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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.

Primary sclerosing cholangitis (PSC) is a rare, chronic, and progressive cholestatic disease involving intra- and/or extrahepatic bile ducts. PSC in many patients results in end-stage liver diseases. Nearly 60% of the PSC patients suffer from concomitant inflammatory bowel diseases (IBDs). Classically, IBDs are divided into two principle types: Crohn's disease (CD) and ulcerative colitis (UC). However, with growing knowledge, PSC-associated IBD (PSC-IBD) seems to be a rather distinct entity with specific genetics, clinical, and microbiota characteristics.In this article, we aim to review the unique characteristics of PSC-IBD from clinical, genetic, and microbiota point of view.PSC-IBD's unique characteristics contribute to the notion that it could be a distinct entity. Acknowledgment of PSC-IBD as a novel entity necessitates designing new clinical guidelines for diagnosis and developing novel therapies.

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.

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.

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: 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.

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.

Despite a huge number of studies towards vaccine development against human immunodeficiency virus-1, no effective vaccine has been approved yet. Thus, new vaccines should be provided with new formulations. Herein, a new DNA vaccine candidate encoding conserved and immunogenic epitopes from HIV-1 antigens of tat, pol, gag and env is designed and constructed. After bioinformatics analyses to find the best epitopes and their tandem, nucleotide sequence corresponding to the designed multiepitope was synthesized and cloned into pcDNA3.1+ vector. Expression of pcDNA3.1-tat/pol/gag/env plasmid was evaluated in HEK293T cells by RT-PCR and western-blotting. Seven groups of BALB/c mice were intramuscularly immunized three times either with 50, 100, 200 µg of plasmid in 2-week intervals or with similar doses of insert-free plasmid. Two weeks after the last injection, proliferation of T cells and secretion of IL4 and IFN-γ cytokines were evaluated using Brdu and ELISA methods, respectively. Results showed the proper expression of the plasmid in protein and mRNA levels. Moreover, the designed multiepitope plasmid was capable of induction of both proliferation responses as well as IFN-γ and IL-4 cytokine production in a considerable level compared to the control groups. Overall, our primary data warranted further detailed studies on the potency of this vaccine.

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 attenuated or non-pathogenic live vectors have been evolved specifically to deliver DNA into cells as efficient delivery tools in gene therapy. Recently, a non-pathogenic protozoan, Leishmania tarentolae (L.tar) has attracted a great attention. In current study, we used Leishmania expression system (LEXSY) for stable expression of HPV16 E7 linked to different mini-chaperones [N-/C-terminal of gp96] and compared their immunogenicity and protective effects in C57BL/6 mice against TC-1 challenge. TC-1 murine model is primary C57BL/6 mice lung epithelial cells co-transformed with HPV16 E6, HPV16 E7 and ras oncogenes. Our results showed that subcutaneous administration of mice with both the recombinant L.tar-E7-NT (gp96) and L.tar-E7-CT (gp96) led to enhance the levels of IFN-γ and also IgG2a before and after challenge with TC-1. Furthermore, L.tar-E7-CT (gp96) live vaccine indicated significant protective effects as compared to control groups as well as group vaccinated with L.tar-E7. Indeed, the recombinant live vector is capable of eliciting effective humoral and cellular immune responses in mice, but however, further studies are required to increase their efficacy.

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.

DNA vaccines have emerged as an attractive approach to induce CTL responses against cancer and infectious agents in recent years. Although CTL induction by DNA vaccination would be a valuable strategy for controlling viral infections, increasing the potency of DNA vaccines is mandatory before DNA vaccines can make it to the clinic. In this study, we developed and characterized a new and safe adjuvanted delivery system for DNA vaccination using cationic influenza virosomes (CIV). CIV were produced by reconstitution of detergent-solubilized influenza virus membranes in the presence of cationic lipids. Plasmid DNA (pDNA) mixed with these virosomes was efficiently transfected into cells of a mouse macrophage cell line (RAW-Blue cells). Moreover, the cells were effectively activated as demonstrated by production of an NF kappa B/AP-1-inducible reporter enzyme. Following three intradermal immunizations, CIV-delivered epitope-encoding pDNA induced equal numbers of IFN gamma- and granzyme B-producing T cells than a 10-fold higher dose of naked pDNA. Virosomes without cationic lipids also improved induction of cellular immunity by pDNA but to a significantly lower extent than CIV. These findings suggest that pDNA-CIV complexes could be an efficacious delivery system suitable for CTL induction by DNA vaccination. (C) 2012 Elsevier B.V. All rights reserved.

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.

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.

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.

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.

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.

Tuberculosis (TB) remains as a major public health problem worldwide. Identification and selection of immunodominant antigens of Mycobacterium tuberculosis (MTB), capable of efficiently inducing a protective immune response is the ultimate goal of TB vaccine development studies. Accordingly, this study was designed to produce a novel M. tuberculosis fusion protein consisted of MTB ESAT-6 (early secreted antigenic target-6 kDa), as a potent immunogenic protein, fused to C-terminus of MTB HSP70 (HSP70 359–610 ), as an appropriate carrier and adjuvant. The constructed gene was inserted into a prokaryotic expression vector (pQE30); consequently, the recombinant fusion protein with a 6xHis-tag was successfully over expressed in Escherichia coli M15. Inclusion bodies from bacterial cell lysates were solubilized and the recombinant fusion protein was easily purified by Ni-NTA affinity chromatography under denaturing conditions followed by urea gradient dialysis. The purified and refolded protein was then applied for immunization of mice that resulted in the detection of high titers of specific antibodies, high level of IFN-γ and cell proliferation. The results of our study could confirm the capability of E6H70C fusion protein, as a potential tuberculosis vaccine candidate, for the efficient induction of specific immune responses in a mouse model. However, further investigation need to evaluate the protectivity of this recombinant protein in host model.

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.

Non-infectious but antigenic human immunodeficiency virus 1 (HIV-1) particles are essential tool for the research on many topics associated with this virus. Here we report the construction of plasmid containing the HIV-1 genome mutated in the pol gene, which was co-transfected with plasmids expressing the pol gene products reverse transcriptase (RT) and integrase (IN), and the glycoprotein G of vesicular stomatitis virus (VSV-G). The virions produced in HEK 293 T cells were antigenic, but able to replicate only for one cycle, e.g. first generation single-cycle replicable (SCR) virions. The presence of VSV-G in the envelope of these virions had to ensure a wider spectrum of susceptible cell types for the replication of SCR. Replication of the first generation SCR virions in HEK 293T, MT-2, and mouse spleen cells was examined by p24-capture ELISA, syncytium formation assay, and electron microscopy (EM). HEK 293T and MT-2 cell lines showed a similar replication capacity, while primary cultures of mouse spleen cells were much less effective. The infection of MT-2 cells with the first generation of SCR virions yielded the second generation SCR virions, which were non-infectious. Summing up, the HIV-1 SCR virions represent the useful tool for HIV-1 research facilitating a better biological safety. Moreover, considering their antigenic composition and limited replication, SCR virions may be a promising candidate for the vaccine studies.

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International audience; An efficient vaccine against hepatitis-C virus (HCV) infection requires vigorous and focused CD8(+) T-cell responses against viral antigens. Due to immunosuppressive effect of HCV antigens, polytope vaccines comprising the minimal CD8(+)CTL epitopes are of peculiar concern. Herein, to provide information for construction of efficient HCV polytope vaccine candidates, one H-2D(d) (E2(405-414):E(2)) and two HLA-A*0201 (E1(363-372):E(1) and Core(35-44):C)-restricted CD8(+) T-cell epitopes of HCV were selected. By employing number of in silico analyses, the E(2)E(1)C linear format was predicted as optimum epitope consecution and after amplification by SOEing-PCR, the corresponding DNA sequence was cloned in pcDNA3.1+ vector. To further evaluate the role of immune-enhancer elements, a universal T-helper epitope (PADRE), endoplasmic reticulum signal sequence (ERss) and hepatitis-B surface-antigen (HBsAg) gene were fused separately or in combination to the E(2)E(1)C minigene. In vitro analyses of polytopes by different DNA/protein-based assays demonstrated proper transcription/expression of constructs in transfected cells. Measurement of the HBsAg-mediated particle secretion by ELISA indicated lack of secretion in the related polytopes. Results of delayed-type hypersensitivity (DTH) as a preliminary in vivo analysis, and confirmatory ELISPOT assays showed the proper processing and presentation of H-2D(d)-restricted-E(2) epitope and approved the enhancing effect of PADRE and ERss sequences but not HBsAg for the immune responses against E(2) in immunized BALB/c mice. Our results pointed to the value of in silico predictions and application of immune-enhancer elements as well as DTH analysis for design and primary in vivo evaluation of HCV polytopes, prior to costly transgenic studies on immunogenicity of HLA-A*0201 epitopes.

Plants transformed by virus-based vectors have emerged as promising tools to rapidly express large amounts and inexpensive antigens in transient condition.We studied the possibility of transient-expression of an HBsAg-fused polytopic construct (HCVpc) [containing H-2d and HLA-A2-restricted CD8+CTL-epitopic peptides of C (Core; aa 132-142), E6 (Envelope2; aa 614-622), N (NS3; aa 1406-1415), and E4 (Envelope2; aa 405-414) in tandem of CE6NE4] in tobacco (Nicotiana tabacum) leaves for the development of a plant-based HCV vaccine.A codon-optimized gene encoding the Kozak sequence, hexahistidine (6×His)-tag peptide, and HCVpc in tandem was designed, chemically synthesized, fused to HBsAg gene, and inserted into Potato virus X (PVX-GW) vector under the control of duplicated PVX coat protein promoter (CPP). The resulted recombinant plasmids (after confirmation by restriction and sequencing analyses) were transferred into Agrobacterium tumefaciens strain GV3101 and vacuum infiltrated into tobacco leaves. The effect of gene-silencing suppressor, p19 protein from tomato bushy stunt virus, on the expression yield of HCVpc-HBsAg was also evaluated by co-infiltration of a p19 expression vector.Codon-optimized gene increased adaptation index (CAI) value (from 0.61 to 0.92) in tobacco. The expression of the HCVpc-HBsAg was confirmed by western blot and HBsAg-based detection ELISA on total extractable proteins of tobacco leaves. The expression level of the fusion protein was significantly higher in p19 co-agroinfiltrated plants.The results indicated the possibility of expression of HCVpc-HBsAg constructs with proper protein conformations in tobacco for final application as a plant-derived HCV vaccine.

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: Hepatitis C virus (HCV) is major cause of liver cirrhosis in humans. HCV capsid (core) protein (HCVcp) is a highly demanded antigen for various diagnostic, immunization and pathogenesis studies. Plants are considered as an expression system for producing safe and inexpensive biopharmaceutical proteins. Although invention of transgenic (stable) tobacco plants expressing HCVcp with proper antigenic properties was recently reported, no data for “transient-expression” that is currently the method of choice for rapid, simple and lower-priced protein expression in plants is available for HCVcp. Objectives: The purpose of this study was to design a highly codon-optimized HCVcp gene for construction of an efficient transient-plant expression system for production of HCVcp with proper antigenic properties in a regional tobacco plant (Iranian Jafarabadi-cultivar) by evaluation of different classes of vectors and suppression of gene-silencing in tobacco. Materials and Methods: A codon-optimized gene encoding the Kozak sequence, 6xHis-tag, HCVcp (1-122) and KDEL peptide in tandem (from N- to C-terminal) was designed and inserted into potato virus-X (PVX) and classic pBI121 binary vectors in separate cloning reactions. The resulted recombinant plasmids were transferred into Agrobacterium tumefaciens and vacuum infiltrated into tobacco leaves. The effect of gene silencing suppressor P19 protein derived from tomato bushy stunt virus on the expression yield of HCVcp by each construct was also evaluated by co-infiltration in separate groups. The expressed HCVcp was evaluated by dot and western blotting and ELISA assays. Results: The codon-optimized gene had an increased adaptation index value (from 0.65 to 0.85) and reduced GC content (from 62.62 to 51.05) in tobacco and removed the possible deleterious effect of “GGTAAG” splice site in native HCVcp. Blotting assays via specific antibodies confirmed the expression of the 15 kDa HCVcp. The expression level of HCVcp was enhanced by 4-5 times in P19 co-agroinfiltrated plants with better outcomes for PVX, compared to pBI121 vector (0.022% versus 0.019% of the total soluble protein). The plant-derived HCVcp (pHCVcp) could properly identify the HCVcp antibody in HCV-infected human sera compared to Escherichia coli-derived HCVcp (eHCVcp), indicating its potential for diagnostic/immunization applications. Conclusions: By employment of gene optimization strategies, use of viral-based vectors and suppression of plant-derived gene silencing effect, efficient transient expression of HCVcp in tobacco with proper antigenic properties could be possible.