Design and Production of a Multiepitope Construct Derived From Hepatitis E Virus Capsid Protein

The aim of this study was to design a high density multiepitope protein, which can be a promising multiepitope vaccine candidate against Hepatitis E virus (HEV). Initially, conserved and antigenic helper T-lymphocyte (HTL) epitopes in the HEV capsid protein were predicted by in silico analysis. Subsequently, a multiepitope comprising four HTL epitopes with high-affinity binding to the HLA molecules was designed, and repeated four times as high density multiepitope construct. This construct was synthesized and cloned into pET-30a (þ) vector. Then, it was transformed and expressed in Escherichia coli BL21 cells. The high density multiepitope protein was purified by Ni-NTA agarose and concentrated using Amicon filters. Finally, the immunological properties of this high density multiepitope protein were evaluated in vitro. The results showed that the high density multiepitope construct was successfully expressed and purified. SDSPAGE and Western blot analyses showed the presence of a high density multiepitope protein band of approximately 33 kDa. Approximately 1mg of the purified protein was obtained from each liter of the culture media. Moreover, the purified multiepitope protein was capable of induction of proliferation responses, IFN-g ELISPOT responses and IFN-g and IL-12 cytokines production in a significant level in peripheral blood mononuclear cells (PBMCs) isolated from HEV-recovered individuals compared to the control group. In conclusion, the newly produced multiepitope protein can induce significant T helper type 1 responses in vitro, and can be considered as a novel strategy for the development of HEV vaccines in the future. J. Med. Virol

Prevalence of Hepatitis E Virus among Adults in South-West of Iran

Background. Knowledge regarding prevalence of HEV in general population can be an indicator of the public health and hygiene. Therefore, this study was conducted to evaluate the prevalence ofHEV among adults in South-West of Iran. Methods. Blood samples were taken from510 participants, 206 (40.4%) males and 304 (59.6%) females fromFebruary to July 2014.Detection of anti-HEVIgG and IgM antibodies was carried out by ELISA test. Results.The overall anti-HEV IgG and IgMprevalence rates were 46.1% and 1.4%, respectively.Anti-HEVIgG and IgMseropositivitywere not statistically associated with gender and race/ethnicity.Meanwhile, there were significant differences between the age groups regarding HEV IgG and IgMseropositivity. HEV IgG seroprevalence increased with age from 14.3% in subjects aged 18–30 years to 71.4%in persons over 71 years old, and considerably individuals aged 61 to 70 years had the highest HEV prevalence (90.9%). Also, 5.7% in the age group 18–30 years and 2.2% in the age group 31–40 years were positive for anti-HEV IgM antibodies and the highest rate was observed in subjects aged 18–30 years. Conclusion. In conclusion, high HEV IgG seroprevalence of 46.1% was observed among adults in South-West of Iran

Development of an indirect sandwich ELISA for detection of urinary antigen, using Legionella pneumophila PAL protein

Legionella pneumophila peptidoglycan-associated lipoprotein (PAL) protein is an extremely conserved antigen among Legionella species. In this study, rabbit and rat anti-PAL immunoglobulin G antibodies were produced by immunization with purified, recombinant PAL (r-PAL) protein of L. pneumophila serogroup 1 and used as capture and detection antibodies in the PAL antigen-based enzyme-linked immunosorbent assay (ELISA) to detect urinary PAL antigen. Urine samples were obtained from rats experimentally infected with L. pneumophila serogroup 1. The PAL antigen was measured in urine samples of 40 infected and 40 uninfected rats. After choosing the cut-off value of 0.192, the sensitivity and specificity of the PAL antigen-based ELISA were 87.5 and 97.5 %, respectively. The results obtained by PAL antigen base ELISA were compared with those obtained by Biotest. The PAL antigen was detected efficiently by both of the assays and all of the control human urine samples were negative by the ELISA test. The PAL antigen-based ELISA assay was relatively simple to perform, precise, highly sensitive and specific, and reproducible. Based on our data the PAL antigen-based ELISA described here is the first indirect sandwich ELISA for urinary antigen detection which could easily be applied for diagnosis of Legionnaires disease

Cloning and periplasmic expression of peptidoglycan-associated lipoprotein (PAL) protein of Legionella pneumophila in Escherichia coli

Abstract Introduction and objective: Legionella pneumophila, the etiological agent of Legionnaires’ disease, is an important cause of both community-acquired and nosocomial pneumonia; therefore, rapid diagnosis and early antibiotic treatment of pneumonia are required. Urinary antigen testing to detect Legionella antigen has proven to be the most powerful diagnostic method. Peptidoglycan-associated lipoprotein (PAL) protein of L. pneumophila, as a component of Legionella antigens, will be detected efficiently by the PAL antigen capture assay and is considered as useful diagnostic antigen to diagnose Legionella infection. Because of the transfer of protein to the periplasmic region of Escherichia coli has numerous advantages including separation from cytoplasmic proteins and the concentration of recombinant proteins in periplasm, the aim of this study was to produce periplasmic PAL protein of L. pneumophila in E. coli. Materials and methods: The pal gene of L. pneumophila serogroup 1 was amplified with specific primers, cloned and expressed under pelB signal sequence and T7 lac promoter in pET26b+ plasmid. Results: The cloning was confirmed with digestion and sequencing of recombinant pET- 26b-pal plasmid. The expression of r-PAL protein in cytoplasm and periplasmic space of E. coli was approved by SDS-PAGE and western blotting. Conclusion: The results of this study demonstrated that the r-PAL protein successfully expressed in E. coli

The Frequency of Human Polyomavirus BK in Patients with Systemic Lupus Erythematosus: A Cross-Sectional Case-Control Study

Background and Aim: Systemic lupus erythematosus (SLE) is an autoimmune disease and human polyomavirus BK (BKV) can be reactivated in patients with SLE due to the changes in the immune system and use of immunosuppressive drugs. In this study, we evaluated the prevalence of BKV infection among patients with SLE referred to Golestan hospital in Ahvaz, Iran between April 2013 to June 2016. Methods: In this cross-sectional study we studied 75 individuals including 40 patients with SLE and 35 normal individuals. Urine and blood samples were taken and DNA was extracted from urine and plasma. Polymerase Chain Reaction (PCR) test was used to detect the BKV genome and positive samples were sequenced to confirm BKV. BioEdit software and MEGA 6.0 software were used for phylogenetic analysis to assemble the viral genome. A phylogenetic tree was constructed by neighbor-joining analysis with 1,000 replicates of the bootstrap resampling test using Mega 6.0. Statistical analysis was done by SPSS version 22. Results: Among the 40 patients, 2 (5%) were men and 38 (95%) were women.  The mean age of the patients was 39±10 years. 2.5% of plasma from patients with SLE were positive for BKV but none of the controls were positive in this regard.0% of control groups (p=0.346). Whereas in urine samples, 17.5% and 11.4% (p=0.458) of patients and the control group, were positive for BKV, respectively. However, there was no statistically significant difference between the patients and controls. Conclusion: BKV reactivation occurs in 17.5% of patients with SLE during immunosuppression therapy. Therefore, more studies on BKV DNA by highly sensitive molecular assays in Patients with SLE seem to be necessary. *Corresponding Author: Gholam Abbas Kaydani; Email: [email protected] Please cite this article as: Behzadi Sheikhrobat S, Kaydani GA, Makvandi M, Rajaee E, Ahmadi Angali K. The Frequency of Human Polyomavirus BK in Patients with Systemic Lupus Erythematosus: A Cross-Sectional Case-Control Study. Arch Med Lab Sci. 2021;7:1-6 (e5). https://doi.org/10.22037/amls.v7.3399

Periplasmic expression and one-step purification of urease subunit B of Helicobacter pylori

UreB is one of the urease subunits of Helicobacter pylori and can be used as an excellent antigen candidate for H. pylori vaccination. Easy access to highly purified UreB protein, facilitate advances in therapeutic or preventive strategies. To achieve a simplified purification procedure, the present report represents a novel method of producing recombinant urease subunit B extracellularly. ureB gene from 26,695 standard strain was amplified by PCR and cloned into pET-26b(+) expression vector. UreB was expressed as a soluble, N-terminal pelB and C-terminal hexahistidine-tagged fusion protein (UreB-6His) and secreted into the periplasmic space of Escherichia coli. Expression of the recombinant UreB in E. coli BL21 (DE3) was induced by isopropylthio-β-d-galactoside (IPTG). Expression of UreB was confirmed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and western blot analysis using anti-His monoclonal antibody. UreB-6His protein was extracted from the periplasm by osmotic shock treatment and was purified in one step by Nickel affinity chromatography. In conclusion, the present protocol is easier to perform; more time effective and low cost than earlier methods. Keywords Helicobacter pylori –Urease subunit B–Cloning–Periplasmic expression Periplasmic expression and one-step purification of urease subunit B of Helicobacter pylori (PDF Download Available). Available from: https://www.researchgate.net/publication/227181089_Periplasmic_expression_and_one-step_purification_of_urease_subunit_B_of_Helicobacter_pylori [accessed Dec 09 2017]

Optimization of gene expression and purification of Legionella pneumophila peptidoglycan associated lipoprotein recombinant protein

زمینه و هدف: اکثر تست های موجود برای تشخیص پنومونی لژیونلایی، با مشکلات زیادی از جمله حساسیت و ویژگی پائین و عدم توانایی در فراهم نمودن نتیجه در یک زمان کلینیکی مناسب مواجه هستند. از آنجایی که لیپوپروتئین مرتبط با پپتیدوگلیکان ( PAL ) لژیونلا پنوموفیلا در درون ادرار ترشح شده و به عنوان یک آنتی ژن در تمام سویه های آن وجود دارد. لذا برای تشخیص بیماری لژیونر از طریق ادرار مورد توجه قرار گرفته است. هدف از این مطالعه بهینه سازی بیان و تخلیص پروتئین نوترکیب PAL باکتری لژیونلا پنوموفیلا بوده است. روش بررسی: در این مطالعه تجربی آزمایشگاهی بیان پروتئین PAL با تغییر در پارامترهای دانسیته سلولی، مدت زمان القاء، دمای رشد، غلظت ایزوپروپیل بتا دی تیوگالاکتوپیرانوزید ( IPTG ) و نوع محیط کشت مورد بررسی قرار گرفت. پس از کلونینگ، عصاره پری پلاسمی تهیه و پروتئین نوترکیب PAL با استفاده از ستون رزین نیکل نیتروتری استیک اسید ( NTA ) تخلیص گردید. در نهایت پروتئین نوترکیب PAL با آزمایش وسترن بلاتینگ بررسی شد. یافته ها: با استفاده از محیط کشت Terrific Broth ، شروع القاء در جذب نوری 6/0 (طول موج 600 نانومتر)، غلظت یک میلی مولار ماده القاء کننده IPTG ، دمای رشد 25 درجه سانتیگراد و مدت زمان 15 ساعت پس از القاء، بهترین بیان پروتئین PAL بدست آمد. پروتئین پری پلاسمی نوترکیب PAL با استفاده از ستون رزین NTA با خلوص بیش از 80 تخلیص گردید. نتایج آزمایش وسترن بلاتینگ نیز نشان داد که پروتئین نوترکیب PAL تخلیص شده به طور اختصاصی با آنتی بادی anti-His6-peroxidase شناسایی گردید. نتیجه گیری: با تخلیص پروتئین PAL به میزان بیش از 80 می توان به منظور بررسی پتانسیل آن در تشخیص بیماری لژیونر و تهیه کیت تشخیصی بر اساس الایزا از آن استفاده نمود

Cytokine Profiles and Cell Proliferation Responses to Truncated ORF2 Protein in Iranian Patients Recovered from Hepatitis E Infection

Background.The aim of this study was to evaluate hepatitis E virus (HEV) specific cellular immune responses to truncated ORF2 protein in Iranian patients recovered from HEV infection. Information about HEV-specific immune responses could be useful in finding an effective way for development of HEV vaccine. Methods. A truncated formof HEVORF2 protein containing amino acids 112-608 was used to stimulate peripheral blood mononuclear cells (PBMCs) separated from HEV-recovered and control groups. Finally, the levels of four cytokines, IFN

Low presence of papillomavirus and its lack of correlation with clinicopathological factors in breast cancer: a case control study

Background and Objectives: Breast cancer is currently the most commonly diagnosed neoplasm in women worldwide. There is evidence that human papillomavirus (HPV) infection may play a key role in breast cancer aggressiveness, but results are conflicting across studies. The aim of this study was to investigate the presence of the HPV viral genome in benign and malignant breast tissue samples and its clinicopathological characteristics of cancer. Materials and Methods: In this case-control study, 100 formalin-fixed paraffin-embedded (FFPE) of breast cancer and 100 blocks of non-cancerous breast tissue were selected as a control group from the pathology department of Imam Khomeini Hospital in Ahvaz from 2020-2022. The presence of HPV was detected using nested PCR including MY09/11 primers and sequencing were performed for virus genotyping. Results: The present study enrolled 100 subjects each in two cancer and control groups with a mean age of 52.81±13.23 and 35.77±11.65, respectively. The risk of cancer in HPV-infected patients is almost 5 times higher than in HPV-negative individuals, it is not statistically significant (OR =4.99, 95% CI 0.35 to 72.15, p=0.238). The prevalence of HPV in the cancer and control groups was 7% and 1%, respectively and HPVs detected in two groups were of the HPV 16 genotype. Although the chance of ER and PR expression, lymphvascular involvement, perineural invasion, and higher tumor grade was higher in HPV-positive subjects than in HPV-negative subjects, this was not statistically significant (OR>1, p>0.05). Conclusion: Based on studies reporting the existence of sequences of different high-risk HPV types (oncogenes) in breast cancer tissues, this study confirmed the hypothesis of a possible infectious cause in the development of breast cancer. So far, however, the results have been controversial and inconclusive. Further studies with large sample sizes are needed to demonstrate the link between HPV and breast cancer

Parechovirus and enteroviruses among young infants with sepsis in Iran

Background and Objectives: Human parechoviruses (HPeV) and Human enteroviruses (EV) frequently cause a sepsis-like illness in young infants (younger than three months). Therefore, this study was conducted to determine the frequency of HPeV and EV among the young infants with clinical signs and symptoms of sepsis in Ahvaz city, Iran. Materials and Methods: The blood specimens were collected from 100 (younger than 90 days hospitalized infants) including 54 (56.25%) males and 46 (43.75%) females with clinical signs and symptoms of sepsis-like disease. The RNA was extracted and tested for detection of VP1 region of HPeV and 5 UTR (Untranslated Region) of EV by RT-PCR. The sequences of positive of HPeV were further analyzed to determine HPeV genotyping. Results: 5/100 (5%) of patients including 2/46 (2%) females and 3/54 (3%) males tested positive for HPeV (P=0.85). The analysis of 5 positive VP1 region of HPeV revealed the genotype 1. The analysis of sequencing and phylogenetic tree revealed that the isolated HPeVs were genotype 1. While 38/100 (38%) specimens including 16 (16%) females and 22 (22%) males were tested positive for EV (P=0.68). Conclusion: The frequency of HPeV genotype 1 was 5% among the young infants with sepsis. While frequency of EV was 38% among the young infants with sepsis. This study showed HPeV genotype 1 and EV are dominant in this region