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High morbidity and mortality caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made coronavirus disease 2019 (COVID-19) the leading challenge for health experts all over the world. Currently, there is no specific treatment for COVID-19; however, thanks to worldwide intense attempts, novel vaccines such as mRNA-1273 (Moderna TX, Inc.) and BNT162b2 (Biontech/Pfizer) were developed very fast and FDA approved them for emergency use. Nanomedicine-based drug delivery can be an advanced therapeutic strategy to deal with clinical complications of COVID-19. Given the fact that SARS-CoV-2 typically affects the respiratory tract, application of inhalable nanoparticles (NPs) for targeted drug delivery to the alveolar space appears to be an effective and promising therapeutic strategy. Loading the medicinal components into NPs enhances the stability, bioavailability, solubility and sustained release of them. This approach can circumvent major challenges in efficient drug delivery such as solubility and any adverse impact of medicinal components due to off-targeted delivery and resulting systemic complications. Inhalable NPs could be delivered through nasal sprays, inhalers, and nebulizers. NPs also could interfere in virus attachment to host cells and prevent infection. Moreover, nanomedicine-based technologies can facilitate accurate and rapid detection of virus compared to the conventional methods. In this review, the nano-based theranostics modalities for the management of respiratory complications of COVID-19 were discussed.

Inflammatory bowel diseases (IBDs) are chronic and relapsing disorders that affect the quality of life in many individuals around the world. Over the past few years, the prevalence of IBDs is substantially rising which might pose a considerable social and economic burden on health systems. Progresses in the management of chronic inflammatory diseases lead to prolonged remission phase and decreased hospitalization rate. However, during treatment, many patients become refractory to conventional therapies. Recently, advanced approaches using somatic cell therapy medicinal products (SCTMPs) including immune and stem cell-based therapies have drawn many researchers' attentions. Promising results from recent trials, alongside with the emerging market indicated that these therapeutic approaches could be an alternative and promising treatment to conventional therapies. In this review, we will discuss recent advances in cell-based therapies, which have been developed for treatment of IBDs. In addition, the global emerging market and the novel products in this field are highlighted.

Highlights • Direct cytopathic effect and immunopathological pathogenesis are as two main underlying mechanisms for severe pulmonary injury in COVID-19 infections. • Repurposed antiviral agents are among the most promising therapeutics for overcoming the COVID-19 pandemic. • Immunomodulatory effects of mesenchymal stem/stromal cells (MSCs) hold the potential for preventing the immune mediated consequences of SARS-CoV-2. • Nucleic acid- based approaches have gained much attention for treating COVID-19 infections., The recent coronavirus disease 2019 (COVID-19) outbreak around the world has an enormous impact on the global health burden, threatening the lives of many individuals, and has severe socio-economic consequences. Many pharmaceutical and biotechnology companies have begun intensive research on different therapeutic strategies, from repurposed antiviral drugs to vaccines and monoclonal antibodies to prevent the spread of the disease and treat infected patients. Among the various strategies, advanced therapeutic approaches including cell- and gene editing-based therapeutics are also being investigated and initial results in in vitro and early phase I studies were promising, however, still further assessments are required. In this paper, we first review the underlying mechanisms for severe acute respiratory syndrome coronavirus (SARS-CoV-2) pathogenesis and further discuss available therapeutic candidates and advanced modalities that are being currently evaluated in in vitro/in vivo models and of note in clinical trials.

Regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) are the two important and interactive immunosuppressive components of the tumor microenvironment that hamper anti-tumor immune responses. Therefore, targeting these two populations together might be beneficial for overcoming immune suppression in the tumor microenvironment. We have recently shown that prophylactic Foxp3 DNA/recombinant protein vaccine (Foxp3 vaccine) promotes immunity against Treg in tumor-free conditions. In the present study, we investigated the immune modulatory effects of a prophylactic regimen of the redesigned Foxp3 vaccine in the B16F10 melanoma model. Our results indicate that Foxp3 vaccination continuously reduces Treg population in both the tumor site and the spleen. Surprisingly, Treg reduction was associated with a significant decrease in the frequency of MDSC, both in the spleen and in the tumor environment. Furthermore, Foxp3 vaccination resulted in a significant reduction of arginase-1(Arg-1)-induced nitric oxide synthase (iNOS), reactive oxygen species (ROS) and suppressed MDSC activity. Moreover, this concurrent depletion restored production of inflammatory cytokine IFN-γ and enhanced tumor-specific CTL response, which subsequently resulted in the reduction of tumor growth and the improved survival rate of vaccinated mice. In conclusion, our results revealed that Foxp3 vaccine promotes an immune response against tumor by targeting both Treg and MDSC, which could be exploited as a potential immunotherapy approach.

The interactions between programmed death-1 (PD-1) and its ligands hamper tumor-specific CD8+ T cell (TCD8) responses, and PD-1-based “checkpoint inhibitors” have shown promise in certain cancers, thus revitalizing interest in immunotherapy. PD-1–targeted therapies reverse TCD8 exhaustion/anergy. However, whether they alter the epitope breadth of TCD8 responses remains unclear. This is an important question because subdominant TCD8 are more likely than immunodominant clones to escape tolerance mechanisms and may contribute to protective anticancer immunity. We have addressed this question in an in vivo model of TCD8 responses to well-defined epitopes of a clinically relevant oncoprotein, large T Ag. We found that unlike other coinhibitory molecules (CTLA-4, LAG-3, TIM-3), PD-1 was highly expressed by subdominant TCD8, which correlated with their propensity to favorably respond to PD-1/PD-1 ligand-1 (PD-L1)-blocking Abs. PD-1 blockade increased the size of subdominant TCD8 clones at the peak of their primary response, and it also sustained their presence, thus giving rise to an enlarged memory pool. The expanded population was fully functional as judged by IFN-γ production and MHC class I–restricted cytotoxicity. The selective increase in subdominant TCD8 clonal size was due to their enhanced survival, not proliferation. Further mechanistic studies utilizing peptide-pulsed dendritic cells, recombinant vaccinia viruses encoding full-length T Ag or epitope mingenes, and tumor cells expressing T Ag variants revealed that anti–PD-1 invigorates subdominant TCD8 responses by relieving their lysis-dependent suppression by immunodominant TCD8. To our knowledge, our work constitutes the first report that interfering with PD-1 signaling potentiates epitope spreading in tumor-specific responses, a finding with clear implications for cancer immunotherapy and vaccination.

HIV-1 transmitted drug resistance (TDR) occurs when primary viruses bear drug resistance mutations (DRMs). TDR causes first-line antiretroviral (ARV) therapy (ART) failure and is becoming more pronounced due to the widespread use of ART. With the absence of routine individual-level drug resistance testing, the World Health Organization (WHO) recommends the tracking of TDR mutations and optimizing the first-line ART following pre-treatment drug resistance (PDR) surveys. Here, we report the PDR frequency for the first time in Hormozgan, a southern province of Iran. In this study, 41 blood samples from HIV-1-positive ART-candidate volunteers were collected across the province between April 2016 and March 2017. Phylogenetic analysis of the sequenced protease (PR) and reverse transcriptase (RT) regions showed that 39 out of 41 samples (95%) were CRF35_AD and the two remaining cases were subtype B (2.5%) and C (2.5%). D67G (2.4%), a mutation that reduces susceptibility to nucleoside reverse transcriptase inhibitors (NRTIs) was the only detectable TDR mutation in this population. Two other DRMs, including E138A (9.7%) and V179T (4.9%), which confer resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs), were also identified. Although no major protease inhibitor (PI) resistance mutations were detected, the minor mutations L10F and L33F (2.5% each) as well as several highly frequent polymorphic mutations were identified. Our results show a PDR frequency of 17% in infected individuals from Hormozgan, classified further as 2.4% NRTIs and 14.6% NNRTIs. These results suggest that first-line ART should be practiced carefully in Hormozgan province, and alternative regimens may become necessary for all starters.

Infection with (SAg)-producing bacteria may precede or follow infection with or vaccination against influenza A viruses (IAVs). However, how SAgs alter the breadth of IAV-specific CD8+ T cell (TCD8) responses is unknown. Moreover, whether recall responses mediating heterosubtypic immunity to IAVs are manipulated by SAgs remains unexplored. We employed wild-type (WT) and mutant bacterial SAgs, SAg-sufficient/deficient Staphylococcus aureus strains, and WT, mouse-adapted and reassortant IAV strains in multiple in vivo settings to address the above questions. Contrary to the popular view that SAgs delete or anergize T cells, systemic administration of staphylococcal enterotoxin B (SEB) or Mycoplasma arthritidis mitogen before intraperitoneal IAV immunization enlarged the clonal size of ‘select’ IAV-specific TCD8 and reshuffled the hierarchical pattern of primary TCD8 responses. This was mechanistically linked to the TCR Vβ makeup of the impacted clones rather than their immunodominance status. Importantly, SAg-expanded TCD8 retained their IFN-γ production and cognate cytolytic capacities. The enhancing effect of SEB on immunodominant TCD8 was also evident in primary responses to vaccination with heat-inactivated and live attenuated IAV strains administered intramuscularly and intranasally, respectively. Interestingly, in prime-boost immunization settings, the outcome of SEB administration depended strictly upon the time point at which this SAg was introduced. Accordingly, SEB injection before priming raised CD127highKLRG1low memory precursor frequencies and augmented the anamnestic responses of SEB-binding TCD8. By comparison, introducing SEB before boosting diminished recall responses to IAV-derived epitopes drastically and indiscriminately. This was accompanied by lower Ki67 and higher Fas, LAG-3 and PD-1 levels consistent with a pro-apoptotic and/or exhausted phenotype. Therefore, SAgs can have contrasting impacts on anti-IAV immunity depending on the naïve/memory status and the TCR composition of exposed TCD8. Finally, local administration of SEB or infection with SEB-producing S. aureus enhanced pulmonary TCD8 responses to IAV. Our findings have clear implications for superinfections and prophylactic vaccination., Author summary Exposure to bacterial superantigens (SAgs) is often a consequence of infection with common Gram-positive bacteria causing septic and toxic shock or food poisoning. How SAgs affect the magnitude, breadth and quality of infection/vaccine-elicited CD8+ T cell (TCD8) responses to respiratory viral pathogens, including influenza A viruses (IAVs), is far from clear. Also importantly, superinfections with IAVs and SAg-producing bacteria are serious clinical occurrences during seasonal and pandemic flu and require urgent attention. We demonstrate that two structurally distinct SAgs, including staphylococcal enterotoxin B (SEB), unexpectedly enhance primary TCD8 responses to ‘select’ IAV-derived epitopes depending on the TCR makeup of the responding clones. Intriguingly, the timing of exposure to SEB dictates the outcome of prime-boost immunization. Seeing a SAg before priming raises memory precursor frequencies and augments anamnestic TCD8 responses. Conversely, a SAg encounter before boosting renders TCD8 prone to death or exhaustion and impedes recall responses, thus likely compromising heterosubtypic immunity to IAVs. Finally, local exposure to SEB increases the pulmonary response of immunodominant IAV-specific TCD8. These findings shed new light on how bacterial infections and SAgs influence the effectiveness of anti-IAV TCD8 responses, and have, as such, wide-ranging implications for preventative vaccination and infection control.

Background: The therapeutic effect of antiretroviral therapy (ART) is adversely influenced by antiretroviral drug resistance, mainly due to mutations (DRMs) in the human immunodeficiency virus (HIV) genome. These mutations are commonly associated with HIV protease and reverse-transcriptase genes. We sought to determine the frequency of DRMs in a population of ART-experienced patients in the South of Iran. Method: A total of 44 HIV-1-positive participants under ART were selected from April 2016 to March 2017. Their DRMs, antiretroviral resistance status, and viral subtypes were determined. Results: At least one DRM was detected in 61.4% of the participants. The highest frequency was related to nucleotide reverse-transcriptase inhibitor (NRTI) mutations (45.45%). In contrast, major protease inhibitor (PI) mutations had the lowest frequency (6.81%). M184V (40.9%) and K103N (25%), respectively related to NRTI and nonnucleoside reverse-transcriptase inhibitor (NNRTI), were the mutations with the highest frequencies. Susceptibility to PI drugs was higher compared to NRTIs and NNRTIs, which was consistent with the results of genotypic DRMs. Conclusion: The highest frequency of antiretroviral DRMs was related to NRTIs and NNRTIs. In contrast, PI resistance mutations had the lowest frequency. Laboratory-guided ART to avoid the expansion of mutants as well as investigating DRMs in other viral regions, such as integrase, are recommended.

Background: Influenza is a highly infectious and acute respiratory disease caused by an infection of the host respiratory tract mucosa by the influenza virus. The use of DNA vaccines that express conserved genes such as nucleoprotein (NP) represents a new method of vaccination against influenza. In this study, the effect of chronic stress on the efficiency of this type of vaccine has been evaluated in a mouse model. Methods: The NP DNA vaccine was administered intradermally 3 times on days 0, 3 and 6 to stressed and nonstressed male BALB/c mice. Two weeks after the last immunization, half of these mice were challenged with A/Puerto Rico/8/34 (PR8) influenza virus and were weighed for 12 days, and their mortality rate was assessed during this period. The cellular immune response of the other half of the mice was evaluated by cytotoxicity assay. Results: The results indicate a significant reduction in the cytotoxic T-lymphocyte response of stressed mice in comparison with unstressed mice. Also, the percentage of weight loss and mortality after the challenge in stressed mice was significantly increased compared to the other group. Conclusion: These results indicate that the NP DNA vaccine is not able to induce any effective cytotoxic T-lymphocyte response against influenza virus in stressed mice and cannot induce protective immunity against influenza infection in this group of mice.

Superantigens (SAgs) are potent exotoxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They target a large fraction of T cell pools to set in motion a “cytokine storm” with severe and sometimes life-threatening consequences typically encountered in toxic shock syndrome (TSS). Given the rapidity with which TSS develops, designing timely and truly targeted therapies for this syndrome requires identification of key mediators of the cytokine storm’s initial wave. Equally important, early host responses to SAgs can be accompanied or followed by a state of immunosuppression, which in turn jeopardizes the host’s ability to combat and clear infections. Unlike in mouse models, the mechanisms underlying SAg-associated immunosuppression in humans are ill-defined. In this work, we have identified a population of innate-like T cells, called mucosa-associated invariant T (MAIT) cells, as the most powerful source of pro-inflammatory cytokines after exposure to SAgs. We have utilized primary human peripheral blood and hepatic mononuclear cells, mouse MAIT hybridoma lines, HLA-DR4-transgenic mice, MAIThighHLA-DR4+ bone marrow chimeras, and humanized NOD-scid IL-2Rγnull mice to demonstrate for the first time that: i) mouse and human MAIT cells are hyperresponsive to SAgs, typified by staphylococcal enterotoxin B (SEB); ii) the human MAIT cell response to SEB is rapid and far greater in magnitude than that launched by unfractionated conventional T, invariant natural killer T (iNKT) or γδ T cells, and is characterized by production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2, but not IL-17A; iii) high-affinity MHC class II interaction with SAgs, but not MHC-related protein 1 (MR1) participation, is required for MAIT cell activation; iv) MAIT cell responses to SEB can occur in a T cell receptor (TCR) Vβ–specific manner but are largely contributed by IL-12 and IL-18; v) as MAIT cells are primed by SAgs, they also begin to develop a molecular signature consistent with exhaustion and failure to participate in antimicrobial defense. Accordingly, they upregulate lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-3 (TIM-3), and/or programmed cell death-1 (PD-1), and acquire an anergic phenotype that interferes with their cognate function against Klebsiella pneumoniae and Escherichia coli; vi) MAIT cell hyperactivation and anergy co-utilize a signaling pathway that is governed by p38 and MEK1/2. Collectively, our findings demonstrate a pathogenic, rather than protective, role for MAIT cells during infection. Furthermore, we propose a novel mechanism of SAg-associated immunosuppression in humans. MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses., Author summary Superantigens (SAgs) are toxins produced by Staphylococcus aureus and Streptococcus pyogenes, microbes that are responsible for a multitude of infectious diseases and conditions. Once released, SAgs activate many immune cells, resulting in a massive inflammatory response that is often followed by a state of immunosuppression, a state that favors opportunistic infections. Using primary human cells as well as wild-type and genetically altered mice, we have now identified a subset of unconventional, innate-like T lymphocytes, called mucosa-associated invariant T (MAIT) cells, as one of the most powerful and quick-acting sources of inflammatory mediators in the aftermath of systemic exposure to SAgs. We also demonstrate that robust activation of MAIT cells by SAgs quickly leads to their exhaustion, and this exhaustion interferes with their ability to participate in antimicrobial host defense and contributes to the immunosuppressive state. Our findings thus define a pathogenic role for MAIT cells during Gram-positive bacterial infections and also uncover a novel mechanism of SAg-mediated immunosuppression. Accordingly, we propose that MAIT cells can be targeted for efficacious treatment of SAg-mediated illnesses.

Introduction: Designing an effective vaccine against human immunodeficiency virus (HIV)-1 is a global health priority. Multiepitope vaccines offer several potential advantages that may be promising in case of mutable divergent pathogens such as HIV-1. Herein, a multiepitopic recombinant protein containing various HIV-1 antigens was expressed in E. coli cells and its immunogenicity in combination with different adjuvants was initially evaluated in BALB/c mouse. Methods: HIVtop4 sequence spanning the junction of six amino acid fragments (Gag 158-186 , Pol 150-190 , ENV 296-323 , ENV 577-610 , Tat 1-20 and Tat 44-61 ) was designed based on immunoinformatic analysis to reduce the creation of junctional epitopes, improve the cleavage of proteasome and avoid the local accumulation of hydrophobic regions. Synthesized nucleotide sequence corresponding to HIVtop4 was cloned into pET23a plasmid. Expression of pET-HIVtop4 plasmid was induced in BL21 (DE3) E. coli cells by addition of 1 mM IPTG during 3 h culture and the protein was purified by Ni-NTA column chromatography and further confirmed against anti-His antibody in western-blotting. Groups of BALB/c mice (n=6) were immunized three times with 2 weeks interval, subcutaneously with 10 μg of candidate vaccine adjuvanted in Complete Freund’s adjuvant, Montanide ISA70 and Alum with suitable control groups. Two weeks after last immunization lymphocyte proliferation was measured with Brdu, IL-4 and IFN-γ cytokines with ELISA, total antibody and IgG1, IgG2a isotypes with indirect ELISA methods. Results: Results showed that Immunization with HIV-1 tat/ pol/gag/env led to a significant increase in the proliferative responses of lymphocytes, IL-4 and IFN- γ cytokine production and humoral immune response in comparison with the control groups. Conclusion: In this study we concluded that Tat, Env, Pol, Gag with adjuvants (Montanide, Alum and CFA) has potentials as a candidate vaccine against the HIV-1 virus. Vac Res, 2014, 1 (1): 10-15

Chitin and its some derivatives are known to be non-allergic and non-toxic substances. It has been shown that chitin microparticles have immunomodulatory activities. In the present study, we investigated the in vivo immunomodulatory activities of chitin microparticles (CMPs) on Leishmania major -infected BALB/c mice. BALB/c mice were infected with L. major promastigotes at their base of the tail. CMPs (100 μg/100 μl) were injected into the site of infection from 3 days before to 2 or 8 weeks after infection at two-day intervals. Cytokine concentrations (TNF-α, IFN-γ, IL-5 and IL-10) were measured using ELISA assays. Compared to the untreated group, production of TNF-α was significantly elevated in the CMPs-treated group. Moreover, the IFN-γ/IL-5 ratio was significantly elevated in CMPs-treated infected mice ( P = 0.023). Notably, the concentration of IL-10 was higher in CMPs-treated mice. These results showed that CMPs have in vivo immunomodulatory effects via the production of IFN-γ and IL-10. We also measured the onset and size of lesions in both treated and untreated mice. The average times taken for the onset of the lesion formation were 35 and 29 days for CMPs-treated and untreated mice ( P = 0.023), respectively. The mean size of the lesions was smaller in CMPs-treated group. Our study serves as a basis for future investigations on the application of CMPs as a prophylactic (vaccine adjuvant) and/or therapeutic modality against leishmaniasis.

The emergence and transmission of drug resistant HIV mutants is a major concern, especially in resource-limited countries with expanding antiretroviral therapy. Studies have recently reported the prevalence of HIV-1 transmitted drug resistance (TDR) mutations in certain Iranian cities; however, no information is currently available about the level of TDR, as well as the nature of the circulating HIV-1 subtypes, in the Southwestern bordering province of Iran, Khuzestan. Herein, we used a WHO-recommended TDR survey method to classify the prevalence of TDR in indigenous people of Khuzestan province. For this purpose, between March 2014 and February 2015, blood samples were collected from 52 newly diagnosed, antiretroviral treatment-naive, HIV-1 infected persons aged from 18 to 30 years. TDR mutations were determined by sequencing the protease (PR) and reverse transcriptase (RT) genes and interpreted using the WHO drug resistance mutations surveillance list. HIV-1 subtypes were characterized by sequencing the PR-RT, C2-V5, and p17 regions of the pol, env and gag genes, respectively. Two participants had non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations, specifically K103N in one individual and K101EK/K103KN/G190AG in the other. No nucleoside reverse transcriptase inhibitor (NRTI) or major protease inhibitor (PI) mutations were identified. HIV-1 subtyping revealed that all participants were infected with HIV-1 CRF35_AD. According to the WHO sequential sampling method, the prevalence of HIV-1 TDR in the sampling area (Khuzestan province) was classified as moderate for NNRTIs and low for NRTIs and PIs. This is the first HIV-1 drug resistance threshold survey in the Khuzestan province of Iran and shows a predominance of NNRTI TDR mutations in this area.

Streptokinase (SK), the heterogeneous protein family secreted by some groups of β-hemolytic streptococci (βHS), is a plasminogen activator and well-known drug for thrombolytic therapy. Differences in plasminogen activation property of streptococcal culture supernatants (SCS) have been traditionally used to identify superior producer strains and SK genes (skc) for recombinant SK (rSK) production. However, the role of SK heterogeneity and whether SK activities in SCS correlate with that of their corresponding rSK is a matter of debate. To address these concerns, SCS of nine group C streptococci (GCS) screened among 252 βHS clinical isolates were compared for plasminogen activation using S-2251 chromogenic assay. The GCS (Streptococcus equisimilis) showing the highest (GCS-S87) and lowest (GCS-S131) activities were selected for PCR-based isolation of skc, cloning and rSK production in Escherichia coli. The 6×His-tagged rSK proteins were purified by NI–NTA chromatography, analyzed by SDS-PAGE and Western blotting and their activities were determined. While SCS of GCS-S87 and GCS-S131 showed different plasminogen activations (95 and 35 %, respectively) compared to that of the reference strain (GCS-9542), but interestingly rSK of all three strains showed close specific activities (1.33, 1.70, and 1.55 × 104 IU mg−1). Accordingly, SKS87 and SKS131 had more than 90 % sequence identity at the amino acids level compared to SK9542. Therefore, SK heterogeneity by itself may not contribute to the differences in plasminogen activation properties of SCS and evaluation of this activity in SCS might not be a proper assay for screening superior skc.

In recent years, the patterns of human immunodeficiency virus 1 (HIV-1) transmission in Iran have been changing gradually from drug injection to unprotected sexual contact. This study sought to investigate the phylogenetic trends and characteristics of transmitted drug resistance (TDR) mutations of HIV-1 in a population that is mainly infected through homo/heterosexual contacts. Sixty newly diagnosed antiretroviral-naive individuals with HIV infection living in Tehran were recruited to this survey, and among them, 42 subjects were established to be infected through sexual intercourse. Following amplification and sequencing of the main part of the HIV-1 pol region, phylogenetic and drug-resistance mutation (DRM) analysis was successfully performed on these 42 patients. Phylogenetic analysis showed that the majority of the subjects were infected with subtype CRF35_AD (88%), followed by subtype B, with 7.1%, and subtype CRF01_AE, with 4.7%. A total of 7.1% of the subjects were found to be infected with HIV-1 variants with surveillance drug-resistant mutations (SDRMs) according to the last world health organisation (WHO) algorithm. All of the identified SDRMs belonged to the non-nucleoside reverse transcriptase inhibitors (NNRTIs) class, including K103 N and V106A, which were found in three patients. Two minor HIV protease-inhibitor-related mutations (L10I and G73S) were detected in two patients, but these mutations are not included in the WHO SDRMs list. The dominance of HIV-1 subtype CRF35_AD was observed among subjects of this study who were infected through sexual contact. The moderate prevalence of SDRMs (7.1%) in this population emphasises the fact that the risk of treatment failure in HIV-infected individuals might increase in the future, and preventive measures should be considered by health authorities.

INTRODUCTION The critical role of regulatory T (Treg) cells in dampening immune responses against tumor cells is apparent. Therefore, several methods have been introduced for eliminating Treg. Among them, inducing immune responses against Treg cells expressing Foxp3 transcription factor is a hopeful approach to decrease the frequency of Tregs. In current study, we used the chimeric FoxP3-Fc(IgG) fusion construct/protein to effectively stimulate the immune responses against Treg cells. MATERIALS AND METHODS Previously constructed FoxP3-Fc(IgG) DNA vaccine and its protein counterpart were injected into C57BL/6 mice in a prime/boost regimen. After 2 weeks, the mice were killed to measure the frequency of Tregs in their spleens, as well as analyze their specific cytokine production, T-cell proliferation, and CD8 T-cell cytotoxicity against FoxP3 protein. RESULTS FACS analysis of FoxP3 CD4 cells in splenocytes revealed the efficiency of FoxP3 DNA-prime protein-boost strategy to decrease the Treg cells and further showed considerable superiority of Fc(IgG) fusion strategy. This significant reduction in Treg frequency was also concomitant with higher FoxP3-specific CTL and Th1 responses in FoxP3-Fc vaccinated animals. CONCLUSIONS Prime/boost vaccination against FoxP3 in addition to enhanced antigen presentation by means of Fc fusion strategy could be successfully considered for Treg depletion studies. Validity of this approach should be experimentally tested in preclinical tumor models.

Genome of the hepatitis C virus (HCV) contains a long open reading frame encoding a polyprotein that is cleaved into 10 proteins. Recently, a novel, so called "ARFP/F", or "core+1", protein, which is expressed through a ribosomal frame shift within the capsid-coding sequence, has been described. Herein, to produce and characterize a recombinant form of this protein, the DNA sequence corresponding to the ARFP/F protein (amino acid 11-161) was amplified using a frame-shifted forward primer exploiting the capsid sequence of the 1b-subtype as a template. The amplicon was cloned into the pET-24a vector and expressed in different Escherichia coli strains. The expressed protein (mostly as insoluble inclusion bodies) was purified under denaturing conditions on a nickel-nitrilotriacetic acid (Ni-NTA) affinity column in a single step with a yield of 5 mg/L of culture media. After refolding steps, characterization of expressed ARFP/F was performed by SDS-PAGE and Western blot assay using specific antibodies. Antigenic properties of the protein were verified by ELISA using HCV-infected human sera and by its ability for a strong and specific interaction with sera of mice immunized with the peptide encoding a dominant ARFP/F B-cell epitope. The antigenicity plot revealed 3 major antigenic domains in the first half of the ARFP/F sequence. Immunization of BALB/c mice with the ARFP/F protein elicited high titers of IgG indicating the relevance of produced protein for induction of a humoral response. In conclusion, possibility of ARFP/F expression with a high yield and immunogenic potency of this protein in a mouse model have been demonstrated.

Summary Objective The main purpose of this study sought to evaluate the frequency of sensitivity of Iranian asthmatic patients to three regional Aspergillus species of fumigatus , flavus and niger , by detection of antigen-specific IgE in the patients’ sera. Patients and methods Crude extracts were prepared following the disruption of fungi cell walls by the application of glass beads and their protein fractions were isolated by SDS-PAGE. After electrotransfer of protein bands into the nitrocellulose membrane, IgE-immunoblotting was performed against the sera from 32 asthmatic patients in addition to 20 healthy controls. Results Our results interestingly showed that all of the studied Iranian asthmatic patients were sensitive to A. fumigatus and A. flavus antigens. This frequency was 65.6% in the case of A. niger , however, all control samples were negative. Age/sex analysis generally indicated higher sensitivities of young patients ( Aspergillus species with a statistical significance in the case of A. niger ( P = 0.02) and additionally more sensitivity of females. Using Immunoblotting assay, 23 IgE-reactive allergenic components from A. fumigatus , 15 from A. flavus and 13 from A. niger in a broad molecular weight spectrum were identified, among which several fragments were not previously reported. Conclusion Overall, this study found a high frequency of sensitivity of Iranian asthmatic patients to regional isolates of A. fumigatus , A. flavus and A. niger , which suggested the importance of these species in development of asthma. Moreover, we reported allergenic profiles of Iranian isolates in different patterns not previously observed.

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.

Background Chitin microparticles (CMPs) are found to be potent macrophage stimulators; however, their immunomodulatory effects on the parasite-infected macrophages have not yet been studied. To address this issue, we used a Leishmania major -infected murine macrophage model and characterized the regulatory effects of CMPs on the parasite-infected cells. Methods Mouse peritoneal macrophages were prepared and infected with L. major (MRHO/IR/1975/ER) standard strain. Following cell treatment with CMPs (500 μg/mL) for 48 h, percent of infected macrophages was determined by Giemsa staining and compared with untreated cells. To find the potential mechanisms of the activity of CMPs, TNF-α and accumulated nitrite in the culture supernatants of the treated and untreated cells were also measured by ELISA and colorimetric Griess assays, respectively. Results According to the obtained results, chitin microparticles reduced the ex vivo parasite infectivity by ∼12%. However, this inhibitory effect was not directly related to the increased biosynthesis and release of nitric oxide (NO) by macrophages. Instead, we observed a significant increase in the level of TNF-α secretion due to cell treatment with CMPs. Interestingly, this overexpression of TNF-α did not impair cell viability, suggesting the anti-apoptotic effects of the CMPs. Conclusions These findings show that chitin microparticles have immunomodulatory effects on L. major- infected macrophages and further provide motivations for future studies on their in vivo effects.

Human immunodeficiency virus infection is a worldwide health problem and a protective vaccine is desperately needed to control the AIDS pandemics. To address this concern, we previously constructed single-cycle replicable (SCR) HIV-1 virions, which completely maintained the antigenic structures of HIV-1. Herein, to optimize a vaccination strategy, we studied the immunogenicity of produced SCR virions and adjuvant-formulated HIV-1 virus-like particles (VLPs) in homologous and heterologous prime-boosting regimens. Accordingly, BALB/c mice received three doses of immunogens in 3-week intervals and their immune responses were evaluated using ELISA, cytokine and IFN-γ ELISpot assays. These analyses not only indicated the superiority of SCR prime-VLP boosting for strong induction of specific IFN-γ producing cells, but also showed the capability of this strategy over the others for better stimulation of humoral response, which was evidenced with the detection of highest titer of total IgG against HIV ENV glycoprotein. Furthermore, determination of IgG subclasses and IFN-γ/IL4 secretion ratio in cultured splenocytes demonstrated the efficient augmentation of mixed responses with the dominancy of Th1 immunity following SCR/VLP immunization strategy. Our results additionally pointed towards the applicability of Montanide ISA 720 + CpG as a potent Th1-directing adjuvant mixture. Overall, this study suggests SCR prime-VLP boosting as a promising approach in HIV vaccine development.

Background Preexisting, donor-specific antibodies (DSAs) are culprits of hyperacute rejection. Donor-specific antibodies are also formed de novo, and their role in acute and chronic rejection is increasingly appreciated. However, it is difficult to assess damage inflicted exclusively by DSAs when alloreactive T cell and B cell responses coincide. We reasoned that allosensitization with "costimulation-deficient" cells should induce DSA synthesis but not naive cytotoxic T lymphocyte (CTL) precursors' priming via direct allorecognition. Accordingly, we have developed a novel model to quantify DSA-mediated cytotoxicity in vivo. Methods C57BL/6 (H-2b) mice were sensitized with H-2 kidney epithelial cells, and a cytofluorimetric killing assay was tailored to the measurement of allocytotoxicity. We took cell/complement depletion, costimulation blockade, and serum transfer approaches to reveal the mediators of cytotoxicity. "Third-party" controls and a skin allotransplantation model were used to confirm DSAs' specificity for allo-major histocompatibility complex. We validated our experimental approach in other mouse strains primed with different allogeneic cell types, including endothelial cells. To demonstrate the usefulness of our model/method for drug efficacy testing, we examined the effect of CTLA4-Ig and rapamycin on DSA-mediated cytolysis. Results Allosensitization of MHC-disparate mouse strains with costimulation-deficient cells led to robust cytotoxicity mediated by complement-fixing DSAs and phagocytic cells. This response was independent of CTLs, natural killer or natural killer T cells. It required CD4 T cell help, CD40 signaling and CD28-based costimulation during allosensitization and could be reversed by sustained rapamycin treatment. Conclusions The unique model described herein should enable mechanistic studies on sensitization and effector phases of humoral alloreactivity as well as efficacy testing of future immunotherapies to prevent DSA-induced pathology.

In the present study we used a fusion peptide from HIV-1 p24 and Nef as vaccine model and adjuvant activity of Naloxone/alum mixture was evaluated in a peptide vaccine model. HIV-1 p24-Nef fusion peptide was synthesized. Female BALB/c mice were divided into five groups. The first group immunized subcutaneously with the p24-Nef fusion peptide adjuvanted with Naloxone/alum mixture and boosted with same protocol. The second was immunized with fusion peptide adjuvanted in alum. The control groups were injected with NLX (Group 3), Alum (Group 4), or PBS (Groups 5) under the same conditions. To determine the type of induced immune response, sera and splenocytes were analyzed by commercial ELISA method for total IgG and isotypes and cytokine secretion (IL-4 & IFN-γ), respectively. We have also used the ELISPOT assay to monitor changes in the frequency of IFN-γ-producing T cells. The proliferation of T cells was assessed using Brdu method and T-cell cytotoxicity was assessed with CFSE method. Immunization of mice with HIV-1 p24-Nef fusion peptide formulated in Naloxone/alum mixture significantly increased lymphocyte proliferation and shifted cytokine responses toward Th1 profile compared to all other groups. Analysis of humoral immune responses revealed that administration of HIV-1 p24-Nef fusion peptide with Naloxone/alum mixture significantly increased specific IgG responses and also increased IgG1,IgG2a, IgG2b, IgG3, and IgM vs. alum-adjuvanted vaccine groups. Naloxone/alum mixture as an adjuvant could improve cellular and humoral immune response for HIV vaccine model and this adjuvant maybe useful for HIV vaccine model in human clinical trial.

Toxic shock syndrome (TSS) and other superantigen-mediated illnesses are associated with 'systemic' immunosuppression that jeopardizes the host's ability to fight pathogens. Here, we define a novel mechanism of 'local' immunosuppression that may benefit the host. Systemic exposure to staphylococcal enterotoxin B (SEB) rapidly and selectively recruited CD11b(+)Gr-1(high)Ly-6C(+) granulocytic myeloid-derived suppressor cells (MDSCs) to the liver of HLA-DR4 transgenic mice. Hepatic MDSCs inhibited SEB-triggered T cell proliferation in a reactive oxygen species-dependent manner, and ex vivo-generated human MDSCs also similarly attenuated the proliferative response of autologous T cells to SEB. We propose a role for MDSCs in mitigating excessive tissue injury during TSS.

The existence of a developed network of suppressory factors and cells against an immune response in different cancers has been proven; regulatory T cells are a typical issue. Therefore their depletion, elimination, or suppression has been assessed in different research studies that were not entirely successful. By applying an improved vaccine against regulatory T cells, we have evaluated the B cell response elicited by the vaccine in an experimental design. A previously described DNA vaccine and recombinant protein of Foxp3-Fc fusion were produced and used in the vaccination regimen. DNA construct and respective protein were injected into C57BL/6 mice. After 2 weeks, serum levels of IgG antibody and its subtypes against Foxp3 were investigated by ELISA. To produce recombinant Foxp3 for ELISA antigen coating, pET24a-Foxp3 vector was transformed into Escherichia coli strain BL21 as host cells. Afterward, protein was expressed and then purified using Ni-NTA agarose. SDS-PAGE and Western blot analysis were carried out to confirm protein expression. The expression analysis of Foxp3 was confirmed by SDS-PAGE followed by Western blot analysis. FOXP3-Fc DNA vaccine/fusion protein vaccination regimen could induce T helper-dependent humoral responses. Due to the effectiveness of Foxp3-Fc(IgG) in inducing humoral responses, it would be expected to be useful in developing vaccines in tumor therapies for the removal of regulatory T cells as a strategy for increasing the efficiency of other means of immunotherapy.

Induction of a strong hepatitis C virus (HCV)-specific immune response plays a key role in control and clearance of the virus. A polytope (PT) DNA vaccine containing B- and T-cell epitopes could be a promising vaccination strategy against HCV, but its efficacy needs to be improved. The N-terminal domain of heat shock protein gp96 (NT(gp96)) has been shown to be a potent adjuvant for enhancing immunity. We constructed a PT DNA vaccine encoding four HCV immunodominant cytotoxic T lymphocyte epitopes (two HLA-A2- and two H2-D(d)-specific motifs) from the Core, E2, NS3 and NS5B antigens in addition to a T-helper CD4+ epitope from NS3 and a B-cell epitope from E2. The NT(gp96) was fused to the C- or N-terminal end of the PT DNA (PT-NT(gp96) or NT(gp96)-PT), and their potency was compared. Cellular and humoral immune responses against the expressed peptides were evaluated in CB6F1 mice. Our results showed that immunization of mice with PT DNA vaccine fused to NT(gp96) induced significantly stronger T-cell and antibody responses than PT DNA alone. Furthermore, the adjuvant activity of NT(gp96) was more efficient in the induction of immune responses when fused to the C-terminal end of the HCV DNA polytope. In conclusion, the NT(gp96) improved the efficacy of the DNA vaccine, and this immunomodulatory effect was dependent on the position of the fusion.

Background: Hypervariability of HCV proteins is an important obstacle to design an efficient vaccine for HCV infection. Multi-epitope vaccines containing conserved epitopes of the virus could be a promising approach for protection against HCV. Objectives: Cellular and humoral immune responses against multi-epitope DNA and peptide vaccines were evaluated in BALB/c mice. Materials and Methods: In this experimental study, multi-epitope DNA- and peptide-based vaccines for HCV infection harboring immunodominant CD8+ T cell epitopes (HLA-A2 and H2-Dd) from Core (132-142), NS3 (1073-1081) and NS5B (2727-2735), a Th CD4+ epitope from NS3 (1248-1262) and a B-cell epitope from E2 (412-426) were designed. Multi-epitope DNA and peptide vaccines were tested in two regimens as heterologous DNA/peptide (group 1) and homologous peptide/peptide (group 2) prime/boost vaccine in BALB/c mice model. Electroporation was used for delivery of the DNA vaccine. Peptide vaccine was formulated with Montanide ISA 720 (M720) as adjuvant. Cytokine assay and antibody detection were performed to analyze the immune responses. Results: Mice immunized with multi-epitope peptide formulated with M720 developed higher HCV-specific levels of total IgG, IgG1 and IgG2a than those immunized with multi-epitope DNA vaccine. IFN-γ levels in group 2 were significantly higher than group 1 (i.e. 3 weeks after the last immunization; 37.61 ± 2.39 vs. 14.43 ± 0.43, P < 0.05). Moreover, group 2 had a higher IFN-γ/IL-4 ratio compared to group 1, suggesting a shift toward Th1 response. In addition, in the present study, induced immune responses were long lasting and stable after 9 weeks of the last immunization. Conclusions: Evaluation of multi-epitope DNA and peptide-vaccines confirmed their specific immunogenicity in BALB/c mice. However, lower Th1 immune responses in mice immunized with DNA vaccine suggests further investigations to improve the immunogenicity of the multi-epitope DNA vaccine through immune enhancers.

Background A supreme vaccine for Hepatitis C virus (HCV) infection should elicit strong Th1-oriented cellular responses. In the absence of a Th1-specific adjuvant, immunizations by protein antigens generally induce Th2-type and weak cellular responses. Objectives To evaluate the adjuvant effect of BCG in comparison with nonionic copolymer-Pluronic F127 (F127) as a classic adjuvant in the formulation of HCV core protein (HCVcp) as a candidate vaccine for induction of Th1 immune responses. Materials and Methods Expression of N-terminally His-Tagged HCVcp (1-122) by pIVEX2.4a-core vector harboring the corresponding gene under the control of arabinose-inducible (araBAD) promoter was achieved in BL21-AI strain of E.coli and purified through application of nitrilotriacetic acid (Ni-NTA) chromatography. Mice were immunized subcutaneously (s.c.) in base of the tail with 100 μl of immunogen (F127+HCVcp or BCG+HCVcp; 5 μgHCVcp/mouse/dose) or control formulations (PBS, BCG, F127) at weeks 0, 3, 6. Total and subtypes of IgG, as well as cellular immune responses (Proliferation, In vivo CTL and IFN-γ/IL-4 ELISpot assays against a strong and dominant H2-d restricted, CD8+-epitopic peptide, core 39-48; RRGPRLGVRA of HCVcp) were compared in each group of immunized animals. Results Expression and purification of core protein around the expected size (21 kDa) was confirmed by Western blotting. The HCVcp + BCG vaccinated mice showed significantly higher lymphocyte proliferation and IFN-γ production but lower levels of cell lysis (45% versus 62% in CTL assay) than the HCVcp+F127 immunized animals. “Besides, total anti-core IgG and IgG1 levels were significantly higher in HCVcp + F127 immunized mice as compared to HCVcp + BCG vaccinated animals, indicating relatively higher efficacy of F127 for the stimulation of humoral and Th2-oriented immune responses”. Conclusions Results showed that HCVcp + BCG induced a moderate CTL and mixed Th1/Th2 immune responses with higher levels of cell proliferation and IFN-γ secretion, indicating that BCG may have a better outcome when formulated in HCVcp-based subunit vaccines.

Medical intervention with fibrinolytic drugs such as tissue plasminogen activator (tPA) and streptokinase (SK) is the principal treatment for life-treating thromboembolic disorders. Contrary to tPA, SK is a heterogenic and non-human (bacterial) protein produced by streptococci and its medical application may elicit sever immune and anaphylactic responses that restrict its utilization. Besides, human plasminogen (HPG) activation by SK is not blood-clot specific and associated with a risk of hemorrhage. Despite these limitations, comparative clinical trials on various thrombolytic agents suggested that SK is the most cost-effective fibrinolytic drug and almost as safe as its other counterparts such as tPA. Therefore, a number of studies were conducted to provide structurally modified SK with reduced immunogenicity, higher blood-clot specificity and half-lives. Although there are extensive overlaps in SK structural domains responsible for functionality, immunogenicity and stability that may limit its modifications, various strategies such as genetic manipulations (amino acid substitution /addition /deletion or domain fusions through production of chimeric SK proteins linked to HPG or hirudin) and chemical modification such as (homogenous/site-specific) PEGylation have been employed to develop a superior SK. In addition, data of the latest studies on SK screened from different streptococcal sources indicated the possibility of retrieving naturally occurring SKs with higher activities, less antigenicity and/or more fibrinspecificity. In the present review, after a survey on structure function relationships of SK domains and different strategies for SK improvement, recent advances and potential application of computer and matrix-based analyses for design and introduction of superior SKs will be presented.

ObjectiveDrug-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.DesignTo monitor transmission of DR HIV, a threshold surveillance based on the world health organization (WHO) guidelines was implemented in Iran.MethodsFor 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.ResultsPhylogenetic 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.ConclusionsOur 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%).

Background : Drug-resistant (DR) HIV emerges during antiretroviral treatment (ART), creating concern about widespread transmission of DR-HIV as ART is expanded in resource-limited countries. The aim of this study was to determine the predominant HIV-1 subtypes and prevalence of drug-resistance mutations among antiretroviral-naive patients in Iran. Design and methods: For this HIV DR threshold surveillance study, blood samples were collected from 50 antiretroviral-naive HIV-1-infected patients. Antiretroviral-resistant mutations were determined by sequencing HIV-1 protease (PR), reverse transcriptase (RT) and integrase (INT) regions. The HIV-1 subtype of each sample was determined by sequencing the p17 and C2-V5 regions of the gag and env genes, respectively. Results: The PR, RT and INT regions were successfully sequenced in 47 samples (94.0%). Phylogenetic analyses of these regions revealed that 45 (95.7%) cases were CRF35_AD. The remaining two cases were subtype B (2.1%) and CRF01_AE (2.1%). Consistent results were obtained also by Env and Gag sequences. Regarding prevalence of transmitted drug-resistant viruses, two cases were found to harbor RT-inhibitor mutations. In addition, nine amino acid differences compared to that of HXB2 were found in protease region as compared to that of HXB2, though none matched with the mutations shown in the WHO list for surveillance of transmitted mutations. No drug-resistant mutations were found in the INT region. Conclusion: Our study clarified that CRF35_AD is the major HIV-1 subtype among Iranian HIV-1-infected patients. According to the WHO surveillance algorithm, the prevalence of transmitted drug resistance in Iran was estimated as moderate (5-15%). (Published: 11 November 2012) Citation: Abstracts of the Eleventh International Congress on Drug Therapy in HIV Infection Jahanbakhsh Sefidi F et al. Journal of the International AIDS Society 2012, 15 (Suppl 4):18225 http://www.jiasociety.org/index.php/jias/article/view/18225 | http://dx.doi.org/10.7448/IAS.15.6.18225

To understand the molecular epidemiology of HIV-1 infection in Iran, we conducted the first study to analyze the genome sequence of Iranian HIV-1 isolates. For this cross-sectional study, we enrolled 10 HIV-1-infected individuals associated with injection drug use from Tehran, Shiraz, and Kermanshah. Near full-length genome sequences obtained from their plasma samples were used for phylogenetic tree and similarity plotting analyses. Among 10 isolates, nine were clearly identified as CRF35_AD and the remaining one as CRF01_AE. Interestingly, five of our Iranian CRF35_AD isolates made two clusters with 10 Afghan CRF35_AD isolates in a phylogenetic tree, indicating epidemiological connections among injection drug users in Iran and Afghanistan. In contrast, our CRF01_AE isolate had no genetic relationship with any other CRF01_AE isolates worldwide, even from Afghanistan. This study provides the first genomic evidence of HIV-1 CRF35_AD predominance and CRF01_AE infection among individuals associated with injection drug use in Iran.

Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for Huh-7 and Vero cells. Different cell confluencies, DNA/reagent ratios and total transfection volumes were optimized for two chemical reagents including jetPEI™ and Lipofectamine™ 2000. Besides, the effects of electric field strength and pulse length were investigated to improve electroporation efficiency. Transfection of cells by pEGFP-N1 vector and tracking the expression of GFP by FACS and Fluorescence Microscopy analysis were the employed methods to evaluate transfection efficiencies. Optimized electroporation protocols yielded 63.73 ± ± 2.36 and 73.9 ± 1.6 % of transfection in Huh-7 and Vero cells respectively, while maximum achieved level of transfection by jetPEI™ was respectively 14.2 ± 0.69 and 28 ± 1.11 % for the same cells. Post transfectional chilling of the cells did not improve electrotransfection efficiency of Huh-7 cells. Compared to chemical based reagents, electroporation showed the superior levels of transfection in both cell lines. The presented protocols should satisfy most of the experimental applications requiring high transfection efficiencies of these two cell lines. Наличие эффективного протокола трансфекции является первым условием успешных исследований по переносу генов в клетки млекопитающих, что достигается экспериментально для каждого конкретного типа клеток. Здесь мы приводим данные сравнительного исследования по оптимизации условий трансфекции клеток Huh-7 и Vero с помощью электропорации и химическими методами. Для двух химических соединений, jetPEI™ и Lipofectamine™ 2000, были оптимизированы сочетания различных клеток, соотношения ДНК/реагент и общие объемы трансфекции. Кроме того, для улучшения эффективности электропорации было изучено влияние силы электрического поля и длины импульса. Трансфекция клеток с помощью вектора pEGFP-N1, определение экспрессии GFP с помощью FACS и флюоресцентная микроскопия были использованы для оценки эффективности трансфекции. В оптимизированных протоколах достигалась трансфекция на уровне 63.73 ± 2.36 и 73.9 ± 1.6 % в клетках Huh-7 и Vero соответственно, в то время как максимальный уровень трансфекции с помощью jetPEI™ составлял 14.2 ± 0.69 и 28 ± 1.11 % для тех же клеток. Охлаждение клеток после трансфекции не улучшало эффективность электротрансфекции клеток Huh-7. В обеих клеточных линиях электропорация позволила достичь более высокого уровня трансфекции по сравнению с использованием химических реагентов. Представленный протокол может быть пригодным для большинства экспериментальных манипуляций, которые требуют высокого уровня трансфекции исследуемых клеточных линий.

There is ample evidence in favor of various immunosuppressive mechanisms that weaken antitumor immune responses and affect currently used immunotherapies. Induction of regulatory T cells (Treg) and secretion of indoleamine 2,3-dioxygenase (IDO) by tumor tissue are considered as two main mechanisms of tumor immune escape. However, little is known about the contribution of these mechanisms on the modulation of dendritic cell vaccine-mediated antitumor response. To address this concern, we assessed Treg's infiltration and the expression of Foxp3 and IDO genes in tumor microenvironment following dendritic cell-based antitumor immunotherapy of mice in different protocols of prophylactic, concurrent, and therapeutic vaccination. According to cytotoxicity assay, the vaccinated mice exposed efficient induction of splenic CTLs in all groups. However, only the mice immunized in prophylactic regimen significantly retarded the growth of tumor cells. Interestingly, the Treg content of tumor samples and transcriptional level of both Foxp3 and IDO genes were reduced in this group, while animals that received the vaccine in concurrent and therapeutic protocols showed increase in tumor-infiltrating Tregs and mRNA levels of Foxp3 and IDO. Accordingly, higher expression of these genes resulted in more inhibition of antitumor response. Our findings indicate that tumor progression may enhance the immunoregulatory response and hence emphasize to the effectiveness of vaccination in early stages of tumor growth for avoiding induction of such regulatory responses.

International audience; Correlation of hepatitis C virus (HCV) spontaneous resolution with Th1 and CD8(+)CTL responses during natural infection implies the potentiality of poly-CTL-epitopic HCV vaccines. We recently reported in silico design and construction of DNA vaccines (pcPOL-plasmids) harboring HCV CTL epitopes. Herein, we provide data of mice immunization by pcPOL, (encoding; core(132-142) [C], E2(405-414) [E(4)], E2(614-622) [E(6)] and NS3(1406-1415) [N] CD8(+)CTL epitopes as CE(4)E(6)N polytope) and its HBsAg-fused counterpart (pcHPOL), compared to the adjuvant-formulated (Montanide+CpG) CE(4)E(6)N synthetic-peptide immunization. All vaccinated groups developed different levels of cellular responses, however, only the pcHPOL-immunized mice elicited strong CTLs and IFN-gamma-secreting cells that were further augmented towards a Th1 response and partial tumor protection by DNA-prime/peptide-boosting regimen. Priming with HBsAg alone could not afford its augmenting effect indicating the importance of priming by polytope itself. Hence, fusion of immunocarriers like HBsAg conjoined with DNA-prime/peptide-boost immunization regimen seems a strategy to enhance the epitope-specific immune responses towards poly-CTL-epitopic vaccines.

International audience; For chronic viral infections like Hepatitis C, CD8-CTLs have emerged as important protective tools. Hence, isolated dominant epitopes arranged as polytope DNA or peptide vaccines represent a promising approach. However, because of controversial rules governing the polytope construction and epitope processing, proper design and primary analysis of such vaccines are prior to the costly transgenic animal studies. In this study, based on in silico epitope selection, four HLA-A2 (C132, E614 and N1406) and H-2d (E405 and C132) immunodominant CD8-epitopes of HCV were selected. The codon optimized nucleotide sequences of the epitopes were assembled by overlap extension PCR in three different sequential tandems for the best proteasomal cleavage predictions and cloned into pcDNA3.1 vector. In addition, to enhance particulate formation, three other plasmids containing the fusion of polytopes with hepatitis B surface antigen gene (HBsAg) were also constructed. Proper expression of all constructs in transfected Cos-7 cells was verified by RT-PCR, immunofluorescence, Western-blot, ELISA and dot blot techniques. Moreover, particle formation of HBsAg-fused polytopes was manifested by their secretion to the culture media albeit in lesser amounts compared to sole HBsAg protein. Finally, the positive delayed-type hypersensitivity (DTH) response of vaccinated mice indicated the in vivo expression of all constructs and efficient stimulation of immune response, which was stronger for HBsAg fusion constructs. In addition, proper processing of the epitopes was evidenced by the DTH response towards H-2d epitopic peptides. These data provide enough support and merit for the further evaluation of the designed constructs in HLA-A2 transgenic mice.

International audience; Due to its conservation, the extracellular domain of the influenza A M2 protein (M2e) has the potential for being applied as a recombinant vaccine candidate against a wide range of strains, though its immunogenicity may need to be improved. The occurrence of several post-translational modifications within the structure of M2 protein may affect its immunopotency for the induction of humoral immune response. Herein, to construct a recombinant M2e-based vaccine candidate with the appropriate structural conformation and immunogenicity the corresponding nucleotide sequence from an H9N2 influenza strain was fused to the N-terminus of the truncated Mycobacterium tuberculosis HSP70(359-610), as a potent adjuvant, and following its cloning into the pPICZ alpha A plasmid the fusion gene was expressed in Pichia pastoris KM71H yeast. The secreted protein was then easily purified from the culture media, based on the presence of polyhistidine tag and used for the production of rabbit polyclonal antisera. This raised antisera could recognize the native M2e protein on the surface of H9N2 influenza virus-infected MDCK cells at a comparable level with the commercial H2N2-specific anti-M2 antibody, which was evidenced with immunofluorescence and cell-ELISA assays. These results not only re-emphasized on the conservancy of the M2e antigen, but also pointed towards the applicability of the M2e-HSP70(359-610) fusion protein for the induction of specific antibodies capable of binding to the native M2e antigen on the infected cells. Collectively, this study implied that purified M2e-HSP70(359-610) represents a promising vaccine candidate; however, its in vivo potency for the induction of protection remains to be evaluated.

Chronic hepatitis B affects nearly 10% of HIV-infected patients. Hepatitis B virus (HBV) infection is a dynamic disease and coinfection with HIV impacts directly on the outcome of HBV infection, considerably complicating its natural history, diagnosis, and management. The aim of this study was to compare two cohorts of HBV monoinfected and HBV/HIV coinfected Iranian patients undergoing long-term lamivudine therapy from the clinical and virological aspects, as well as the frequency of detected mutations in HBV genome. To this end, HBV Pol/S regions from 72 patients were PCR-amplified and directly sequenced. Phylogenetic analysis indicated a 40-times higher risk of coinfection with ayw3 subtype of HBV genotype D rather than ayw2 subtype [P < 0.001, odd: 40.66, CI: 95% (4.69-352.23)]. While no resistance mutation was detected in HBV/HIV coinfected cohort, LAM-resistance mutations (rtM204I/V in YMDD and rtL180M in FLLA polymerase motifs) were identified in 30% (9 out of 30) and 16.66% (5 out of 30) of HBV monoinfected patients (P < 0.05). Moreover, several mutations (sP105A, sI110S/L, sS136Y and sP127T/L) with significant differences in the frequency were identified in the S region of both cohorts. Finally, this study found strong correlation between the type of infection (mono or coinfection) and characteristics like patient gender, ALT levels, HBV-DNA levels and HBV subtypes. These results pointed to the importance of determination of HBV variants in the management of patients and suggested that in contrary to HBV monoinfections, LAM may be still an appropriate drug for the treatment of HBV in HBV/HIV coinfected patients; however, further studies to clarify the role of HIV in HBV LAM-resistance mutations are required..