سید مهدی سادات

Immunodeficiency virus-1 (HIV-1) is a global health problem and it has affected more than 75 million individuals since the epidemic started. Various approaches have been investigated to some up with a preventive or therapeutic formulation against this virus. However, none of them has been achieved. The aim of the study was the immunological evaluation of HIV-1 Nef-MPER-V3 Harboring IMT-P8 penetrating peptide in BALB/c mice to induce effective immune responses.

In current study, to evaluate of the antigen immunogenicity of IMT-P8-Nef-MPER-V3, 11 different groups of female BALB/c mice were immunized with three doses of the antigen with or without Hsp27 and Hp91 adjuvants formulation. The immune responses were assessed using IgG ELISA assay and its isotype determination. IL-10 and IFN-γ were also evaluated by sandwich ELISA.

The data showed that the recombinant protein developed different levels of humoral and cellular responses. IMTP8-Nef-MPER-V3+Hp91 groups reached highest IgG2a, and elicited strong IFN-γ production towards a Th1 response compared to the other groups. Cytokine assay indicated that the immunized mice with the antigen formulation containing IMT-P8 CPP applied with Hp91 has a high potency in immune induction.

These results demonstrated that application of IMTP8-Nef-MPER-V3 combining the adjuvant-formulated (Hp91) provides strong responses which must be considered as an effective formulation towards a potential HIV vaccine candidate.

In spite of the improvements in developing antiretroviral treatments, there are no approved HIV vaccines. To achieve an effective vaccine against HIV-1 requires induction of strong humoral and cellular immune responses, so developing an effectual vaccine is required. The aim of this study was the immunological assessment of HIV-1 Nef-MPER-V3 harboring LDP12 penetrating peptide in BALB/c mice in order to induce effective immune responses. Materials &

In the current study, presenting 55 female mice were utilized for immunization with LDP12-NefMPER-V3. The mice were divided into 11 groups with 5 mice each group. Immunizations were performed three times at three week intervals and subcutaneously in a volume of 100 μl per mouse. Two weeks after final injection, humoral and cellular immune responses were evaluated in blood serum and splenocytes respectively, by using ELISA method. Finally, the data analysis was performed, using Mann-Whitney u test.

Although the level of total antibody production was observed in all main groups with different titrations, but the antibody level was higher in the mice groups that injected with the LDP12 antigen with adjuvants immunity than in the control group (p=0.045). Also, IgG2a was the predominant isotype (Th1-biased response) in mice immunized group that had LDP12 antigen with Hsp27 adjuvant.

The data indicated that the high immune system stimulating capabilities of LDP12-Nef-MPER-V3 injection regime accompanied by Hp91 adjuvant, which provides the potential for an efficient HIV vaccine.

Nef regulatory protein as well as structural proteins MPER and V3 of HIV-1 is important targets in vaccine studies. Therefore, in the current study, the Nef-MPER-V3 fusion protein was cloned and expressed in prokaryotic expression system that will be considered as a candidate for future vaccine studies.

The Nef-MPER-V3 sequence was synthesized and inserted into pET-28a(+) vector using NotI/HindIII enzymes. Then, the recombinant construct was expressed in BL21 (DE3) strain of E.coli. Several factors including time course, IPTG concentration and optical density (OD) were evaluated for optimization of protein expression. The protein expression was purified on a Ni-NTA agarose column and analyzed by 12% SDS-PAGE. Finally, the fusion protein was identified and confirmed by western blotting.

The target gene was cloned successfully into the recombinant vector and after selection of the best condition for the protein expression such as; 2 mM of IPTG, OD= 0.7 at 600nm and 5 hours after induction, purification of the recombinant protein by affinity chromatography in denaturing condition by urea yielded about 300 µg/ mL. The purified fusion protein migrated as a clear band of around 35 kDa in SDS-PAGE and was detectable using anti-Nef antibody in western blotting.

Our results showed that the Nef-MPER-V3 protein was successfully expressed in prokaryotic system and purified protein may provide the antigen for future experiments for immunogenicity evaluation in mice are currently undertaken.

Aims: Developing an effective vaccine against Human Immunodeficiency Virus (HIV) is necessary. The aim of this study was the immunological evaluation of HIV-1 VLP harboring MPER-V3 in BALB/c mice model.
In the present experimental research, presenting 40 female mice, which were between 6 and 8 weeks old, were used for immunization with VLP MPER-V3. The mice were divided into 8 groups and in each group, 5 mice were considered. Injections were performed three times at three weeks intervals and subcutaneously in a volume of 100μl per mouse. Two weeks after last injection, mouse blood samples were collected by retro-orbital bleeding and immune responses were evaluated in serum for levels of total IgG and splenocytes for cytokine assay, using ELISA method. The data analysis was performed, using Mann-Whitney test and one-way analysis of variance.
Findings: The level of total antibody production was very high in all groups that had VLP alone or with adjuvant immunity, having a significant difference with the control group (p VLPs can stimulate the humoral immune system in mice immunized with these particles alone or formulated with adjuvant. Also, the level of production of IL-5 in the presence of the vaccine candidate as VLP increased significantly.

Aim and Recent studies have claimed that the genetic variation in cytokine production systems has a major effect on immune system potency and strongly associated with the antiviral treatment response. On the other hand, tumor necrosis factor-alpha (TNF-α), a multifunctional pro-inflammatory cytokine, plays an essential role in host immune response to HCV infection. Therefore, current study was done in order to determine the distribution of the rs1800629 (A/G) polymorphism in the promoter of TNF-α gene in Iranian population.
Material and This cross-sectional study, performed on 165 blood samples based on 89 HCV infected patients including 68 SVR positive and 21 negative and 76 healthy individual controls. After DNA extraction the frequency of rs1800629 (A/G) polymorphism was analyzed using PCR-RFLP method. For detection and analysis of the PCR products we used 20% Polyacrylamide gel electrophoresis.
Based on analysis of the data, the distribution of the A/G polymorphism between healthy individuals and patients were obtained as AA: 1.1%, AG: 52.6%, GG: 46.1%, and AA: 1.3%, AG: 20.2%, GG: 78.7% respectively. The GG genotype was identified in 70 patients of whom 55 achieved SVR, while the AG heterozygous was found in 18 patients and SVR was achieved in 12. Finally, the AA was detected only in one patient with positive SVR.
Conclusion In this study, significant difference between the TNF-α-308 locus SNP (rs1800629) and susceptibility to chronic HCV infection in Iranian population (P value= 0.002) was observed. Moreover, the presence of G allele among SVR positive people and non-SVR group did not show strong statistic difference (P value=0.476). However, further studies with more samples seems to be necessary.

Aim and Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are two forms of non-melanoma skin cancer. Recent research indicated that Merkel cell polyomavirus (MCPyV) is associated with Merkel cell Carcinoma (MCC). So, the aim of current study was presence of MCPyV in the tumor tissue and role of large T antigen (LT Ag) mutations that associated with MCPyV oncogenic properties in LTAg region in positive samples that referred to Razi Hospital in Tehran within 2015-2016.
In this cross-sectional study, 55 tissue samples containing: 30 BCC, 10 SCC and 15 individual healthy samples were collected and genomic DNA was extracted. To finding positive samples, quantitative Real-Time PCR was done and the full-length of Large T antigen region was amplified and sequenced directly for detection of probable mutations.
MCPyV genome was detected only 10% of BCC samples. There is no correlation between sex (P-Value=0.33), age (P-Value=0.5), or stage of cancer (P-Value=0.25) and the presence of MCPyV genome in our populations of study.
Viral infections are one of the factors in causing cancer. Although, several studies were reported that frame shift mutation at the N-terminus of LT Ag region is associated with tumorigenesis of the virus, but in current study, no any mutations causing stop codons or frame shifting were observed. As our knowledge this is the first study presenting data on the prevalence of MCPyV in non-melanoma skin cancer from Iranian patients. However, further studies with more samples are necessary.

Nef protein is one of the HIV regulatory proteins. This protein has various conserved epitopes inducing the efficient immune responses in HIV-1 infected individuals. Thus, Nef protein has been proposed as a suitable candidate for vaccine design. In current study, our goal was the cloning and expression of Nef protein in prokaryotic expression system and its purification using the reverse staining method.
The coding sequence of Nef protein was amplified from pUC19-nef vector by PCR. Then, nef gene was inserted into the pGEX6p2 expression vector. This construct was transformed into the E.coli BL21 E.coli strain and subsequently protein expression was induced by IPTG anti-repressor. The protein expression was confirmed by SDS-PAGE and Western blot using anti-Nef antibody. Protein purification was performed by reverse staining method.
The PCR and digestion analysis showed a clear band of 648 bp in agarose gel indicating the correct cloning of HIV-nef in pGEX6p2 expression vector. In addition, the detection of a clear 50 kDa band in Western blotting using Anti-Nef antibody suggests the Nef protein expression induced by IPTG. Finally, the purified protein was obtained by reverse staining method.
The recombinant Nef protein expressed in E.coli was purified by reverse staining method. The Nef protein has the potential of antigenicity for vaccine designing against HIV infections.

Nef is one of the HIV-1 critical proteins, because it is essential for viral replication and AIDS disease progression and induction of immune response against it can partially inhibit viral infection. Moreover, a domain of the HIV-1 Trans-Activator of Transcription (Tat, 48-60 aa) could act as a cell penetrating peptide (CPP). In current study, cloning and expression of Tat-Nef fusion protein was performed in E. coli for the first time. The protein expression was confirmed by western blot analysis and was purified using reverse staining method.
In this experimental study, primarily, cloning of Tat-Nef fusion gene was done in pGEX6p2 expression vector. Then, the expression of Tat-Nef recombinat protein in E.coli BL21 (DE3) strain was performed by using IPTG inducer. The protein expression was confirmed by SDS-PAGE and western blotting using anti-Nef monoclonal antibody. Then, the recombinant fusion protein was purified from gel using reverse staining method.
The results of PCR analysis and enzyme digestion showed a clear band of ~ 726 bp in agarose gel indicating the correct Tat-Nef fusion cloning in pGEX6p2 prokaryotic expression vector. In addition, a 54 kDa band of Tat-Nef on SDS-PAGE revealed Tat-Nef protein expression that western blot analysis using anti-Nef monoclonal antibody confirmed it.
The purified Tat-Nef recombinant fusion protein will be used as an antigen for protein vaccine design against HIV infection.

Aim and The envelope glycoprotein E2 in Hepatitis C virus is required for host-cell entry. The nucleotide sequence diversity in carboxyl-terminus (C-terminal) of this gene results in induction of neutralizing antibody that makes this gene an important target for vaccine development studies. In this study we tried to make a recombinant pET28a () containing full length of the E2 gene from genotype 2a (JFH1) followed by being transformed into the E-coli (DH5a).
Material and Full-length of HCV E2 region from genotype 2a (JFH1) was amplified by PCR reaction using the specific primers including the restriction sites for EcoR1 and Nde1. The confirmed PCR product was cloned into the pET28a () vector and transformed into the E-coli (DH5a) using the heat shock method.
The HCV E2 gene was successfully cloned into the pET28a () vector in full length size. Transformation of the E-coli strain DH5a by this recombinant vector was confirmed.
Transformation of the E-coli (DH5a) using the recombinant vector pET28a- HCV 2a (JFH1) E2 gene provides useful tools for further expression of this gene in full length size in different expression system.

Background and The purpose of this study was to design a recombinant vector pEFGP- N1 containing the full length of HIV-1Vpr gene. To the best of our knowledge, the cloning of Vpr gene in pEGFP_N1 is not previously done.
As a source of Vpr gene the pUC19-Vpr recombinant vector was confirmed by digestion with restriction enzymes BglII and NotI in order to separate the Vpr gene. The specific primers for Vpr gene including the restriction sites of XhoI and KpnI were designed according to the multiple cloning site of pEGFP-N1 and PCR reaction was performed using the pUC 19-Vpr vector as a template. The PCR product was undergone electrophoresis and gel extraction. Digestion reaction was done on both the extracted PCR product and the pEGFP-N1vector. The recombinant pEGFP-N1-Vpr vector was achieved by the ligation reaction using the T4 DNA ligase and it was transformed into the E-coli (DH5α) and propagated. Finally, confirmation was done through the restriction enzyme digestion and PCR amplification.
The recombinant vector pUC19-Vpr was confirmed using the restriction enzymes digestion, and then Vpr gene was successfully amplified using its specific primers including restriction sites for XhoI and KpnI. The PCR product was confirmed by electrophoresis. Finally, a recombinant vector pEGFP-N1 containing the full length of human immunodeficiency virus-1 Vpr gene (pEGFP-N1-Vpr) was successfully constructed.
HIV-1 Vpr gene in its full length size could be ligated into the pEGFP-N1.

Salmonella spp. are enteric pathogen with a worldwide distribution comprising a large number of serovars. Many virulence factors of Salmonella are encoded by located genes on Salmonella pathogenicity islands (SPIs). This study was conducted to investigate the distribution of two SPI-3 related genes in among three serotypes of Iranian salmonella: typhi, paratyphi B and paratyphi C.
A total of 25 salmonella strains were isolated from Iranian patients with clinical symptoms of salmonellosis. All isolated belong to salmonella serotypes of typhi, paratyphi B and paratyphi C. Salmonella isolates were screened for the presence of mgtC and misL genes by PCR amplification method.
Among 25 salmonella strains, 20 (80%) were positive for SPI-3 region. The presence of misL gene in these serotypes of salmonella was as follows: typhi (100%), Paratyphi C (33%), Paratyphi B (25%) and for mgtC: typhi(97%), paratyphC(33%), paratyphiB(0%).
the widely distribution of SPI-3 among Iranian salmonella isolates provide the basic information on the genetic background of virulence as well as molecular properties of three different serotypes of Salmonella. This is the first report on the distribution of SPI-3 in Salmonella isolates from Iran.

The current medical treatment for hepatitis C is a combination of antiviral therapy along with pegylated interferon alpha and ribavirin. Recent studies have demonstrated that single nucleotide polymorphisms near the interleukin 28B gene coding for IFN-λ3 were associated with the antiviral response. Therefore, this study aimed to determine the frequency of G/T polymorphism of rs8099917 among the Iranian population. This cross-sectional study was performed on 93 blood samples (71 sensitive and 22 resistant to treatment) were collected from individuals suffering from chronic HCV, and 57 healthy controls. DNA was extracted from the samples and the frequency of the polymorphism was analyzed using the PCR-RFLP method. Finally, the products were detected on 3.5% agarose gel electrophoresis. The frequency of the G/T polymorphism between the healthy individuals and patients were TT: 75%, TG: 23%, GG: 2%, and TT: 57%, TG: 35%, GG: 8%, respectively. Moreover, the TT genotype was identified in 46 patients of whom 71 achieved SVR, while the GT heterozygous was found in 33 patients and SVR was achieved in 19. Finally, the GG was detected in 7 patients and only one patient was resistant to treatment. Results show a significant effect of G allele on susceptibility to HCV compared to the other allele T (P=0.013). Although no correlation was seen between the polymorphism and SVR among the patients, further studies with more samples are necessary.

Uterine myomas are benign tumors of the uterus and the most common solid pelvic tumors causing symptoms in approximately 25% of women in their reproductive years. However, its etiology and pathogenesis remain obscure; there is increasing evidence that endometriosis is inherited as a complex genetic trait. Recent studies indicated the involvement of glutathione S-transferase M1 (GSTM1) gene in the pathogenesis of this disease and current investigations are devoted to the other members of phase II detoxification system genes such as glutathione S-transferase T1 (GSTT1). Therefore, current study was carried out to investigate the distribution of GSTM1 and GSTT1polymorphisms in Iranian population in order to estimate possible impact of null-alleles of each gene in development of this disease. In this study, 50 patients with endometriosis diagnosed by both pathology and laparoscopic findings according to the revised American Fertility Society classification of endometriosis were recruited from subjects referred to the Pasteur Institute of Iran between November 2012 to September 2013. Accordingly, controls (n=50) were subjects without any of aforementioned gynecologic conditions. The genomic DNA was extracted from peripheral blood leucocytes using the salting out method and GSTM1 and GSTT1 genotyping for gene deletions were carried out using Gap-polymerase chain re-action. Logistic regression analysis was applied to assess whether there was any significant risk increase between the case group with higher null genotypes compared to control group. The level of statistical significance was set at 0.05 and all analyses were conducted using the SPSS version 18.0 (SPSS Inc., Chicago, IL). There was significant evidence that the distribution of the GSTM1 and GSTT1 genotypes differed between the patients and the controls with an allelic odds ratio (OR) of 3.56 (95%CI: 1.35-9.37, P=0.01) and 3.92 (95%CI: 1.4-10; P=0.009) respectively. Data analysis also revealed that individuals with both GSTM1 and GSTT1 null genotypes (-/-) had higher risk to develop the disease in comparison to the people with the both present (+/+) genotype (OR:19.23, P=0.007). The findings suggest that the GSTM1 and GSTT1 genetic polymorphisms are associated with the development of endometriosis in Iranian women which is in agreement with previous results obtained in other populations. However, the ethnic variations of polymorphisms should be evaluated in detail and differences should be incorporated into investigations of susceptibility variants for this disease.

Uterine leiomyoma is one of the most common benign smooth muscle tumors occurring in 20-40% of women worldwide in their reproductive years. Recent studies revealed that estrogen plays an important role in the pathogenesis of uterine leiomyoma. Since, Glutathione s-transferase (GST) gene families are involved in the biosynthesis of estrogen, the prior probability that variants at this locus are associated with uterine leiomyoma is likely to be above the null. Therefore, this study was carried out to examine whether GSTP1 Ile105Val polymorphism is associated with uterine leiomyoma in Iranian population. In this case-control study, 50 women affected with uterine leiomyoma and 50 healthy controls were recruited among participants at the Pasteur Institute of Iran from November 2012 to September 2013. The genomic DNA was extracted from peripheral blood leucocytes using the standard phenol-chloroform method and subsequently the GSTP1 polymorphism was genotyped using Amplification Refractory Mutation System (ARMS-PCR). Association of Ile105Val polymorphism with uterine leiomyoma was examined using the SPSS statistical software package, version 18.0 after age adjustment. The results showed significant differences between case and control groups in terms of genotype frequency (P<0.0001). In addition, the results indicated that the presence of the valine allele significantly increased risk of uterine leiomyoma approximately 3 times more in individuals carrying the mutant allele compared to control group (Odds Ratio: 3.34; 95%CI: 1.82-6.15; P<0.0001). To our knowledge, this is the first study performed in Iranian population assessing the association between GSTP1 Ile105Val polymorphism and risk of uterine leiomyoma. However, further extensive researches with a large number of samples from different populations and ethnicities are required to validate the results obtained in this study.

Background and The standard of care treatment for infected patients with HCV is based on a combination of pegylated interferon alpha and ribavirin. Recently, the rs12979860 SNP polymorphism, located upstream of the interleukin 28B gene, was shown to be strongly associated with response to anti-HCV therapy. This study investigated the distribution of the (C/T) polymorphism with sustained virologic response (SVR) to chronic Hepatitis C virus infection among Iranian population. This cross-sectional study was performed in 108 blood samples including 71 SVR positive and 37 negative samples from individuals suffering from chronic hepatitis C, and 50 healthy controls. DNA was extracted from the samples and the frequency of the polymorphism was analyzed using PCR-RFLP method. Finally, the products were detected on polyacrylamide gel electrophoresis. In the analysis of the data for C/T polymorphism, the CC genotype was identified in 29 patients of whom 28 (39.4%) achieved SVR, while the CT heterozygous was found in 75 patients and SVR was achieved in 42 (59.2%). Finally, the TT was detected in 14 patients and only one (1.4%) responded to treatment. Patients with C allele had significantly higher SVR rate than those with T allele. These data suggest that genotype detection of rs12979860 SNP may be useful as an important predictive biomarker for SVR in patients infected with HCV. However, further studies with more samples lead to more validated results.

Schizophrenia is a widespread neurodegenerative disorder, which affects approximately 1% of the world population. It is a multifactorial and a highly heritable disease to which genetic factors contribute up to approximately 80%. Nowadays, multitude of genes have been discovered that relate to this disorder mostly by affecting the performance and levels of neurotransmitters in neural systems. Since PAI-1 is a considerable gene in the performance of neural systems, the present study dealt with the relationship between -675 4G/5G polymorphism in PAI-1 gene and schizophrenia among Iranian patients. This case-control study was carried out on 106 blood samples collected from individuals suffering from schizophrenia and 122 healthy controls. DNA was extracted from the samples and the frequency of the polymorphisms was analyzed using ARMS-PCR method. Finally, the products were detected on 2% agarose gel electrophoresis. The analysis of the data for -675 4G/5G polymorphism showed that 17.9% of the patients and 1.6% of the controls were mutant homozygous and 65.1% of the patients and 45.9% of controls were heterozygous. Also, 17% of the patients and 52.5% of the controls were normal homozygous. There was a significant relationship between PAI-1 4G/5G polymorphism and the incidence of schizophrenia. To the best of our knowledge, this is first study in Iran that assesses the frequency of the polymorphism among Iranian patients. However, further studies with more samples are necessary.

Due to the lack of efficient anti-HIV vaccine، anti-HIV pharmaceuticals play an important role in controlling HIV infection. Also significant rise in drug resistance and drug toxicity has caused increased interest in finding new anti-HIV agents. In this study، a nano-sized version of lamivudine based on PEGylated chitosan was synthesized.

In this research، nanoparticles of chitosan were efficiently PEGylated for increasing their stability in water and then the anti-HIV drug، lamivudine، was loaded on these PEGylated nanoparticles. After purification and lyophilization of new synthesized nanoparticle، the raw materials and final product were sampled and FTIR، HNMR and CHN analyses were done.

Results of HNMR spectroscopy showed that chitosan nanoparticle was successfully PEGylated. HNMR data confirmed FTIR results and indicated that lamivudine was conjugated on chitosan nanoparticle. In addition، CHN analysis data also confirmed both HNMR and FTIR data، and demonstrated that a high yield of chitosan nanoparticle PEGylation (approximately 97%) was done and illustrated a high capacity of lamivudine conjugation on nano-sized PEGylated chitosan (30% W/W chitosan).

In this study، lamivudine drug was successfully synthesized، based on PEGylated chitosan nanoparticle.

Gastroenteritis is one of the most common symptoms in AIDS patients. Although gastroenteritis in these patients is caused by several factors, the role of viral agents, especially rotavirus, is still unknown. The aim of this study was to evaluate the prevalence of rotavirus infection among the HIV-positive cases. In this cross-sectional study, 75 fecal samples were collected from HIV-positive patients with gastroenteritis referred to Imam Khomeini hospital. After viral RNA extraction, the sixth conserved segment of the virus genome (VP6) was amplified using RT-PCR method. Finally, the products were detected on 1.5% agarose gel electrophoresis and confirmed by sequencing. RT-PCR products with the expected size (433bp) were obtained for all rotavirus- positive as well as the wild-type standard viral isolates. Among the samples taken from 75 HIV-positive cases, 19 (25.3%) were rotavirus-positive and confirmed using direct sequencing. Although in this study the anticipated prevalence of rotavirus among the HIV-positive cases is about 25%, further studies are required to characterize the genotype of rotavirus in HIV- positive cases with gastroenteritis.

Background and The need to find new antiviral compounds is increasing due to the emergence of resistant strains and toxicity of currently available HIV drugs. In this research the inhibitory activity of a number of novel iron chelating compounds derived from 3-Hydroxy-4-pyridinosnes was studied. Single cycle replicable (SCR) virion was produced by transfection of HEK293T cells with pmzNL4-3, pSPAX2 and pMD2G plasmids. HEK cells were infected with SCR virus and the production of new virions was evaluated by p24 ELISA. Also, cytotoxicity of these compounds was investigated by XTT method and then the effect of compounds on viral infection was examined by GFP-expressing virions. PhH, PhHB, DNB, TsB, AbphE and Adlb compounds had the ability of inhibition of virus production insofar as PhH, PhHB, DNB, TsB, AbphE and Adlb inhibited the viral production with IC50 of 63, 43, 10, 73, 12 and78µ M, respectively. DNB and AbphE showed high toxicity for target cells (CC50 of 113 and 112µM). Similar activity was found in single round infection assay compared to the results of replication study. This study found derivatives of 3-Hydroxy-4-pyridinosnes effective in preventing the HIV virus. Compounds with high anti-viral potential and low cytotoxicity are promising candidate for more anti-viral studies.

Molecular typing is an important tool in surveillance and outbreak investigations of human Salmonella infections. In this study, Subtyping of Salmonella Paratyphi B and C isolates derived from Iranian patients was carried out by RAPD-PCR to assess the extent of genetic diversity of these isolates. Fourteen Salmonella isolates including 6 strains of Salmonella paratyphi B and 8 strains of Salmonella paratyphi C were characterized using RAPD-PCR. Two arbitrary primers, namely OPP-16 and P1254 were used for RAPD analysis and the dendrograms were constructed with NTsys 2.0 computer software. Both primers showed high discriminatory power in differentiating of the related strains of Salmonella. The dendrograms constructed based on RAPD-PCR profiles (with both primers) involving 14 salmonella strains revealed 4 distinct patterns, indicating that these isolates are genetically heterogeneous. Furthermore, a good correlation was not observed between the serotype and the molecular profiles obtained from RAPD data of the Salmonella isolates. The findings of the present study verify the usefulness of RAPD-PCR in characterizing and comparing strains of Salmonella Paratyphi B and C.

Animal studies show that vaccination with epitope-based peptides results in protective immunity. However, immunodominance should be regarded as a major challenge in this area. Considering the advantages of epitopic-vaccines against hepatitis C virus (HCV) infection, herein, we compared the occurrence of immunodominance following mice immunization with three different HCV epitopic-peptide formulations. We synthesized four CD8+ epitopic-peptides (C1,E6,N,E4) that were derived from HCV-antigens. A polytope-peptide (C1E6NE4) spanning fusion of epitopes was designed based on immunoinformatics analyses for optimum proteasomal cleavage. BALB/c mice received three subcutaneous injections that contained 10 µg of peptide (minimal epitopes, or mixture of four epitopes or long-polytope) formulated with CpG (50 µg) and Montanide-ISA720 (70%) adjuvants in the tail-base at three-week intervals. Considering the H2-Dd (BALB/c)-restriction of C1 and E4-epitopes, three weeks after the last injection splenocytes from vaccinated animals were subjected to IFNγ/IL4 ELISpot assays in the presence of C1 and E4-peptides. All vaccinated animals promoted Th1-oriented responses as evidenced by detection of IFNγ-secreting cells and a low-level of IL4 secretion. Mice injected with minimal CTL-epitopes provoked stronger responses, however, due to the higher affinity of E4-epitope for H2-Dd, frequency of E4-specific cells was considerably higher than C1-specific ones, showing some level of immunodominance. Interestingly, animals vaccinated with polytope-peptide developed high-quality balanced responses against both C1and E4-epitopes, however at a lower intensity. These results supported the superiority of polytope-peptides over minimal epitopes, yet emphasized the key role of polytope design and optimization to avoid epitope dominancy.

Hepatitis C virus (HCV) genome contains a large open reading frame coding for a polyprotein that is cleaved into ten proteins. Recently, a new protein, named core+1, has been described to be expressed through a ribosomal frame shift within the capsid-encoding sequence. To address these possibilities, core+1 was produced in E.coli and the purified protein was evaluated for the immunological properties. The core+1corresponding nucleotide sequence was created by PCR-based induction of a +1 frame shift mutation within the core gene template. The amplicon was cloned into the pET-24a vector and expressed in E.coli host. The expressed protein was purified under denaturing condition and after refolding steps was characterized by SDS-PAGE and Western Blotting. The immunization potential of core+1 with various adjuvant (Freunds (C/IFA), Montanide ISA 206 and IMS 1312, pluronic acid (F127) and imiquimod (IMIQ) was assessed in Balb/c mice. ELISA-based assays were used to analyze the humoral immune responses. The yield of E.coli-derived core+1 was 5 mg/ L of culture media and antigenicity of this protein was confirmed by western blotting. All the core+1/adjuvant formulations significantly developed the anti- core+1 IgG responses in the immunized mice but C/IFA and ISA206 elicited the highest antibody titers. ISA 206 and IMS 1312 formulation of core+1 induced strong Th1/Th2 responses. Our results indicated that core+1 formulation with various adjuvants may elicit the different immune response profiles (Th1/Th2). Thus core+1 might be a potential component of future HCV vaccine too.

HIV virions with replication capacity are needed for HIV researches, like investigating for new anti HIV agents. Here HIV-1 replication assay was optimized with HIV-1 single cycle replicable (SCR) virions to improve biological safety condition. pSPAX2, pmzNL4-3 and pMD2G plasmids were co-transfected to the HEK293T by using polyfect reagent to produce the SCR HIV-1 virions. Virions were quantified using capture ELISA P24. Different MOI of SCR virions were used for infecting of Target cells (HEK) and the load of the supernatant P24 was monitored days after infection. Single cycle replication assay (SCRA) was developed using kinetic studies data. The P24 load of the infected cells supernatant has linear relation to the beginning infectious MOI. 24 hours post infection with HIV-1 SCR virions the viral particle production was detectable. The highest load of P24 in infected cells supernatant was detected 48 hours after infection. Using this developed method, the 50 and 95 percent inhibitory concentration of (IC95 and IC50) Indinavir and Nevirapine were calculated as 25nM and 50nM. In this study, using SCR HIV-1 virions the SCRA was developed. SCR HIV-1 virions are replicable only for one cycle and this improves the safety of developed assays. The accuracy of assay was examined by quantifying the anti HIV-1 potential of two commercial anti-AIDS drugs and the calculated activity for test agents was equal to previously known amounts.

Several studies have been conducted to explore anti-HIV drugs. Discovery and study of novel anti-HIV-1 compounds need live viruses and has serious biosafety concerns. In this research we reported a novel and safe system for assaying the cytopathic effects of HIV by using single cycle replicable (SCR) HIV-1 virions. To produce the SCR HIV-1 virions, pMD2G, pmzNL4-3 and pSPAX2 plasmids were co-transfected into HEK293T cells. Different amount of SCR virions were used to infect target cells (MT-2). Within the infected cells, the number of formed syncytia was counted under the light microscopy. The lethal effects of the SCR HIV virions were measured using XTT proliferation assay. Formation of syncytia among SCR HIV infected cells was detectable 24 hours after infection. Highest amount of syncytia was seen 72 hours after infection. Increase in the amount of virions caused increasing of syncytia and the cytopathic effects of SCR HIV-1. Infection with more than 1600ng P24 SCR HIV decreased the syncytium formation and viability of all cells. The calculated IC50 (50 percent inhibitory capacity) for nevirapine and BMS806 using this method was 50nM and 30nM, respectively. SCR HIV-1 virions are replicable only for one cycle. Using these virions can improve the safety of HIV researches. Herein, we optimized the assaying of HIV induced cytopathic effects by using SCR HIV-1 (NL4-3) virions. The accuracy of this method was accepted by quantifying the anti-HIV-1 effects of nevirapine and BMS806 by (SCR) HIV-1 virions.

Background and Finding an efficient vaccine against HÏV infection is still a major concern and a great challenge. Ïn our previous study, HÏV-1 virions with capability of a single replication were produced. Ïn the present study their immunological evaluation has been carried out to study their induction of cellular and humoral responses in mice model. Ïn this study, first and second virion generations were produced in human HËK293T cells, and were injected to three different mice groups in three doses with and without adjuvants. Âfter the last injection, immune responses were evaluated in serum and spleen cells of mice using ËLÏSÂ. Âlthough all of mice vaccinated by different immunogen regimens did develop cellular responses, in the main group injected with novel adjuvants the highest levels of specific antibody, ÏFN-γ, ÏL-4 an also most ÏFN-γ excreting cells were evident, which is suggestive of a strong cellular response in these cells.Çonclusion: Ân effective vaccine against HÏV requires induction of strong humoral and cellular responses. Hence, these results demonstrate the high immune system stimulating capabilities of virion injection regime accompanied by M70 and ÇpG adjuvants, which provides the potential for an efficient HÏV vaccine.