Mohammad Reza Aghasadeghi

Polymeric nanoparticles: potent vectors for vaccine delivery targeting cancer and infectious diseases.

Nanocarriers with various compositions and biological properties have been extensively applied for in vitro/in vivo drug and gene delivery. The family of nanocarriers includes polymeric nanoparticles, lipid-based carriers (liposomes/micelles), dendrimers, carbon nanotubes, and gold nanoparticles (nanoshells/nanocages). Among different delivery systems, polymeric carriers have several properties such as: easy to synthesize, inexpensive, biocompatible, biodegradable, non-immunogenic, non-toxic, and water soluble. In addition, cationic polymers seem to produce more stable complexes led to a more protection during cellular trafficking than cationic lipids. Nanoparticles often show significant adjuvant effects in vaccine delivery since they may be easily taken up by antigen presenting cells (APCs). Natural polymers such as polysaccharides and synthetic polymers have demonstrated great potential to form vaccine nanoparticles. The development of new adjuvants or delivery systems for DNA and protein immunization is an expanding research field. This review describes polymeric carriers especially PLGA, chitosan, and PEI as vaccine delivery systems.

Serum Bactericidal Antibody Response 1 Year after Meningococcal Polysaccharide Vaccination of Patients with Common Variable Immunodeficiency

ABSTRACT Some patients with common variable immunodeficiency (CVID) can generate an antibody response following vaccination with Neisseria meningitidis polysaccharide, but the duration of this protection is unknown. In this study, serum bactericidal antibody (SBA) responses to serogroup C N. meningitidis of 23 patients with CVID and 23 sex- and age-matched controls were measured 1 year after vaccination with the plain A/C meningococcal polysaccharide vaccine. The fold rise in serum bactericidal antibody geometric mean titers of the control group from prevaccination to 1 year postvaccination was significantly higher than that of the patient group (5.41- versus 2.96-fold, P = 0.009). Of 23 CVID patients, 8 had a poor response to vaccine (<4-fold rise) 3 weeks after vaccination, and low titers remained when measured 1 year later. Of the 15 CVID patients who had a normal response to vaccine (≥4-fold rise) 3 weeks after vaccination, 6 cases failed to maintain protective SBA titers, whereas the remaining 9 had protective titers 1 year after vaccination. Only one of the 23 controls, who developed protective SBA titers after 3 weeks, lost the protective titers after 1 year. Among the patients, the presence of bronchiectasis and/or splenomegaly at enrollment was associated with poor SBA response to vaccine at 3 weeks and/or failure to maintain protective levels at 1 year. The results of this study demonstrate that a number of CVID patients can produce protective antibody titers that can persist for 1 year after vaccination, which lends strong support to the inclusion of polysaccharide vaccine in the immunization program for CVID patients.

Gd(3+)-DTPA-DG: novel nanosized dual anticancer and molecular imaging agent.

Background: Difficulties in the use, preparation, and cost of radioactively-labeled glycosylated compounds led to this research and development study of a new gadolinium-labeled glucose compound that does not have a radioactive half-life or difficulties in its synthesis and utilization. Methods: Based on the structure of the 2-fluoro-2-deoxy-D-glucose molecule (18FDG), a new compound consisting of D-glucose (1.1 nm) conjugated to a well-known chelator, diethylenetriamine penta-acetic acid (DTPA), was synthesized, labeled with Gd3+, and examined in vitro and in vivo. Results: This novel compound not only demonstrated excellent and less costly imaging capability, but also showed anticancer effects on treated cells. Our results demonstrated that the new Gd3+-DTPA-DG compound (GDD, with GDD conjugate aggregation of about 8 nm at 0.02 mg/mL concentration) significantly decreased HT1080 and HT29 tumor cell numbers. Application of GDD to cancer cells also increased levels of tumor necrosis factor alpha, but did not alter blood glucose levels. Interestingly, no toxicological findings were seen in normal human kidney cells. Conclusion: Dual application of GDD for both imaging and treatment of tumor cells could be remarkably advantageous in both the diagnosis and treatment of cancer.

HIV-1 Vpr Protein Enhances Proteasomal Degradation of MCM10 DNA Replication Factor through the Cul4-DDB1[VprBP] E3 Ubiquitin Ligase to Induce G2/M Cell Cycle Arrest

Background: MCM10 regulates initiation of eukaryotic genome replication. Results: HIV-1 Vpr enhances proteasomal degradation of MCM10. Conclusion: HIV-1 Vpr-mediated degradation of MCM10 is essential for induction of G2/M cell cycle arrest. Significance: Our study reveals a novel protein target for HIV-1 Vpr whose degradation is involved in the induction of G2/M cell cycle arrest by the virus. Human immunodeficiency virus type 1 Vpr is an accessory protein that induces G2/M cell cycle arrest. It is well documented that interaction of Vpr with the Cul4-DDB1[VprBP] E3 ubiquitin ligase is essential for the induction of G2/M arrest. In this study, we show that HIV-1 Vpr indirectly binds MCM10, a eukaryotic DNA replication factor, in a Vpr-binding protein (VprBP) (VprBP)-dependent manner. Binding of Vpr to MCM10 enhanced ubiquitination and proteasomal degradation of MCM10. G2/M-defective mutants of Vpr were not able to deplete MCM10, and we show that Vpr-induced depletion of MCM10 is related to the ability of Vpr to induce G2/M arrest. Our study demonstrates that MCM10 is the natural substrate of the Cul4-DDB1[VprBP] E3 ubiquitin ligase whose degradation is regulated by VprBP, but Vpr enhances the proteasomal degradation of MCM10 by interacting with VprBP.

Prevalence of Transmitted HIV Drug Resistance in Iran between 2010 and 2011

Objective Drug-resistant (DR) HIV emerges during combined antiretroviral treatment (cART), creating concern about widespread transmission of DR-HIV as cART is expanded in resource-limited countries. The aim of this study was to determine the predominant HIV-1 subtypes and prevalence of transmitted DR mutations among antiretroviral-naïve patients in Iran. Design To monitor transmission of DR HIV, a threshold surveillance based on the world health organization (WHO) guidelines was implemented in Iran. Methods For this HIVDR threshold surveillance study, blood samples were collected from 50 antiretroviral-naïve HIV-1-infected patients. Antiretroviral-resistant mutations were determined by sequencing HIV-1 protease, reverse transcriptase and integrase regions. The HIV-1 subtype was determined by sequencing the p17 and C2-V5 regions of the gag and env genes, respectively. Results Phylogenetic analyses of the sequenced regions revealed that 45 (95.7%) of 47 samples that were successfully obtained were CRF35_AD. The remaining two cases were subtype B (2.1%) and CRF01_AE (2.1%). Consistent results were obtained also from Env and Gag sequences. Regarding prevalence of transmitted DR viruses, two cases were found to harbor reverse transcriptase-inhibitor-resistant mutations (4.3%). In addition, although not in the WHO list for surveillance of transmitted mutations, 13 minor protease-inhibitor-resistant mutations listed in the International AIDS Society-USA panel of drug resistance mutations were found. No DR mutations were detected in the integrase region. Conclusions Our study clarified that CRF35_AD is the major subtype among HIV-1-infected patients in Iran. According to the WHO categorization method of HIVDR threshold survey, the prevalence of transmitted drug resistant HIV in Iran was estimated as moderate (5–15%).

Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin

Background: Recombinant human erythropoietin (rhEPO) is considered to be one of the most pivotal pharmaceutical drugs in the market because of its clinical application in the treatment of anemia-associated disorders worldwide. However, like other therapeutic proteins, it does not have suitable pharmacokinetic properties for it to be administrated at least two to three times per week. Chemoselective cysteine PEGylation, employing molecular dynamics and graphics in in silico studies, can be considered to overcome such a problem. Methods: A special kind of EPO analog was elicited based on a literature review, homology modeling, molecular dynamic simulation, and factors affecting the PEGylation reaction. Then, cDNA of the selected analog was generated by site-directed mutagenesis and subsequently cloned into the expression vector. The construct was transfected to Chinese hamster ovary/dhfr− cells, and highly expressed clones were selected via methotrexate amplification. Ion-immobilized affinity and size exclusion (SE) chromatography techniques were used to purify the expressed analog. Thereafter, chemoselective PEGylation was performed and a nanosize PEGylated EPO was obtained through dialysis. The in vitro biologic assay and in vivo pharmacokinetic parameters were studied. Finally, E31C analog Fourier transform infrared, analytical SE-high-performance liquid chromatography, zeta potential, and size before and after PEGylation were characterized. Results: The findings indicate that a novel nanosize EPO31-PEG has a five-fold longer terminal half-life in rats with similar biologic activity compared with unmodified rhEPO in proliferation cell assay. The results also show that EPO31-PEG size and charge versus unmodified protein was increased in a nanospectrum, and this may be one criterion of EPO biologic potency enhancement. Discussion: This kind of novel engineered nanosize PEGylated EPO has remarkable advantages over rhEPO.

MPG-based nanoparticle: An efficient delivery system for enhancing the potency of DNA vaccine expressing HPV16E7

DNA vaccines against human papillomavirus (HPV) type 16 have not been successful in clinical trials, due to the lack of an appropriate delivery system. In this study, a peptide-based gene delivery system, MPG, which forms stable non-covalent nanoparticles with nucleic acids, was used for in vitro and in vivo delivery of HPV16 E7 DNA as a model antigen. The results demonstrated that at Nitrogen/Phosphate (N/P) ratio over 10:1, this peptide can effectively condense plasmid DNA into stable nanoparticles with an average size of 180-210nm and a positive surface charge. The transfection efficiency of MPG-based nanoparticles was shown to be comparable with Polyethyleneimine (PEI). The efficient protein expression detected by western blotting and flow cytometry supports the potential of MPG-based nanoparticles as a potent delivery system in DNA vaccine formulations. Immunization with MPG/E7DNA nanoparticles at an N/P ratio of 10:1 induced a stronger Th1 cellular immune response with a predominant interferon-γ (IFN-γ) profile than those induced by E7DNA alone in a murine tumor model. These findings suggest that MPG peptide as a novel gene delivery system could have promising applications in improving HPV therapeutic vaccines.

HIV-1 Vpr Protein Induces Proteasomal Degradation of Chromatin-associated Class I HDACs to Overcome Latent Infection of Macrophages.

Mechanisms underlying HIV-1 latency remain among the most crucial questions that need to be answered to adopt strategies for purging the latent viral reservoirs. Here we show that HIV-1 accessory protein Vpr induces depletion of class I HDACs, including HDAC1, 2, 3, and 8, to overcome latency in macrophages. We found that Vpr binds and depletes chromatin-associated class I HDACs through a VprBP-dependent mechanism, with HDAC3 as the most affected class I HDAC. De novo expression of Vpr in infected macrophages induced depletion of HDAC1 and 3 on the HIV-1 LTR that was associated with hyperacetylation of histones on the HIV-1 LTR. As a result of hyperacetylation of histones on HIV-1 promotor, the virus established an active promotor and this contributed to the acute infection of macrophages. Collectively, HIV-1 Vpr down-regulates class I HDACs on chromatin to counteract latent infections of macrophages.

APPLICATION OF OUTER MEMBRANE VESICLE OF NEISSERIA MENINGITIDIS SEROGROUP B AS A NEW ADJUVANT TO INDUCE STRONGLY TH1-ORIENTED RESPONSES AGAINST HIV-1

Despite the worldwide efforts made in the field of HIV vaccine development, an efficient AIDS vaccine strategy is still vague. Virus-like particles (VLPs) are one of the introduced aspects for HIV vaccine development since the non-replicative nature of HIV VLPs, resulting from the lack of viral genomic RNA, makes them suitable for broad applications. We have previously designed and introduced non-infectious VLPs (mzNL4-3) by introduction of a deletion mutation in the reverse transcriptase and integrase coding regions of HV-1. There are evidences suggesting that an effective cellular immune response against HIV-1 is able to control and suppress viremia during primary and chronic HIV infections. In the present study we have evaluated the potency of mzNL4-3 VLPs mixed with Neisseria meningitidis serogroup B outer-membrane vesicle (OMV), which is among the microbial components with proved adjuvant properties, to induce humoral and cellular responses against HIV-1. Analysis of anti-HIV-1 responses elicited in immunized BALB/c mice following different immunization regimens indicated OMV+VLP as an immunopotent combination which significantly induced anti-HIV-1 IgG with IgG2a dominancy. Results of cytokine and ELISpot assays also showed the capability of VLP+OMV immunogen for effective induction of IFN-gamma; and IL4 secreting cells and further suggested the promotion of Th1-oriented response that was evidenced with the increased IFN-γ/IL4 secretion ratio. According to our study, HIV-1 VLPs combined with N. meningitidis B OMVs seem to be a promising approach in vaccine development against HIV-1.

HIV-1 Vpr reactivates latent HIV-1 provirus by inducing depletion of class I HDACs on chromatin

HIV-1 Vpr is an accessory protein that induces proteasomal degradation of multiple proteins. We recently showed that Vpr targets class I HDACs on chromatin for proteasomal degradation. Here we show that Vpr induces degradation of HDAC1 and HDAC3 in HIV-1 latently infected J-Lat cells. Degradation of HDAC1 and HDAC3 was also observed on the HIV-1 LTR and as a result, markers of active transcription were recruited to the viral promoter and induced viral activation. Knockdown of HDAC1 and HDAC3 activated the latent HIV-1 provirus and complementation with HDAC3 inhibited Vpr-induced HIV-1 reactivation. Viral reactivation and degradation of HDAC1 and HDAC3 was conserved among Vpr proteins of HV-1 group M. Serum Vpr isolated from patients or the release of virion-incorporated Vpr from viral lysates also activated HIV-1 in latently infected cell lines and PBMCs from HIV-1 infected patients. Our results indicate that Vpr counteracts HIV-1 latency by inducing proteasomal degradation of HDAC1 and 3 leading to reactivation of the viral promoter.