scholarly journals Development of Delivery Systems Enhances the Potency of Cell-Based HIV-1 Therapeutic Vaccine Candidates

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Amin Hadi ◽  
Abbas Rastgoo ◽  
Maryam Eskandarian ◽  
Nooshin Haghighipour ◽  
Azam Bolhassani
Keyword(s):  
Gene Transfection ◽  
Therapeutic Vaccine ◽  
Cyclic Stretch ◽  
Mouse Bone Marrow ◽  
Transfection Efficacy ◽  
Protein Boost ◽  
Mechanical Methods ◽  
Protein Immunization ◽  
Hiv 1

An effective therapeutic vaccine to eradicate HIV-1 infection does not exist yet. Among different vaccination strategies, cell-based vaccines could achieve in clinical trials. Cell viability and low nucleic acid expression are the problems related to dendritic cells (DCs) and mesenchymal stem cells (MSCs), which are transfected with plasmid DNA. Thus, novel in vitro strategies are needed to improve DNA transfection into these cells. The recent study assessed immune responses generated by MSCs and DCs, which were derived from mouse bone marrow and modified with Nef antigen using novel methods in mice. For this purpose, an excellent gene transfection approach by mechanical methods was used. Our data revealed that the transfection efficacy of Nef DNA into the immature MSCs and DCs was improved by the combination of chemical and mechanical (causing equiaxial cyclic stretch) approaches. Also, chemical transfection performed two times with 48-hour intervals further increased gene expression in both cells. The groups immunized with Nef DC prime/rNef protein boost and then Nef MSC prime/rNef protein boost were able to stimulate high levels of IFN-γ, IgG2b, IgG2a, and Granzyme B directed toward Th1 responses in mice. Furthermore, the mesenchymal or dendritic cell-based immunizations were more effective compared to protein immunization for enhancement of the Nef-specific T-cell responses in mice. Hence, the use of chemical reagent and mechanical loading simultaneously can be an excellent method in delivering cargoes into DCs and MSCs. Moreover, DC- and MSC-based immunizations can be considered as promising approaches for protection against HIV-1 infections.

The Effect of Anti-GPIIIa49-66 Antibody on Megakaryocyte Differentiation.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1434-1434
Author(s):  
Jianhui Wang ◽  
Ruimin Pan ◽  
Michael A. Nardi ◽  
Zongdong Li
Keyword(s):  
Nadph Oxidase ◽  
In Vitro Culture ◽  
Conflicts Of Interest ◽  
Signal Pathways ◽  
Mouse Bone Marrow ◽  
Colony Forming Units ◽  
Sequential Activation ◽  
12 Lipoxygenase ◽  
Hiv 1

Abstract Abstract 1434 We previously reported that patients with early-onset HIV-1 ITP develop a unique anti-platelet integrin GPIIIa antibody against the GPIIIa49-66 epitope. Anti-GPIIIa49-66 antibody-induced platelet fragmentation requires sequential activation of the platelet 12-lipoxygenase (12-LO) and NADPH oxidase to release reactive oxygen species (ROS). 12-LO is upstream of the NADPH oxidase pathway and 12(S)-HETE, the product of 12-LO, induces the same oxidative platelet fragmentation as anti-GPIIIa49-66. Since the megakaryocyte (MK) is the progenitor cells for platelets and may contain similar signal pathways, we have investigated the effect of anti-GPIIIa49-66 on MK differentiation and, in particular, the potential role of anti-GPIIIa49-66 induced ROS in this process. We first show that polyclonal anti-GPIIIa49-66 antibody isolated from HIV-1 ITP patients inhibits MK proliferation 2.5 fold in in vitro culture of mouse bone marrow Lin-/- cells driven by thrombopoietin (TPO). We also observed a 3 fold decrease in the number of MK colony-forming units in the presence of a human monoclonal anti-GPIIIa49-66 antibody we generated. However, we could not detect ROS release in DCFH-loaded MEG-01 cells treated with anti-GPIIIa49-66 antibody. In addition, 12(S)-HETE does not inhibit the in vitro differentiation of MK cell line L8057 induced by TPO. In fact, we found a dose dependent increasing of the percentage of αIIb integrin positive cells (from 17.1% to 48.7%) in in vitro culture of L8057 treated by various concentration of H2O2 (from 5 to 20μM). Thus, our data suggests that ROS is not involved in the inhibition of MK differentiation induced by anti-GPIIIa49-66, in contrast to the effect that this antibody has on mature platelets. We therefore conclude that the anti-GPIIIa49-66 antibody dysregulates ROS independent β3 integrin signaling to inhibit MK differentiation. Disclosures: No relevant conflicts of interest to declare.

Production of Recombinant HIV-1 p24-Nef Protein in Two Forms as Potential Candidate Vaccines in Three Vehicles

2020 ◽  
Vol 17 (5) ◽  
pp. 387-395
Author(s):  
Mona Sadat Larijani ◽  
Mohammad Hassan Pouriayevali ◽  
Seyed Mehdi Sadat ◽  
Amitis Ramezani
Keyword(s):  
Fusion Protein ◽  
Western Blotting ◽  
Highly Active Antiretroviral Therapy ◽  
Fusion Proteins ◽  
Therapeutic Vaccine ◽  
Potential Candidate ◽  
Conserved Regions ◽  
Bioinformatics Tools ◽  
Hiv 1

Background: Different approaches have been investigated to develop a preventive or therapeutic vaccine, although none of them has been fully practical. Therapeutic vaccines against HIV-1 have been studied with the aim of eliminating the virus from reservoir cells with or without HAART (Highly Active Antiretroviral Therapy). Fusion proteins with the most immunogenic features among conserved regions can facilitate this achievement in such a variable virus. To achieve the most immunogenic and also conserved regions, bioinformatics tools are widely used to predict antigens’ features before applying them. Objective: This study aimed at the in vitro evaluation of p24 -Nef fusion protein based on the previous in silico design to achieve a potential therapeutic subunit vaccine against HIV-1. Methods: The truncated form of p24-Nef using AAY flexible linker and the full protein were expressed and evaluated in the prokaryotic system and confirmed by western blotting. We also used pcDNA3.1 to transfect Lenti-X 293T cells. Moreover, lentiviral vectors were applied to produce recombinant virions harboring the genes of interest and cell transduction. Results: Both fusion proteins in a truncated and a full form were expressed and confirmed by Anti Nef polyclonal antibody in western blotting. Recombinant virions were generated and transduced Lenti-X 293T cells confirming by immunofluorescence microscope and p24 ELISA assay kit. Transduced cells were analyzed by SDS-PAGE and western blotting, which resulted in approved protein expression. Conclusion: Fusion protein of p24 and Nef is well expressed in eukaryotic cell lines according to its pre-evaluated features by bioinformatics tools.

scholarly journals ART-Treated Patients Exhibit an Adaptive Immune Response against the HFVAC Peptides, a Potential HIV-1 Therapeutic Vaccine (Provir/Latitude45 Study)

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1256
Author(s):  
Hervé Fleury ◽  
Sabrina Caldato ◽  
Patricia Recordon-Pinson ◽  
Patricia Thebault ◽  
Gwenda-Line Guidicelli ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Cd8 T Cells ◽  
Cytotoxic T Lymphocyte ◽  
Therapeutic Vaccine ◽  
Adaptive Immune Response ◽  
Immune Reactivity ◽  
Subtype B ◽  
Hiv 1

We proposed a new HIV-1 therapeutic vaccine based on conserved cytotoxic T lymphocyte (CTL) epitopes of archived HIV-1 DNA according to their affinity to the dominant HLA-A and -B alleles of the population investigated. Our proposal (Hla Fitted VAC, HFVAC) was composed of 15 peptides originating from the RT, gag and nef parts of proviral DNA. Our aim was to investigate baseline immune reactivity to the vaccine in HIV-1 chronically infected patients at success of antiretroviral therapy (ART) who would be eligible for a therapeutic vaccine. Forty-one patients were tested. Most of them had been infected with HIV-1 subtype B and all had been receiving successful ART for 2 to 20 years. The predominant HLA-A and -B alleles were those of a Caucasian population. ELISPOT was carried out using the HFVAC peptides. In 22 patients, the PD-1 marker was investigated on CD4+ and CD8+ T cells by flow cytometry in order to evaluate global T cell exhaustion. ELISPOT positivity was 65% overall and 69% in patients exhibiting at least one HLA allele fitting with HFVAC. The percentages of CD4+ and CD8+ T cells expressing PD-1 were high (median values 23.70 and 32.60, respectively), but did not seem to be associated with an impairment of the immune response investigated in vitro. In conclusion, reactivity to HFVAC was high in this ART-treated population with dominant HLA alleles, despite potential cellular exhaustion associated with the PD-1 marker.

Abstract 15404: Efficient and Persistent Transgene Expression by Minicircle Plasmid versus Standard Plasmid Using Ultrasound Targeted Microbubble Destruction

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Dmitriy Rudenko ◽  
Pratiek N Matkar ◽  
Saleh A Alghadeer ◽  
Christine Liao ◽  
Hao H Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hind Limb ◽  
Gene Transfection ◽  
Fluorescent Microscopy ◽  
Rt Pcr ◽  
Gfp Expression ◽  
Transfection Efficacy ◽  
Minicircle Dna

Background: Ultrasound-targeted microbubble destruction (UTMD) is a non-invasive gene transfection technique using carrier microbubbles and targeted high power ultrasound, primarily used to deliver plasmid DNA (pDNA). Minicircle-DNA (mcDNA) is a novel gene vector, which has recently been shown to have an improved and persistent transfection, compared to conventional pDNA. Hypothesis: We hypothesized that UTMD of mcDNA in a hind-limb model would exhibit a more potent and prolonged gene expression compared to conventional pDNA. Methods: In vitro , HUVECs, fibroblasts (3T3) and neonatal cardiomyocytes were transfected with molar equivalents of GFP-minicircle and GFP-plasmid. GFP expression was measured by RT-PCR and fluorescent microscopy for 28 days. We then performed a comparison of bubble binding capacities of both vectors to cationic lipid microbubbles. In vivo , for UTMD to the left proximal hind-limb adductor muscle, 500μg and 214μg of GFP-plasmid and GFP-minicircle respectively were charge-coupled with 1x10 9 cationic microbubbles and delivered via UMGD into Sprague-Dawley rats (n=30). The animals were followed for 28 days with GFP measured by RT-PCR and immunohistochemistry. Results: In vitro results showed greater GFP expression by mcDNA across all cell lines, with 7-10 fold transfection efficacy compared to pDNA. mcDNA demonstrated greater binding capacity to cationic microbubbles compared to pDNA. For plasmid and minicircle DNA, binding saturations were reached at ~6000 copies per microbubble, and ~20000 copies per microbubble respectively, suggesting a higher minicircle bubble binding efficiency. In vivo results showed higher GFP levels as early as 6 hours post minicircle UTMD, demonstrating minicircle to be a faster acting therapeutic agent over conventional plasmid. A significantly greater (p<0.01) expression of GFP was also evident at day 28 in minicircle UTMD-treated group, proving mcDNA to be a better choice for longer-term gene expression. Conclusions: In summary, UTMD using mcDNA results in more rapid and sustained transfection compared to conventional pDNA, and may be a more effective vector for translational studies of UTMD.

scholarly journals Viral Vectors for the Induction of Broadly Neutralizing Antibodies against HIV

Vaccines ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 119 ◽  
Author(s):  
Sarah Wilmschen ◽  
Joern E. Schmitz ◽  
Janine Kimpel
Keyword(s):  
Neutralizing Antibodies ◽  
Viral Vectors ◽  
Adenoviral Vectors ◽  
Tier 2 ◽  
Broadly Neutralizing Antibodies ◽  
Protein Boost ◽  
Tier 1 ◽  
Protein Immunization ◽  
Env Antigens ◽  
Hiv 1

Extensive research on generating an efficient HIV vaccine is ongoing. A major aim of HIV vaccines is the induction of long-lasting, broadly neutralizing antibodies (bnAbs) that can confer sterile immunity for a prolonged period of time. Several strategies have been explored to reach this goal, i.e. protein immunization, DNA, or viral vectors, or a combination thereof. In this review, we give an overview of approaches using viral vectors for the induction of HIV-specific bnAbs. Many pre-clinical studies were performed using various replication-competent and -incompetent vectors. Amongst them, poxviral and adenoviral vectors were the most prevalent ones. In many studies, viral vectors were combined with a DNA prime or a protein boost. However, neutralizing antibodies were mainly induced against the homologous HIV-1 vaccine strain or tier 1 viruses, and in rare cases, against tier 2 viruses, indicating the need for improved antigens and vaccination strategies. Furthermore, we also review next generation Env antigens that are currently being used in protein vaccination approaches and point out how they could be utilized in viral vectors.

scholarly journals Gene Therapy with HSV1-sr39TK/GCV Exhibits a Stronger Therapeutic Efficacy Than HSV1-TK/GCV in Rat C6 Glioma Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Lei-qing Li ◽  
Fang Shen ◽  
Xiao-yan Xu ◽  
Hong Zhang ◽  
Xiao-feng Yang ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Therapeutic Efficacy ◽  
Gene Transfection ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
Transfection Efficacy ◽  
Rat C6 Glioma

Although the combination of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) with ganciclovir (GCV) has been shown as a promising suicide gene treatment strategy for glioma, the almost immunodepressive dose of GCV required for its adequatein vivoefficacy has hampered its further clinical application. Therefore, In order to reduce the GCV dose required, we aim to compare the therapeutic efficacy of HSV1-sr39TK, an HSV1-TK mutant with increased GCV prodrug catalytic activity, with wildtype TK in C6 glioma cells. Accordingly, rat C6 glioma cells were first transfected with pCDNA-TK and pCDNA-sr39TK, respectively, and the gene transfection efficacy was verified by immunocytochemistry and western blot analysis. Then thein vivosensitivity of these transfected C6-TK and C6-sr39TK cells to GCV was determined by 3-(4,5)-dimethylthiahiazo-(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) colorimetric assay and Hoechst-propidium iodide (PI) staining. Finally, a subcutaneously C6 xenograft tumor model was established in the nude mice to test thein vitroefficacy of TK/GCV gene therapy. Our results showed that, as compared with wildtype TK, HSV1-sr39TK/GCV demonstrated a stronger therapeutic efficacy against C6 glioma bothin vitroandin vivo, which, by reducing the required GCV dose, might warrant its future use in the treatment of glioma under clinical setting.

scholarly journals MVA-B AS HIV/AIDS VACCINE CANDIDATE: FROM BASIC SCIENCE TO PROPHYLACTIC AND THERAPEUTIC CLINICAL TRIALS

2020 ◽  
pp. 29-60
Author(s):  
Carmen Elena Gómez Rodríguez
Keyword(s):  
Clinical Trials ◽  
Vaccine Candidate ◽  
Therapeutic Vaccine ◽  
Basic Knowledge ◽  
Antigen Delivery ◽  
Modified Vaccinia Virus Ankara ◽  
Hiv 1 ◽  
Hiv Aids

The highly attenuated poxvirus strain modified vaccinia virus Ankara (MVA) has reached maturity as antigen delivery system and as a vaccine candidate against a broad spectrum of infectious diseases. This has been largely recognized from research on virus–host cell interactions, gene expression profiling, virus distribution and immunological studies in preclinical and clinical trials. This review includes our main contributions from the basic knowledge of the biology of the MVA vector, both in vitro and in vivo, in comparison with the attenuated NYVAC strain, to its evaluation as a vaccine candidate against HIV/AIDS in clinical trials. We will detail the generation and characterization of the recombinant poxvirus vector MVA expressing the HIV-1 Env, Gag, Pol and Nef antigens from clade B (referred as MVA-B) and review the preclinical data that supported the evaluation of MVA-B as the first in human HIV-1 prophylactic and therapeutic vaccine in Spain. In addition, we will assess the results of clinical trials and discuss the research projects we are currently working on considering the latest scientific advances in the HIV vaccine field.

DEVELOPMENT OF APPROACHES FOR DETECTION OF GENOME-INTEGRATED PROVIRAL DNA OF THE HUMAN IMMUNODEFICIENCY VIRUS (HIV-1) IN ULTRA LOW CONCENTRATIONS USING THE CRISPR/CAS SYSTEM

2020 ◽  
Author(s):  
M.A. Tyumentseva ◽  
◽  
A.I. Tyumentsev ◽  
V.G. Akimkin ◽  
◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Health Monitoring ◽  
Biological Samples ◽  
Proviral Dna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Low Concentrations ◽  
Immunodeficiency Virus ◽  
Hiv 1

For the effective functioning of supervisory and health monitoring services, it is necessary to introduce modern molecular technologies into their practice. Therefore, the task of developing new effective methods for detecting pathogen, for example HIV, based on CRISPR/CAS genome editing systems, remains urgent. In the present work, guide RNAs and specific oligonucleotides were developed for preliminary amplification of highly conserved regions of the HIV-1 genome. The developed guide RNAs make it possible to detect single copies of HIV-1 proviral DNA in vitro as part of CRISPR/CAS ribonucleoprotein complexes in biological samples after preliminary amplification.

In vitro binding activity between HCK SH3 domain and HIV-1 Nef protein

2011 ◽  
Vol 31 (3) ◽  
pp. 262-265
Author(s):  
Xiao-lin QIN ◽  
Chao-qi LIU ◽  
Dong-ming REN ◽  
Yong-qin ZHOU
Keyword(s):  
Sh3 Domain ◽  
Binding Activity ◽  
Hiv 1
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