potency of an aPV vaccine produced in our laboratory consisting of pertussis toxin (PT), filamentous hemagglutinin (FHA), and pertactin (PRN) in mice. The aPV components were produced and purified from the supernatant and pellet of the bacterial culture. Two doses of the formulated vaccine, in parallel with two commercial vaccines, were administered intraperitoneally to mice at 3-week intervals. Antibody titers against aPV antigens were measured by enzyme-linked immunosorbent assay (ELISA) after primary and booster vaccinations. To assess the protective efficacy, an intranasal challenge with a live pathogenic B pertussis strain was conducted 2 weeks after the booster vaccination, and bacterial counts (colony-forming units [CFUs]) in the lungs were determined 2 hours and 10 days after the challenge. The results demonstrated a significant increase in antibody titers against all pertussis antigens in the serum of the vaccinated groups compared with the negative control group, following both the primary and booster doses. No significant differences were observed between our formulation and the commercial vaccines. Furthermore, the CFU results after the challenge showed complete eradication of infection 10 days after the challenge in all immunized groups, in contrast to the control group. Our aPV formulation, the first aPV candidate developed in Iran, exhibits immunogenicity and protective efficacy comparable to those of commercial vaccines. Further investigation of human subjects is warranted.

Producing therapeutic proteins can be done quickly and on a large scale through Transient Gene Expression (TGE). Chinese hamster ovary (CHO) cell lines are commonly used to achieve this. Although there are few comparative studies, TGE has been observed in suspension-adapted CHO cells. We tested TGE’s effectiveness in DG-44, CHO-S, and ExpiCHO-S cell lines with four transfection reagents. A design of experiments (DoE) was followed to optimize transfection using a recombinant monoclonal antibody (mAb) construct. To evaluate the efficacy, flow cytometry and ELISA were used. Feeding strategies and temperature shifts were implemented to enhance transfection effectiveness. The quality of the mAb was assessed through ELISA, SDS-PAGE, and proliferation inhibition assays. We adapted all cell lines to grow in suspension using a serum-free medium. Our findings from flow cytometry and ELISA tests indicate that PEI and Pmax reagents had a higher rate of transfection and mAb production than the ExpiCHO commercial transfection reagent. While DG-44 cells had better transfection efficiency than CHO-S and ExpiCHO-S, there was no significant difference between CHO-S and ExpiCHO-S. Our TGE system was more productive at 32 °C than at 37 °C. In the optimized TGE of Pmax-based transfection in DG-44 at 37 and 32 °C, the production level of mAb was more than half of the amount of the commercial ExpiCHO-S expression system. Still, the number of transfected cells was three times higher, making it more efficient. The purified mAb from all transfected cell lines had similar structural and functional properties under different conditions. Our research shows that using Pmax and DG-44 cells in the TGE system is a cost-effective and efficient way to produce humanized monoclonal antibodies. We discovered that this method outperforms the ExpiCHO-S kit.

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.

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.

The coronavirus disease 2019 (COVID‐19) pandemic is transmitted by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and has affected millions of people all around the world, leading to more than 6.5 million deaths. The nucleocapsid (N) phosphoprotein plays important roles in modulating viral replication and transcription, virus‐infected cell cycle progression, apoptosis, and regulation of host innate immunity. As an immunodominant protein, N protein induces strong humoral and cellular immune responses in COVID‐19 patients, making it a key marker for studying N‐specific B cell and T cell responses and the development of diagnostic serological assays and efficient vaccines. In this review, we focus on the structural and functional features and the kinetic and epitope mapping of B cell and T cell responses against SARS‐CoV‐2 N protein to extend our understanding on the development of sensitive and specific diagnostic immunological tests and effective vaccines.

SLE is a multisystem autoimmune disease characterized by multiple immunological abnormalities including production of autoantibodies. While the etiology of SLE is largely unknown, it is generally accepted that both genetic and environmental factors contribute to disease risk and immune dysregulation. Production of IFN-α is important for protecting the host against infections; however, over stimulation of innate immune pathways can induce autoimmune disease. Environmental factors, particularly Epstein-Barr virus (EBV), have been proposed to play an important role in SLE disease. Improper engagement of Toll-like receptor (TLR) pathways by endogenous or exogenous ligands may lead to the initiation of autoimmune responses and tissue injury. EBV is shown to be a potent stimulant of IFN-α by TLR signaling cascades. Given the highlighted role of IFN-α in SLE pathogenesis and potential role of EBV infection in this disease, the present study is aimed at exploring the in vitro effects of EBV infection and CPG (either alone or in combination) on IFN-α. We also examined the expression level of CD20 and BDCA-4 and CD123 in PBMCs in 32 SLE patients and 32 healthy controls. Our results showed PBMCs treated with CPG-induced higher levels of IFN-α and TLR-9 gene expression fold change compared to cells treated with either EBV or EBV-CPG. Moreover, PBMCs treated with CPG produced significantly higher IFN-α concentration in supernatant compared to cells treated with EBV but not EBV-CPG. Our results further highlight the potential role of EBV infection and TLRs in SLE patients although more studies are warranted to ascertain the global imprint that EBV infection can have on immune signature in patients with SLE.

Hepatitis B virus (HBV) infection is a major health problem worldwide and causes almost one million deaths annually. The HBV core gene codes for two related antigens, known as core antigen (HBcAg) and e-antigen (HBeAg), sharing 149 residues but having different amino- and carboxy-terminals. HBeAg is a soluble variant of HBcAg and a clinical marker for determining the disease severity and patients' screening. Currently available HBeAg assays have a shortcoming of showing cross-reactivity with HBcAg. In this study, for the first time, we evaluated whether HBcAg-adsorbed anti-HBe polyclonal antibodies could specifically recognize HBeAg or still show cross-reactivity with HBcAg. Recombinant HBeAg was cloned in pCold1 vector and successfully expressed in Escherichia coli and after purification by Ni-NTA resin was used to generate polyclonal anti-HBe antibodies in rabbit. Purified HBeAg was further characterized by assessing its reactivity with anti-HBe in the sera of chronically infected patients and HBeAg-immunized rabbit. Sera from patients with chronic HBV infection, containing anti-HBe, specifically reacted with recombinant HBeAg, implying antigenic similarity between the prokaryotic and native HBeAg in the serum of HBV-infected patients. In addition, the designed enzyme-linked immunosorbent assay (ELISA) with rabbit anti-HBe polyclonal antibodies could detect recombinant HBeAg with high sensitivity, while high cross-reactivity with HBcAg was observed. It is noteworthy that HBcAg-adsorbed anti-HBe polyclonal antibodies still showed high cross-reactivity with HBcAg, implying that due to the presence of highly similar epitopes in both antigens, HBcAg-adsorbed polyclonal antibodies cannot differentiate between the two antigens.

AIM:Although people with HER2-positive breast cancer benefit from approved HER2-targeted therapy, acquiring resistance to the therapies occurs. Animal models can play a part in gaining a deep understanding of such a process and addressing questions concerning developing and improving immunotherapy approaches. MATERIALS AND METHODS:To develop such a model, we transfected murine 4T1 cells with the pCMV6-Neo-HER2 construct and evaluated HER2 expression and its effects on the established cell line behavior in vitro and in vivo. RESULTS:Data illustrated that human HER2 protein was expressed on isolated 4T1-HER2 clones in vitro and in vivo. Except for proliferation over 48 hours, such expression did not change 4T1-HER2 characteristics compared to 4T1 in vitro. Notwithstanding the reduction in proliferation, the rate of tumorigenicity was 90% in challenged mice and Herceptin therapy significantly decreased tumors' growth and metastasis compared to the control group. CONCLUSION:We describe a murine model for HER2-positive breast cancer not only helping shed light on the mechanisms by which the tumor evades antitumor immunity but also playing a key role in making breast cancer more sensitive to novel immunotherapy modalities.