فهرست مطالب fazel shokri

Acellular pertussis vaccines (aPVs) have been developed as an alternative to whole-cell pertussis vaccines (wPVs) due to their similar efficacy but reduced reactogenicity. The aPV contains three or more immunogenic components of BP.  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 formulated vaccine in parallel with two commercial vaccines, were administered intraperitoneally (IP) in mice at 3-week intervals. Antibody titers against aPV antigens were measured by ELISA after primary and booster vaccinations. To assess the protective efficacy, an intranasal challenge with a live pathogenic BP strain was conducted two weeks after the booster vaccination, and bacterial count (colony-forming unit, CFU) in the lungs was conducted two hours and ten days after the challenge. The results demonstrated a significant increase in antibody titers against all pertussis antigens in the serum of vaccinated groups compared to 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 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 commercial vaccines. Further investigation in human subjects is warranted.

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.

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.

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.

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.

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.

Hepatitis B, as a major healthcare problem and a potentially life-threatening infection, is caused by Hepatitis B virus (HBV). Nucleocapsid of HBV is composed of a single polypeptide chain known as core antigen, HBcAg. As a highly immunogenic subviral particle, HBcAg has been used as a carrier platform for heterologous proteins. Anti-HBc antibody, as one of the serological markers for HBV infection, is the best marker for documenting prior exposure to HBV. Here, we produced, purified and characterized HBcAg. pColdI vector, containing a histidine tag, was used for the first time to clone HBcAg gene. The highest level of HBcAg expression was achieved in Tuner strain, followed by BL21 and Rosetta-gami. Histidine-tagged HBcAg was purified using Ni-NTA resin, and its structural and antigenic similarity with natural HBcAg was confirmed by ELISA and western blot. Then, recombinant HBcAg was used to produce anti-HBc polyclonal antibodies in rabbits, and specific anti-HBcAg polyclonal antibodies were purified by the HBcAg-CNBr sepharose column. Specific anti-HBcAg polyclonal antibodies were able to detect recombinant HBcAg up to 0.15 ng/ml in an optimized sandwich ELISA. The results of this study provide valuable information for the production of recombinant HBcAg and specific anti-HBcAg polyclonal antibodies for further research and diagnostic applications.

Coronavirus disease 2019 (COVID-19), described as World War 3, is the current worldwide health challenge and nearly all countries have so far faced this disaster. There is still no cure because of the complicated pathogenesis, however, there are several studies on track investigating different aspects of the immune response to the virus. In this review, we will provide an overview of recent investigations that have analyzed immune cells in patients with COVID-19. We will then discuss the differences in immune profiles between healthy controls and various clinical presentations, including asymptomatic, mild, moderate, and severe cases.

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. To generate and characterize novel monoclonal antibodies (mAbs) against human Ki67 and P53 antigens for IHC purposes. 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. 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. 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.

Human polyclonal plasma-derived hepatitis B immunoglobulin (HBIG) is currently used for immunoprophylaxis of HBV infection. The development of virus-neutralizing monoclonal antibodies (MAbs) requires the use of optimized cell culture systems supporting HBV infection.

This study aims to optimize the hepatitis B virus infectivity of NTCP-reconstituted HepG2 (HepG2-NTCP) cells to establish an efficient system to evaluate the HBV-neutralizing effect of anti-HBs MAbs.

Serum-derived HBV (sHBV) and cell culture-derived HBV (ccHBV) were simultaneously used for the optimization of HBV infection in HepG2-NTCP cells by applying different modifications.

Our results for the first time showed that in addition to human serum, monkey serum could significantly improve ccHBV infection, while fetal and adult bovine serum as well as duck and sheep serum did not have a promotive effect. In addition, sHBV and ccHBV infectivity are largely similar except that adding 5% of PEG, which is commonly used to improve in vitro infection of ccHBV, significantly reduced sHBV infection. We showed that a combination of spinoculation, trypsinization, and also adding human or monkey serum to HBV inoculum could significantly improve the permissivity of HepG2-NTCP cells to HBV infection compared with individual strategies. All anti-HBs MAbs were able to successfully neutralize both ccHBV and sHBV infection in our optimized in vitro system.

Our study suggests different strategies for improving ccHBV and sHBV infection in HepG2-NTCP cells. This cell culture-based system allows assessment of HBV neutralizing MAbs and may also prove to be valuable for the analysis of other HBV neutralizing therapeutics.

 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.

 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.

 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.

 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.

Systemic lupus erythematous (SLE) is a multisystem autoimmune immune disorder. While the pathogenesis of SLE is so popular, both infectious and non-infectious elements are regarded to exert an important impact on the disease's development.

To explore the overall status of EBV, TLR7, TLR9, and IFN-α gene expression in 32 patients suffering from SLE and 32 healthy controls.

Plasma and PBMCs were separated from fresh whole blood. To measure EBV DNA load and mRNA levels of IFN-a, TLR-7,9 in PBMCs, molecular techniques were employed. The production of IFN-α, ds-DNA IgG antibody, and EBNA-1 IgG levels were also measured in plasma.

SLE patients showed significantly higher EBV load (p=0.001) and transcriptional levels of TLR7 (p=0.0001), IFN-α (p=0.0001), and TLR9 (p=0.0001) than in the controls. Moreover, the plasma levels of IFN-α (p=0.0002) and EBNA-1specific IgG antibody (p=0.01) were significantly higher in SLE patients.

The results stressed the potential role of EBV infection and TLRs in SLE patients although more research is needed to determine the global impact that EBV infection can have on immune signature in patients with SLE.

Incidence and severity of SARS-CoV2 infection are significantly lower in children and teenagers proposing that certain vaccines, routinely administered to neonates and children may provide cross-protection against this emerging infection.

To assess the cross-protection induced by prior measles, mumps and rubella (MMR) vaccinations against COVID-19.

The antibody responses to MMR and tetanus vaccines were determined in 53 patients affected with SARS-CoV2 infection and 52 age-matched healthy subjects. Serum levels of antibodies specific for NP and RBD of SARS-CoV2 were also determined in both groups of subjects with ELISA.

Our results revealed significant differences in anti-NP (p <0.0001) and anti-RBD (p <0.0001) IgG levels between patients and healthy controls. While the levels of rubella- and mumps specific IgG were not different in the two groups of subjects, measles-specific IgG was significantly higher in patients (p <0.01). The serum titer of anti-tetanus antibody, however, was significantly lower in patients compared to healthy individuals (p <0.01).

Our findings suggest that measles vaccination triggers those B cells cross-reactive with SARS-CoV2 antigens leading to the production of increased levels of measles-specific antibody.

The serological measurement of the anti-hepatitis C virus antibody is a widely used tool in the first-line diagnosis of HCV infection. Therefore, increasing the testing criteria of these tests is of crucial importance for screening HCV infection.

The current study aimed to optimize a novel enzyme-linked immuno assay model to detect E2 antigen with or without sample pretreatment in combination with antibodies against core, NS3, NS4, and NS5 antigens of the hepatitis C virus and to compare the performances of these assays with indirect antigen (Ag), biotin/HRP labeled Antigen Sandwich andmethods of enzymelinked immunosorbent assay (ELISA) for their ability to detect HCV.

A total of 107 positive and 415 negative controls from volunteer whole blood donors in Blood TransfusionOrganization and 204 blood samples from patients under hemodialysis treatment in Tehran and Bandar Abbas hemodialysis centers are investigated. Six different methods of ELISA test were used to detect anti-HCV antibodies and/or HCV antigens in serum samples.

Regarding sensitivity, specificity, and accuracy, E2 Antigen detection alone or combined with antibody detection have the highest accuracy value (99% and 98%, respectively) compared to othermethods for antibodies detection. The results of the combined Ag/Ab ELISA test were closer to the results of real-time PCR.

This new approach to the detection of antigen and antigen/antibody has better performance criteria concerning the serologic detection of HCV, especially in HD patients who might experience a longer window period.

We have recently produced an inhibitory mouse anti-human HER2 mAb (2A8) which displayed potent anti-tumor activity in combination with trastuzumab. To describe chimerization and functional characterization of 2A8 mAb. The VH and VL genes of 2A8 mAb were amplified from cDNA of the mouse hybridoma, ligated to constant regions of human immunoglobulin, and expressed in CHO cell line. Reactivity with four members of human HER family, the inhibitory effects and antibody-dependent cell cytotoxicity (ADCC) of purified chimeric mAb (c2A8) were assessed by ELISA, XTT, H3-tymidine incorporation and lactate dehydrogenase assays. Inhibition of ERK and AKT downstream signaling pathways by the chimeric antibody were analyzed by Western blotting. Chimeric 2A8 mAb bound to recombinant human HER2 and did not cross-react with the other members of HER family. Moreover, c2A8 was able to recognize HER2-overexpressing cancer cell line and inhibited growth and proliferation of these cells. The binding affinity of c2A8 was comparable to the mouse parental mAb. ADCC and Western blotting results showed that the mouse 2A8 mAb was successfully chimerized and could significantly inhibit phosphorylation of AKT in combination with trastuzumab. The c2A8 mAb is potentially a valuable tool for targeted immunotherapy of HER2 positive cancers.

Prostate cancer is the second most common cancer in men. Prostate-Specific Antigen (PSA) is a tumor-associated glycoprotein with enzymatic activity which is secreted by the prostate gland. Following entry to the blood, 70-90% of PSA forms complexes with protease inhibitors and its enzymatic activity is inhibited. The serum level of PSA is increased and the rate of free PSA (fPSA) to total PSA is decreased in prostate cancer patients. Therefore, measurement of PSA and fPSA in serum is very valuable for diagnosis and prognosis of prostate cancer. In the present study, five anti PSA monoclonal Antibodies (mAb) were characterized by Enzyme-Linked Immunosorbent Assay (ELISA) and immunoblotting. For designing a sandwich ELISA, epitope specificity of these antibodies was studied by a competition ELISA. Free PSA was purified by electroelution technique from seminal plasma and used to produce polyclonal anti-fPSA antibody in rabbit. Purified polyclonal antibody (pAb) and mAbs were conjugated with HRP enzyme and Biotin (Bio) to set up the sandwich ELISA. Three of the mAbs were found to recognize PSA similarly. One of these mAbs (2G3) was paired with anti-fPSA pAb to detect fPSA in serum. Eventually, serum fPSA concentration of 356 subjects was measured and compared by our designed ELISA and a commercial ELISA kit. Our results demonstrated a significant correlation (r=0.68; p<0.001) between the two assays. Sensitivity and specificity of our designed ELISA was 72.4 and 82.8%, respectively. These results imply suitability of our designed ELISA for detection of fPSA in patients with prostate cancer.

Among the many pneumococcal antigens, choline-binding proteins (CPBs) display high immunogenicity in animal models. This study aims to determine the immunogenicity of CbpM, CbpG and CbpL proteins in a mice model. The genes were cloned into pET21a expression vector and the recombinant proteins were produced. Mice and rabbits were immunized with the purified recombinant proteins. Subsequently, the mice were challenged with S. pneumoniae and their survival as well as bacterial clearances were followed. The antibody responses of the mice immunized with recombinant proteins were determined by ELISA assay. The opsonophagocytosis assay was performed using rabbit’s sera. Passive immunization was also carried out in another group of mice using two doses of anti-CbPs antibodies. Finally, these mice were challenged and their survival as well as bacterial clearance were determined. The mice actively immunized with CbpM, CbpG and CbpL recombinant proteins showed survival rate of 100%, 85% and 75%, respectively. The survival rates among passively immunized mice groups which received100µg/ml dose of anti-CbpM, anti-CbpG and anti- CbpL were 50%, 50% and 25%, respectively. For all groups, however, only 25% of mice which received 10µg/ml dose of antibody survived after bacterial challenge. The rates of opsonization with anti-CbpM, anti-CbpG, and anti-CbpL antibodies at 100 and 10µg/ml doses, were found to be 45.6 and 14.7%, 82.3 and 12.9% and 12.2 and 9.35%, respectively. Our findings suggest that the recombinant proteins particularly CbpM and CbpG can protect the mice against pneumococcus19F serotype and effectively induce a protective antibody response. Thus, CbpG and CbpM proteins might be used as suitable vaccine candidate in pneumococcal vaccine formulations.

It is more than sixty years that the concept of the fetal allograft and immunological paradox of pregnancy was proposed and in this context, several regulatory networks and mechanisms have been introduced so far. It is now generally recognized that mesenchymal stem cells exert potent immunoregulatory activity. In this study, for the first time, the potential impact of Menstrual blood Stem Cells (MenSCs), as surrogate for endometrial stem cells, on proliferative capacity of CD4 T cells was tested.
MenSCs and Bone marrow Mesenchymal Stem Cells (BMSCs) were isolated and assessed for their immunophenotypic features and multi-lineage differentiation capability. BMSCs and MenSCs with or without IFNγ pre-stimulation were co-cultured with purified anti-CD3/CD28-activated CD4 T cells and the extent of T cell proliferation at different MenSCs: T cell ratios were investigated by CSFE flow cytometry. IDO activity of both cell types was measured after stimulation with IFNγ by a colorimetric assay.
MenSCs exhibited dual mesenchymal and embryonic markers and multi-lineage differentiation capacity. MenSCs significantly increased proliferation of CD4 cells at ratios 1:2, 1:4 and 1:8. IFNγ pre-treated BMSCs but not MenSCs significantly suppressed CD4 T cells proliferation. Such proliferation promoting capacity of MenSCs was not correlated with IDO activity as these cells showed the high IDO activity following IFNγ treatment.
Although augmentation of T cell proliferation by MenSCs can be a basis for maintenance of endometrial homeostasis to cope with ascending infections, this may not fulfill the requirement for immunological tolerance to a semi-allogeneic fetus. However, more investigation is needed to examine whether or not the immunomodulatory properties of these cells are affected by endometrial microenvironment during pregnancy.

Human epidermal growth factor receptor 2 (HER2) has a crucial role in several malignancies. The extracellular domain of HER2 (HER2-ECD) has been extensively employed as an important target in passive and active immunotherapy. Isolated recombinant prokaryotic HER2-ECD subdomains were previously found to be ineffective in inducing anti-tumor antibody response.
To employ recombinant eukaryotic HER2-ECD subdomains to raise anti-HER2 antibodies and determine their anti-tumor activity in vitro.
Two paired subdomains of HER2-ECD (DI and DIII), representing Pertuzumab and Trastuzumab binding domains, respectively, along with the full extracellular domain of HER2 were generated in CHO-K1 cells. Polyclonal antibodies were raised against these subdomains and characterized using ELISA, flow cytometry, and immunoblot and their anti-tumor activity was assessed by XTT assay. The cross-reactivity of these antibodies was specified along with other members of the human HER family.
Similar to Trastuzumab and anti-HER2-ECD antibody, anti-DI and DIII polyclonal antibodies reacted with recombinant HER2-ECD and native HER2 expressed on tumor cells. These two polyclonal antibodies were able to inhibit the binding of Pertuzumab and Trastuzumab to HER2, respectively, and did not cross-react with other members of HER family. These antibodies were able to inhibit tumor cell growth in vitro, similar to Trastuzumab.
The high immunogenicity of human HER2 DI and DIII subdomains in rabbits and the tumor inhibitory activity of the purified specific antibodies imply that they might be suitable for active immunotherapy in formulation with appropriate adjuvants and in combination with other HER2 specific therapeutics.

Designing a vaccine against a defined tumor is a promising but complicated process that can be used as tumor immunotherapy. The presence of human telomerase reverse transcriptase (hTERT) has been proved in about 85% of tumors; therefore, this enzyme is a promising vaccine target. Most studies in tumor immunotherapy have focused on induction and reinforcement of CD8 T cell responses against tumor antigens, but some evidence indicates that CD4 T cell responses are important for induction of CD8 T cell response and memory. Therefore, in this in vitro study, we combined an hTERT peptide with HIV peptides restricted to HLA-DRB1*11:03/04, the most common MHC class II molecule in Iran, to induce CD4 T cell responses in healthy individuals and lung cancer patients.
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation from nine healthy individuals and six lung cancer patients who were HLA-DRB1*11:03/04 positive. PBMCs were incubated with the tumoral and viral peptides for five days and then exposed to A549 cells to evaluate specific proliferative responses. The proliferation of PBMCs was examined by the MTT assay. Then, levels of secreted interferon-γ in culture media were analyzed by enzyme-linked immunosorbent assay (ELISA).
Proliferation of PBMCs was significantly greater in the presence of the tumoral peptide than in controls (P = 0.001). In addition, PBMC proliferation was greater when the viral peptides were added than with the tumoral peptide alone (P = 0.000). The IFN-γ secretion assay results supported the MTT assay (P = 0.000).
In this study, we demonstrated that the hTERT-derived peptide induced CD4 T cell proliferation in healthy individuals and patients with lung cancer. Both PBMC proliferation and IFN-γ secretion were further induced by the addition of tumor and viral peptides in combination.

Choline-binding proteins (CBPs) are a group of surface-exposed proteins, which play crucial and physiological roles in Streptococcus pneumoniae. The novel member of CBPs, choline-binding protein M (CbpM) may have binding activity to plasma proteins. This study aimed to clone and express CbpM and demonstrate its interaction with plasma proteins and patients’ sera.
The total length of cbpM gene was cloned in pET21a vector and expressed in BL21 expression host. Verification of recombinant protein was evaluated by Western blot using anti-His tag monoclonal antibody. Binding ability of the recombinant protein to plasma proteins and the interaction with patients’ sera were assessed by Western blot and ELISA methods.
The cbpM gene was successfully cloned into pET21a and expressed in BL21 host. Binding activity to fibronectin and fibrinogen and antibody reaction of CbpM to patients’ sera was demonstrated by Western blot and ELISA methods, respectively.
CbpM is one of the pneumococcal surface-exposed proteins, which mediates pneumococcal binding to fibronectin and fibrinogen proteins.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are major health problem in the world. Hairdressers (barbers) are in continuous contact with scissors and blades, and are considered a high-risk group for these infections.
The aim of this study was to analyze the prevalence of hepatitis B and C infections in barbers in Tehran and to evaluate their attitudes and knowledge about the occupational risk of these infections.
Six hundred eleven barbers were included in this study. A group of 556 bakers were also selected from the same regions, as a low-risk control group. Serum levels of hepatitis B surface antigen (HBsAg), HBsAg-specific antibody (HBsAb), hepatitis B core antigen-specific antibody (HBcAb), and hepatitis C virus-specific (anti-HCV) antibody markers were measured with the enzyme-linked immunosorbent assay (ELISA). Participants were interviewed using a questionnaire consisting of four sections: demographic information, awareness, behavior, and personal attitudes.
There were no significant differences in the frequency of HBsAg between the two groups. However, the frequency of HCV Ab in barbers was significantly higher than that in bakers (P Being a barber alone is not a potential risk factor for HBV infection, while HCV infection is still an occupational health hazards for barbers. We suggest more extensive case-control studies with regard to rates of hepatitis B and C markers among barbers in other Iranian cities to assess the incidence of hepatitis B and C infections among this population.

In addition to passive immunotherapy using anti-HER2 monoclonal
antibodies, active immunotherapy via HER2 targeting is an interesting approach to
inducing specific anti-tumor immune responses. We have recently reported the
immunogenicity of HER2 subdomains following DNA immunization and HER2 protein
boosting. In the present study, we evaluated the immunogenicity of different HER2
extracellular subdomains for the induction of anti-HER2 antibody response in BALB/c
mice.
To investigate and characterize antibody responses to human
recombinant proteins of HER2 extracellular subdomains in immunized mice. Four subdomains of HER2 extracellular domain were expressed in E.coli; subsequently,
purified recombinant proteins were intraperitoneally injected in BALB/c mice with
Freund's adjuvant. The anti-HER2 antibody response was detected by ELISA,
immunoblotting and flow cytometry.
All the four HER2 subdomains along with the full extracellular domain (fECD) were able to induce specific anti-HER2 antibodies. Although anti-HER2 subdomains antibodies could not react with eukaryotic recombinant fECD protein by ELISA, they were able to recognize this protein by immunoblotting under both reduced and non-reduced conditions. Furthermore, only the sera of mice immunized with fECD protein could recognize native HER2 on HER2 overexpressing tumor cells (>99%) by flow cytometry. Moreover, fECD immunized mice sera inhibited the proliferation of tumor cells by XTT assay.
The prokaryotic recombinant proteins of HER2 extracellular subdomains are immunogenic, yet the induced specific antibodies do not react with the native HER2 protein due to the paucity of post-translation modifications and /or distortion of the native conformation of isolated HER2 extracellular subdomains which might be potentially effective for induction of cell mediated immune response against HER2.

Antibodies have a wide application in diagnosis and treatment. In order to maintain optimal stability of various functional parts of antibodies such as antigen binding sites, several approaches have been suggested. Using additives such as polysaccharides and polyols is one of the main methods in protecting antibodies against aggregation or degradation in the formulation. The aim of this study was to evaluate the protective effect of various additives on the specific reactivity of monoclonal antibodies (mAbs) against recombinant HBsAg (rHBsAg) epitopes. To estimate the protective effect of different additives on the stability of antibody against conformational epitopes (S3 antibody) and linear epitopes (S7 and S11 antibodies) of rHBsAg, heat shock at 37°C was performed in liquid and solid phases. Environmental factors were considered to be constant. The specific reactivity of antibodies was evaluated using ELISA method. The data were analyzed using SPSS software by Mann-Whitney nonparametric test with the confidence interval of 95%. Our results showed that 0.25 M sucrose, 0.04 M trehalose and 0.5% BSA had the most protective effect on maintaining the reactivity of mAbs (S3) against conformational epitopes of rHBsAg. Results obtained from S7 and S11 mAbs against linear characteristics showed minor differences. The most efficient protective additives were 0.04 M trehalose and 1 M sucrose. Nowadays, application of appropriate additives is important for increasing the stability of antibodies. It was concluded that sucrose, trehalose and BSA have considerable effects on the specific reactivity of anti rHBsAg mAbs during long storage.

Hepatitis B virus (HBV) infection and its sequelae such as cirrhosis and hepatocellular carcinoma has remained a serious public health problem throughout the world. The WHO strategy for effective control of HBV infection and its complications is mass vaccination of neonates and children within the framework of Expanded Programme on Immunization (EPI). Vaccination with hepatitis B surface antigen (HBsAg) induces protective antibody response (anti-HBs ≥ 10 IU/L) in 90-99% of vaccinees.The lack of response to HBsAg has been attributed to a variety of immunological mechanisms, including defect in antigen presentation, defect in HBsAg-specific T and/or B cell repertoires, T-cell suppression, increase in the regulatory T cell count, lack of necessary help of T-cells for production of anti-HBs by B cells, defect in Th1 and/or Th2 cytokine production and selective killing of HBsAg-specific B-cells by human leukocyte antigen (HLA)-restricted cytotoxic T lymphocytes. The HLA complex plays an important role in many of these immunological processes.A variety of HLA class I, II, and III alleles and antigens have been reported to be associated with antibody response to HBsAg vaccination in different ethnic populations. Moreover, some HLA haplotypes were also associated with responsiveness to HBsAg.In this review the association of the HLA specificities with antibody response to hepatitis B (HB) vaccine is discussed.

bovine serum albumin (BSA) nanoparticles. Concerning two different crosslinkers for conjugation of 1F2, Maleimide-poly (ethylene glycol)-Succinimidyl carbonate (Mal-PEG5000-NHS) was selected due to its higher conjugation efficiency (23±4 %) obtained in comparison to N-succinimidyl 3-(2-Pyridyl Dithio) Propionate (SPDP) (8±2 %). A slight increase in the average particle size with a negligible prolongation of the 5-FU release was observed after 1F2 coupling. The 1F2-coupled 5-FU-loaded BSA nanoparticles interacted with nearly all HER2 receptors available on the surface of HER2-positive SKBR3 cells. No cellular uptake was observed for HER2-negative MCF7 cells. Physicochemical and biological properties of the mAb-modified nanoparticles did not significantly alter after three months of storage at room temperature. The in vitro cytotoxicity evaluation by MTT assay, demonstrated lower SKBR3 viability (50.7±9 %) after 5 hours contact with 1F2-coupled 5-FU-loaded BSA nanoparticles in comparison with the other control systems due to their cell attachment and internalization after washing. In addition, no significant toxicity was observed on MCF7 cells. This novel system can efficiently be employed for targeted delivery of 5-FU to HER2-positive cancerous cells.

Re-emergence of pertussis has been reported in Iran despite a high rate of vaccination coverage. Low efficacy of the vaccine might be due to the genetic divergence between clinical versus vaccine strains. In the current study، the genetic profiles of clinical isolates and vaccine strains of Bordetella pertussis (B. pertussis) were assessed by using Pulsed Field Gel Electrophoresis (PFGE). Following phenotypic and molecular identification of isolates، XbaI-digested genomic DNA of 5 clinical isolates، 2 vaccine strains and a Tohama I strain were analyzed by PFGE along with B. parapertussis as a control. Seven distinct PFGE profiles were found among all examined isolates/strains. In 5 clinical isolates، 4 profiles were identified whereas the vaccine strains displayed 2 distinct profiles. The reference strain، Tohama I had a distinct profile. Vaccine and clinical profiles had low similarity، with relatedness of approximately 40%. The genetic profiles of B. pertussis were different between circulating isolates and vaccine strains used in the national vaccination programs. Since new genetic profiles of B. pertussis can be disseminated periodically، the profiles of isolates circulating in the population should be monitored over the course of the re-emergence.

The antibody response to hepatitis B surface antigen (HBsAg) controls hepatitis B virus infection. The "a" determinant of HBsAg is the most important target for protective antibody response, diagnosis and immunoprophylaxis. Mutations in this area may induce immune escape mutants and affect the performance of HBsAg assays. To construct clinically relevant recombinant mutant forms of HBsAg and assessment of their reactivity with anti-HBs monoclonal antibodies (MAbs). Wild type (wt) and mutant (mt) HBsAg genes were constructed by site directed mutagenesis and SEOing PCR. The amplified genes were inserted into pCMV6-neo plasmid and transfected in CHO cell line. The expression of wt- and mtHBsAg was assessed by commercial ELISA assays and stable cells were established and cloned by limiting dilution. The recombinant mutants were further characterized using a panel of anti-HBs monoclonal antibodies (MAbs) and the pattern of their reactivity was assessed by ELISA. Ten HBsAg mutants having single mutation within the "a" determinant including P120E, T123N, Q129H, M133L, K141E, P142S, D144A, G145R, N146S and C147S together with a wt form were successfully constructed and expressed in CHO cells. Reactivity of anti-HBs MAbs with mtHBsAgs displayed different patterns. The effect of mutations on antibody binding differed depending on the amino acid involved and its location within the ‘‘a’’ determinant. Mutation at amino acids 123 and 145 resulted in either complete loss or significant reduction of binding to all anti-HBs MAbs. Our panel of mtHBsAgs is a valuable tool for assessment of the antibody response to HBV escape mutants and may have substantial implications in HBV immunological diagnostics.

The Fc receptor like (FCRL) molecules belong to the immunoglobulin (Ig) superfamily with potentially immunoregulatory function. Among the FCRL family FCRL2 and 4 are predominantly expressed on memory B cells and FCRL1 is a pan- B cell marker. To date، no ligand has been identified for the human FCRL1، 2 and 4 molecules.. Cloning، expression، purification and structural analysis of the extracellular domain of human FCRL1، 2 and 4 proteins.. In this study، the extracellular part of human FCRL1، 2 and 4 were subcloned into prokaryotic expression vectors pET-28b (+) and transformed into BL21-DE3 E. coli strain. Protein expression was optimized by fine adjustments such as induction time، incubation temperature and expression hosts. Recombinant FCRL proteins were purified by metal affinity chromatography using Ni-NTA resin. Purified FCRL proteins were further characterized by SDS-PAGE and immunoblotting using His-tag and FCRL specific polyclonal antibodies.. Our results demonstrated that FCRL1، 2 and 4 were successfully expressed in pET-28b (+) vector. Optimization of the expression procedure showed that IPTG induction at OD600 = 0. 9 and overnight incubation at 37˚C resulted in the highest expression levels of FCRL proteins ranging from approximately 15% (FCRL1) to 25% (FCRL2 and 4) of the total bacterial lysate proteins.. These purified recombinant proteins are potentially a valuable tool for investigating the immunoregulatory function of FCRL molecules and the production of specific mAbs for immunotherapeutic interventions..