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1
Popov, Yu A., and N. I. Mikshis. "Genetic (DNA) Vaccines." Problems of Particularly Dangerous Infections, no. 3(105) (June 20, 2010): 20–24. http://dx.doi.org/10.21055/0370-1069-2010-3(105)-20-24.
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With the development of various branches of medicine and biology the classical ideas about means to prevent infectious diseases have changed. Nowadays in different countries of the world, investigations are carried out intensively in the sphere of genetic vaccines. Distinctive feature of DNA-vaccination is long lasted expression in eukaryotic cell cytoplasm of nucleic acids encoding synthesis of immunogenic proteins. Genetic vaccines induce both humoral and cellular responses accompanied by production of large pool of immunological memory cells. A number of questions regarding features of gene-engineered construction and transfer of DNA-vaccines into the cells of macroorganism, structure of DNA-vaccines and mechanisms of immune response generation are considered in the review. Attention is paid on the safety of gene vaccination and ways to improve its efficiency.
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Starostina, E. V., S. V. Sharabrin, A. P. Rudometov, V. R. Litvinova, M. B. Borgoyakova, S. I. Bazhan, A. A. Ilyichev, and L. I. Karpenko. "Immune response against DNA- and mRNA vaccines encoding artificial influenza virus immunogens." Russian Journal of Immunology 25, no. 3 (September 20, 2022): 321–26. http://dx.doi.org/10.46235/1028-7221-1103-ira.
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Constant antigenic drift of circulating influenza viruses leads to inefficiency of seasonal influenza vaccines, thus requiring annual re-design of these vaccines. Therefore, the development of a universal influenza vaccine is of particular relevance. A promising line of research in this area is to design the immunogens consisting of conserved protein fragments from different influenza viral strains. The aim of this work was to assess immunogenicity of DNA vaccines and mRNA vaccines encoding artificial antigens consisting of conserved hemagglutinin stem fragments and conserved M2 protein. We have obtained DNA vaccine constructs encoding artificial immunogens AgH1, AgH3, and AgM2, which contained conserved fragments of the hemagglutinin stalk from the two subtypes of influenza A H1N1 and H3N2, and conserved M2 protein. These DNA vaccines were used as templates for the synthesis of mRNA vaccines. To assess immunogenicity of the obtained constructs, BALB/c mice were immunized with DNA and mRNA vaccines by i/m administration. Assessment of the humoral immune response was carried out by ELISA, using influenza viruses A/Aichi/2/68(H3N2), A/California/07/2009 as antigens and the ULTRIX vaccine containing purified antigens of H1N1 and H3N2 influenza viruses. T cell immune response was assessed using two methods: intracellular cytokine staining (ICS) and ELISpot. ICS was performed to determine CD8+ and CD4+T-lymphocytes producing IFN. ELISpot was carried out using the mouse IFN ELISpot kit (BD). A peptide mixture which included composition of the target antigens, was used for cell stimulation. The results showed that the designed DNA vaccine constructs induce virus-specific humoral and cellular responses in immunized BALB/c mice. Intramuscular administration of the naked mRNA vaccine constructs induced a weak humoral immune response, thus suggesting a need for further work to improve the delivery approaches.
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Kravchenko, L. M., K. V. Kudzin, and U. A. Prakulevich. "Design of genetic construction for creation DNA vaccine against porcine reproductive and respiratory syndrome." Proceedings of the National Academy of Sciences of Belarus, Biological Series 63, no. 4 (October 30, 2018): 419–25. http://dx.doi.org/10.29235/1029-8940-2018-63-4-419-425.
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The porcine reproductive and respiratory syndrome (PRRS) caused the serious economic damage to swine breeding around the world. It is a viral infective disease against which live attenuated and inactivated vaccines are not always successful. Development of new types of drugs such as DNA vaccines is necessary for improving the protection against the virus. DNA vaccines induce the development of both a cellular and humoral immune response. Such vaccines consist of a plasmid or viral vector with genes of potentially immunogenic proteins. The expression of these genes realized in cells of the vaccinated animal. It leads to the synthesis of antigen proteins triggering the immune response. The purpose of this work is to create a genetic construction that can be used as DNA vaccine against PRRS virus. The construction consists of the commercial vector pVAX1 and open reading frame of two structural proteins of PRRS virus, a lysosomal localization signal sequence of the invariant chain gene and regulatory elements necessary for the expression of cloned genes in mammalian cells.
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Ilyichev, A. A., L. A. Orlova, S. V. Sharabrin, and L. I. Karpenko. "mRNA technology as one of the promising platforms for the SARS-CoV-2 vaccine development." Vavilov Journal of Genetics and Breeding 24, no. 7 (December 6, 2020): 802–7. http://dx.doi.org/10.18699/vj20.676.
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After the genome sequence of SARS-CoV-2 (Severe acute respiratory syndrome-related coronavirus 2) was published and the number of infected people began to increase rapidly, many global companies began to develop a vaccine. Almost all known approaches to vaccine design were applied for this purpose, including inactivated viruses, mRNA and DNA-vaccines, vaccines based on various viral vectors, synthetically generated peptides and recombinant proteins produced in cells of insects and mammals. This review considers one of the promising vaccine platforms based on messenger RNA. Until recent years, mRNA-vaccination was out of practical implementation due to high sensitivity to nuclease degradation and consequent instability of drugs based on mRNA. Latest technological advances significantly mitigated the problems of low immunogenicity, instability, and difficulties in RNA-vaccine delivery. It is worth noting that mRNA-vaccines can efficiently activate both components of the immune system, i. e. T-cell and humoral responses. The essential advantage of mRNA-vaccines includes fast, inexpensive, scalable and uniform production providing a large output of desirable products in vitro. Synthesis and purification processes significantly simplify the process technology of mRNA drugs with injectable purity. Thus, mRNA production via in vitro transcription is more advantageous as compared with DNA-vaccines since it is a chemical process without the use of cells. mRNA techniques make it possible to pass all the phases of vaccine development much faster in comparison with the production of vaccines based on inactivated viruses or recombinant proteins. This property is critically important when designing vaccines against viral pathogens as the main problem of disease control includes a time gap between an epidemic and vaccine development. This paper discusses studies on the development of vaccines against coronaviruses including SARS-CoV-2 with special attention to the mRNA technique.
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Bazhan, Sergei I., Denis V. Antonets, Larisa I. Karpenko, Svetlana F. Oreshkova, Olga N. Kaplina, Ekaterina V. Starostina, Sergei G. Dudko, Sofia A. Fedotova, and Alexander A. Ilyichev. "In silico Designed Ebola Virus T-Cell Multi-Epitope DNA Vaccine Constructions Are Immunogenic in Mice." Vaccines 7, no. 2 (March 29, 2019): 34. http://dx.doi.org/10.3390/vaccines7020034.
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Background: The lack of effective vaccines against Ebola virus initiates a search for new approaches to overcoming this problem. The aim of the study was to design artificial polyepitope T-cell immunogens––candidate DNA vaccines against Ebola virus and to evaluate their capacity to induce a specific immune response in a laboratory animal model. Method: Design of two artificial polyepitope T-cell immunogens, one of which (EV.CTL) includes cytotoxic and the other (EV.Th)––T-helper epitopes of Ebola virus proteins was carried out using original TEpredict/PolyCTLDesigner software. Synthesized genes were cloned in pcDNA3.1 plasmid vector. Target gene expression was estimated by synthesis of specific mRNAs and proteins in cells transfected with recombinant plasmids. Immunogenicity of obtained DNA vaccine constructs was evaluated according to their capacity to induce T-cell response in BALB/c mice using IFNγ ELISpot and ICS. Results: We show that recombinant plasmids pEV.CTL and pEV.Th encoding artificial antigens provide synthesis of corresponding mRNAs and proteins in transfected cells, as well as induce specific responses both to CD4+ and CD8+ T-lymphocytes in immunized animals. Conclusions: The obtained recombinant plasmids can be regarded as promising DNA vaccine candidates in future studies of their capacity to induce cytotoxic and protective responses against Ebola virus.
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Serra, Ana Sofia, Dalinda Eusébio, Ana Raquel Neves, Tânia Albuquerque, Himanshu Bhatt, Swati Biswas, Diana Costa, and Ângela Sousa. "Synthesis and Characterization of Mannosylated Formulations to Deliver a Minicircle DNA Vaccine." Pharmaceutics 13, no. 5 (May 7, 2021): 673. http://dx.doi.org/10.3390/pharmaceutics13050673.
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DNA vaccines still represent an emergent area of research, giving rise to continuous progress towards several biomedicine demands. The formulation of delivery systems to specifically target mannose receptors, which are overexpressed on antigen presenting cells (APCs), is considered a suitable strategy to improve the DNA vaccine immunogenicity. The present study developed binary and ternary carriers, based on polyethylenimine (PEI), octa-arginine peptide (R8), and mannose ligands, to specifically deliver a minicircle DNA (mcDNA) vaccine to APCs. Systems were prepared at various nitrogen to phosphate group (N/P) ratios and characterized in terms of their morphology, size, surface charge, and complexation capacity. In vitro studies were conducted to assess the biocompatibility, cell internalization ability, and gene expression of formulated carriers. The high charge density and condensing capacity of both PEI and R8 enhance the interaction with the mcDNA, leading to the formation of smaller particles. The addition of PEI polymer to the R8-mannose/mcDNA binary system reduces the size and increases the zeta potential and system stability. Confocal microscopy studies confirmed intracellular localization of targeting systems, resulting in sustained mcDNA uptake. Furthermore, the efficiency of in vitro transfection can be influenced by the presence of R8-mannose, with great implications for gene expression. R8-mannose/PEI/mcDNA ternary systems can be considered valuable tools to instigate further research, aiming for advances in the DNA vaccine field.
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Mir, Iqra, Sania Aamir, Syed Rizwan Hussain Shah, Muhammad Shahid, Iram Amin, Samia Afzal, Amjad Nawaz, Muhammad Umer Khan, and Muhammad Idrees. "Immune-related therapeutics: an update on antiviral drugs and vaccines to tackle the COVID-19 pandemic." Osong Public Health and Research Perspectives 13, no. 2 (April 30, 2022): 84–100. http://dx.doi.org/10.24171/j.phrp.2022.0024.
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The coronavirus disease 2019 (COVID-19) pandemic rapidly spread globally. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19, is a positive-sense single-stranded RNA virus with a reported fatality rate ranging from 1% to 7%, and people with immune-compromised conditions, children, and older adults are particularly vulnerable. Respiratory failure and cytokine storm-induced multiple organ failure are the major causes of death. This article highlights the innate and adaptive immune mechanisms of host cells activated in response to SARS-CoV-2 infection and possible therapeutic approaches against COVID-19. Some potential drugs proven to be effective for other viral diseases are under clinical trials now for use against COVID-19. Examples include inhibitors of RNA-dependent RNA polymerase (remdesivir, favipiravir, ribavirin), viral protein synthesis (ivermectin, lopinavir/ritonavir), and fusion of the viral membrane with host cells (chloroquine, hydroxychloroquine, nitazoxanide, and umifenovir). This article also presents the intellectual groundwork for the ongoing development of vaccines in preclinical and clinical trials, explaining potential candidates (live attenuated-whole virus vaccines, inactivated vaccines, subunit vaccines, DNA-based vaccines, protein-based vaccines, nanoparticle-based vaccines, virus-like particles and mRNA-based vaccines). Designing and developing an effective vaccine (both prophylactic and therapeutic) would be a long-term solution and the most effective way to eliminate the COVID-19 pandemic.
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Zadorozhny, Aleksey M., Maria B. Borgoyakova, Ekaterina V. Starostina, and Larisa I. Karpenko. "Cellular immune response to combined DNA-protein constructs carrying SARS-CoV-2 antigens." Medical academic journal 2, no. 2 (November 6, 2022): 177–81. http://dx.doi.org/10.17816/maj108654.
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BACKGROUND: One approach that makes it possible to create vaccines relatively quickly and inexpensively is the creation of DNA vaccine constructs. They are of particular interest for the prevention of COVID-19, as they induce both types of immune response humoral and T cell. Previously, we have created two DNA vaccines based on the pVAX vector: one encodes the full gene of the S protein of the SARS-CoV-2 virus, the second only encodes the receptor-binding domain (RBD) of the S protein of the SARS-CoV-2 virus. Next, the naked DNA was wrapped in a polycationic polyglucin-spermidine complex conjugated with the RBD protein. The resulting combined DNA-protein constructs were named CCV-RBD and CCV-S, respectively.
AIM: To investigate the induction of the T cell immune response of the developed combined DNA protein candidate vaccines in an animal model.
MATERIALS AND METHODS: BALB/c mice were immunized with constructs CCV-RBD and CCV-S, after which their spleens were removed from which splenocytes were isolated. The cellular response was assessed by the ability of splenocytes to secrete cytokines in response to stimulation with viral peptides. The intensity of the response was recorded using the intracellular cytokine staining (ICS) method using flow cytometry.
RESULTS: BALB/c mice were immunized twice with an interval of three weeks with a dose of 100 g of DNA (8 animals per group): 1) CCV-RBD 2) CCV-S and 3) intact animals. It has been shown that both T helper lymphocytes (CD4+) and cytotoxic lymphocytes (CD8+) of animals immunized with CCV-S and CCV-RBD respond with the release of cytokines in response to stimulation with viral peptides.
CONCLUSIONS: In the case of CCV-RBD, a trend towards a higher response in both CD4+ and CD8+ was observed compared to the CCV-S group. Possibly, this difference may be due to more efficient synthesis of the RBD protein than the S protein, providing a DNA vaccine.
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Samantaray, Utkalendu Suvendusekhar, and Rudra Prasad Khuntia. "mRNA vaccines against emerging infectious diseases; A challenging approach of novel vaccine discovery." International Journal of Medical, Pharmacy and Drug Research 6, no. 2 (2022): 52–57. http://dx.doi.org/10.22161/ijmpd.6.2.7.
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Basic human biology is dealt with by mRNA, which creates instructions for making proteins that may aid in the fight against infectious illnesses using our bodies' own mechanisms. mRNA therapies are neither tiny compounds nor huge biological such as recombinant proteins or monoclonal antibodies. These are a series of instructions that assist our cells' machinery in producing proteins that protect us against a certain virus. Our bodies would be unable to perform their activities if mRNA was not introduced. mRNA, or messenger ribonucleic acid, is an important component of the living world, especially in the process of protein synthesis. mRNA is a single-stranded molecule that transmits genetic instructions from a cell's nucleus DNA to the ribosomes, which are the cell's protein-making machinery. The synthesis of an RNA copy from the coded sequence of DNA leads in the production of a particular protein. This copy of mRNA moves from the nucleus of the cell to the cytoplasm, where ribosomes reside. Ribosomes are a sort of sophisticated machinery organelle that aids and begins protein synthesis in cells. Ribosomes ‘read' the mRNA sequence and follow the instructions, progressively adding on various needed amino acids to make the intended protein during the translation process. The protein is subsequently expressed by the cell, and it goes on to execute its role in the cell or in the body. The use of mRNA as a medication offers up a whole new universe of possibilities in terms of illness treatment and prevention. This review contributes to the growing body of knowledge in the field of mRNA therapeutic delivery and the identification of appropriate antigens for mRNA target locations. Two major mRNA vaccines for protection against SARS-CoV-2 have recently been developed and approved for use in the general population by international health authorities. They've been demonstrated to defend against the SARS-CoV-2 virus, which is still active and evolving. This will draw attention to a variety of mRNA vaccines now being evaluated for infectious diseases in clinical studies. mRNA vaccines offer a number of advantages, including speedy design, fabrication, manufacturing, and administration, and they hold a lot of potential for future use against a wide range of diseases.
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Dmitrieva, M. V., M. A. Baryshnikovа, O. L. Orlova, and V. S. Kosorukov. "Technological aspects of creating neopeptide vaccines." Russian Journal of Biotherapy 21, no. 4 (December 10, 2022): 10–21. http://dx.doi.org/10.17650/1726-9784-2022-21-4-10-21.
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Personalized neoantigen vaccines are a group of individually designed cancer vaccines that enhance patients’ own antigen-specific immune responses. These include vaccines based on dendritic cells, DNA, mRNA and synthetic peptides. An analysis of 98 clinical trials of neoantigenic vaccines from the ClinicalTrials.gov database found that peptide vaccines are one of the most popular cancer vaccines, accounting for about 50 % of clinical trials. They usually consist of a mixture of long or short peptides, dissolved depending on their properties in an appropriate solvent, and an adjuvant that stabilizes and increases their effectiveness. The most used immunoadjuvants in the formulation of neopeptide vaccines are Toll-like receptor agonists (poly-ICLC) and granulocyte-macrophage colony-stimulating factor. The development of neoantigenic vaccines presents a number of distinctive challenges compared to other types of vaccines. The process should cover and validate the various steps in the development, production and administration processes in order to maximize the efficacy and safety of vaccines. In the technology for the production of peptide vaccines, 3 main stages can be distinguished: 1) screening and identification of neoepitopes using the approaches of computer prediction, co-immunoprecipitation, mass spectrometry and cytotoxic experiments; 2) synthesis of peptides by methods of standard solid-phase synthetic peptide chemistry; 3) actually obtaining a vaccine preparation suitable for storage, transportation and administration to the patient. Taking into account the specificity of the drug, the manufacturing process must be carried out strictly according to the Good Manufacturing Practice standard with mandatory quality control of intermediate and finished products
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Kim, Carol H., Marc C. Johnson, John D. Drennan, Benjamin E. Simon, Estela Thomann, and Jo-Ann C. Leong. "DNA Vaccines Encoding Viral Glycoproteins Induce Nonspecific Immunity and Mx Protein Synthesis in Fish." Journal of Virology 74, no. 15 (August 1, 2000): 7048–54. http://dx.doi.org/10.1128/jvi.74.15.7048-7054.2000.
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ABSTRACT Protective immunity by vaccination with plasmid DNA encoding a viral glycoprotein (G) has long been assumed to result from the induction of a specific immune response. We report here that the initial protection may be due to the induction of alpha/beta interferon, with long-term protection due to a specific response to the encoded viral G. DNA vaccines encoding the Gs of three serologically unrelated fish rhabdoviruses were used to vaccinate rainbow trout against a lethal challenge with infectious hematopoietic necrosis virus (IHNV). All three vaccines, each encoding the G gene of either IHNV (IHNV-G), snakehead rhabdovirus (SHRV) (SHRV-G), or spring viremia of carp virus (SVCV) (SVCV-G), elicited protective immunity against IHNV. Vaccinated fish were challenged at 30 or 70 days postvaccination with lethal doses of IHNV. At 30 days postvaccination, only 5% of fish that had received any of the G vaccines died, whereas more than 50% of the control fish succumbed to virus challenge. When fish were vaccinated and challenged at 70 days postvaccination, only 12% of the IHNV-G-vaccinated fish died compared to 68% for the SHRV-G- and 76% for the SVCV-G-vaccinated fish. Assays for trout Mx protein, an indicator of alpha/beta interferon induction, showed that only fish vaccinated with a G-containing plasmid produced high levels of Mx protein in the kidneys and liver. Interestingly, at day 7 after virus challenge, all of the fish vaccinated with the IHNV-G plasmid were negative for Mx, but the SHRV-G- and SVCV-G-vaccinated fish still showed detectable levels of Mx. These results suggest that DNA vaccines in fish induce an early, nonspecific antiviral protection mediated by an alpha/beta interferon and, later, a specific immune response.
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Bazhan, Sergei, Denis Antonets, Ekaterina Starostina, Tatyana Ilyicheva, Olga Kaplina, Vasiliy Marchenko, Alexander Durymanov, Svetlana Oreshkova, and Larisa Karpenko. "Immunogenicity and Protective Efficacy of Influenza A DNA Vaccines Encoding Artificial Antigens Based on Conservative Hemagglutinin Stem Region and M2 Protein in Mice." Vaccines 8, no. 3 (August 9, 2020): 448. http://dx.doi.org/10.3390/vaccines8030448.
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Background: Development of a universal vaccine capable to induce antibody responses against a broad range of influenza virus strains attracts growing attention. Hemagglutinin stem and the exposed fragment of influenza virus M2 protein are promising targets for induction of cross-protective humoral and cell-mediated response, since they contain conservative epitopes capable to induce antibodies and cytotoxic T lymphocytes (CTLs) to a wide range of influenza virus subtypes. Methods: In this study, we generated DNA vaccine constructs encoding artificial antigens AgH1, AgH3, and AgM2 designed on the basis of conservative hemagglutinin stem fragments of two influenza A virus subtypes, H1N1 and H3N2, and conservative M2 protein, and evaluate their immunogenicity and protective efficacy. To obtain DNA vaccine constructs, genes encoding the designed antigens were cloned into a pcDNA3.1 vector. Expression of the target genes in 293T cells transfected with DNA vaccine constructs has been confirmed by synthesis of specific mRNA. Results: Immunization of BALB/c mice with DNA vaccines encoding these antigens was shown to evoke humoral and T-cell immune responses as well as a moderated statistically significant cross-protective effect against two heterologous viruses A/California/4/2009 (H1N1pdm09) and A/Aichi/2/68 (H3N2). Conclusions: The results demonstrate a potential approach to creating a universal influenza vaccine based on artificial antigens.
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Francis, Jasmine E., Ivana Skakic, Chaitali Dekiwadia, Ravi Shukla, Aya C. Taki, Anna Walduck, and Peter M. Smooker. "Solid Lipid Nanoparticle Carrier Platform Containing Synthetic TLR4 Agonist Mediates Non-Viral DNA Vaccine Delivery." Vaccines 8, no. 3 (September 21, 2020): 551. http://dx.doi.org/10.3390/vaccines8030551.
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There is a growing demand for better delivery systems to improve the stability and efficacy of DNA vaccines. Here we report the synthesis of a non-viral DNA vaccine delivery system using a novel adjuvanted solid lipid nanoparticle (SLN-A) platform as a carrier for a DNA vaccine candidate encoding the Urease alpha (UreA) antigen from Helicobacter pylori. Cationic SLN-A particles containing monophosphoryl lipid A (adjuvant) were synthesised by a modified solvent-emulsification method and were investigated for their morphology, zeta potential and in vitro transfection capacity. Particles were found to bind plasmid DNA to form lipoplexes, which were characterised by electron microscopy, dynamic light scattering and fluorescence microscopy. Cellular uptake studies confirmed particle uptake within 3 h, and intracellular localisation within endosomal compartments. In vitro studies further confirmed the ability of SLN-A particles to stimulate expression of pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) in human macrophage-like Tohoku Hospital Pediatrics-1 (THP-1) cells. Lipoplexes were found to be biocompatible and could be efficiently transfected in murine immune cells for expression of recombinant H. pylori antigen Urease A, demonstrating their potential as a DNA vaccine delivery system.
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MAJID, MARINA ABDUL. "A GENE SYNTHESIS REGIME FOR MALAYSIA TO EMULATE IN SECURING FUTURE BIOPRINTED VACCINES." MALIM: JURNAL PENGAJIAN UMUM ASIA TENGGARA (SEA JOURNAL OF GENERAL STUDIES) 21, no. 1 (November 10, 2020): 79–101. http://dx.doi.org/10.17576/malim-2020-2101-07.
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The merger between synthetic biology and bioprinting will someday enable vaccines to be bioprinted utilising genetic material. Unregulated gene synthesis companies may unwittingly supply genetic material to a terrorist if there is no verification of purchasers’ personal identity and affiliation with a legitimate research organisation. This study has the objective of focusing on whether Malaysia regulates and conducts Deoxyribonucleic Acid (DNA) sequence screening among gene synthesis companies which are meant for bioprinting vaccines that can be misused for bioterrorism. This study is qualitative. Gene synthesis guidelines from the United States (US), the International Gene Synthesis Consortium (IGSC), the Nuclear Threat Initiative-World Economic Forum (NTI-WEF) Biosecurity Innovation and Risk Reduction report were referred as examples for changes in Malaysia’s draft National Code of Conduct for Biosecurity [thereafter known as Code]. These soft law documents constitute the regime for gene synthesis and a form of transnational new governance. The findings indicate that in the absence of a specific binding regulation, Malaysia’s draft Code must be amended to incorporate the need to screen customers, genetic sequences and address the cyberbiosecurity of biological life in digitalised form besides the physical biosecurity of laboratories which houses seedstocks from being stolen for malicious intent.
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Starostina, Ekaterina V., Sergei V. Sharabrin, Denis N. Antropov, Grigory A. Stepanov, Georgiy Yu Shevelev, Anna E. Lemza, Andrey P. Rudometov, et al. "Construction and Immunogenicity of Modified mRNA-Vaccine Variants Encoding Influenza Virus Antigens." Vaccines 9, no. 5 (May 3, 2021): 452. http://dx.doi.org/10.3390/vaccines9050452.
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Nucleic acid-based influenza vaccines are a promising platform that have recently and rapidly developed. We previously demonstrated the immunogenicity of DNA vaccines encoding artificial immunogens AgH1, AgH3, and AgM2, which contained conserved fragments of the hemagglutinin stem of two subtypes of influenza A—H1N1 and H3N2—and conserved protein M2. Thus, the aim of this study was to design and characterize modified mRNA obtained using the above plasmid DNA vaccines as a template. To select the most promising protocol for creating highly immunogenic mRNA vaccines, we performed a comparative analysis of mRNA modifications aimed at increasing its translational activity and decreasing toxicity. We used mRNA encoding a green fluorescent protein (GFP) as a model. Eight mRNA-GFP variants with different modifications (M0–M7) were obtained using the classic cap(1), its chemical analog ARCA (anti-reverse cap analog), pseudouridine (Ψ), N6-methyladenosine (m6A), and 5-methylcytosine (m5C) in different ratios. Modifications M2, M6, and M7, which provided the most intensive fluorescence of transfected HEK293FT cells were used for template synthesis when mRNA encoded influenza immunogens AgH1, AgH3, and AgM2. Virus specific antibodies were registered in groups of animals immunized with a mix of mRNAs encoding AgH1, AgH3, and AgM2, which contained either ARCA (with inclusions of 100% Ψ and 20% m6A (M6)) or a classic cap(1) (with 100% substitution of U with Ψ (M7)). M6 modification was the least toxic when compared with other mRNA variants. M6 and M7 RNA modifications can therefore be considered as promising protocols for designing mRNA vaccines.
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Chiu, Sheng-Kang, Kuo-Wang Tsai, Chia-Chao Wu, Cai-Mei Zheng, Chung-Hsiang Yang, Wan-Chung Hu, Yi-Chou Hou, Kuo-Cheng Lu, and You-Chen Chao. "Putative Role of Vitamin D for COVID-19 Vaccination." International Journal of Molecular Sciences 22, no. 16 (August 20, 2021): 8988. http://dx.doi.org/10.3390/ijms22168988.
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Severe acute respiratory syndrome coronavirus 2 is a new, highly pathogenic virus that has recently elicited a global pandemic called the 2019 coronavirus disease (COVID-19). COVID-19 is characterized by significant immune dysfunction, which is caused by strong but unregulated innate immunity with depressed adaptive immunity. Reduced and delayed responses to interferons (IFN-I/IFN-III) can increase the synthesis of proinflammatory cytokines and extensive immune cell infiltration into the airways, leading to pulmonary disease. The development of effective treatments for severe COVID-19 patients relies on our knowledge of the pathophysiological components of this imbalanced innate immune response. Strategies to address innate response factors will be essential. Significant efforts are currently underway to develop vaccines against SARS-CoV-2. COVID-19 vaccines, such as inactivated DNA, mRNA, and protein subunit vaccines, have already been applied in clinical use. Various vaccines display different levels of effectiveness, and it is important to continue to optimize and update their composition in order to increase their effectiveness. However, due to the continuous emergence of variant viruses, improving the immunity of the general public may also increase the effectiveness of the vaccines. Many observational studies have demonstrated that serum levels of vitamin D are inversely correlated with the incidence or severity of COVID-19. Extensive evidence has shown that vitamin D supplementation could be vital in mitigating the progression of COVID-19 to reduce its severity. Vitamin D defends against SARS-CoV-2 through a complex mechanism through interactions between the modulation of innate and adaptive immune reactions, ACE2 expression, and inhibition of the renin-angiotensin system (RAS). However, it remains unclear whether Vit-D also plays an important role in the effectiveness of different COVID-19 vaccines. Based on analysis of the molecular mechanism involved, we speculated that vit-D, via various immune signaling pathways, plays a complementary role in the development of vaccine efficacy.
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Miggiano, Riccardo, Castrese Morrone, Franca Rossi, and Menico Rizzi. "Targeting Genome Integrity in Mycobacterium Tuberculosis: From Nucleotide Synthesis to DNA Replication and Repair." Molecules 25, no. 5 (March 7, 2020): 1205. http://dx.doi.org/10.3390/molecules25051205.
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Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis (TB), an ancient disease which still today causes 1.4 million deaths worldwide per year. Long-term, multi-agent anti-tubercular regimens can lead to the anticipated non-compliance of the patient and increased drug toxicity, which in turn can contribute to the emergence of drug-resistant MTB strains that are not susceptible to first- and second-line available drugs. Hence, there is an urgent need for innovative antitubercular drugs and vaccines. A number of biochemical processes are required to maintain the correct homeostasis of DNA metabolism in all organisms. Here we focused on reviewing our current knowledge and understanding of biochemical and structural aspects of relevance for drug discovery, for some such processes in MTB, and particularly DNA synthesis, synthesis of its nucleotide precursors, and processes that guarantee DNA integrity and genome stability. Overall, the area of drug discovery in DNA metabolism appears very much alive, rich of investigations and promising with respect to new antitubercular drug candidates. However, the complexity of molecular events that occur in DNA metabolic processes requires an accurate characterization of mechanistic details in order to avoid major flaws, and therefore the failure, of drug discovery approaches targeting genome integrity.
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Lim, JaeHyun, Steven C. Derrick, Kristopher Kolibab, Amy Li Yang, Steven Porcelli, William R. Jacobs, and Sheldon L. Morris. "Early Pulmonary Cytokine and Chemokine Responses in Mice Immunized with Three Different Vaccines against Mycobacterium tuberculosis Determined by PCR Array." Clinical and Vaccine Immunology 16, no. 1 (November 26, 2008): 122–26. http://dx.doi.org/10.1128/cvi.00359-08.
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ABSTRACT In this study, the early pulmonary cytokine and chemokine responses in mice immunized with either BCG vaccine, a ΔsecA2 mutant of Mycobacterium tuberculosis, or a DNA vaccine expressing an ESAT6-antigen 85B fusion protein and then aerogenically challenged with a low dose of M. tuberculosis were evaluated by PCR array. The cellular immune responses at day 10 postchallenge were essentially equivalent in the lungs of mice immunized with either the highly immunogenic BCG vaccine or the ΔsecA2 M. tuberculosis mutant strain. Specifically, 12 immune biomolecules (including gamma interferon [IFN-γ], interleukin-21 [IL-21], IL-27, IL-17f, CXCL9, CXCL10, and CXCL11) were differentially regulated, relative to the levels for naïve controls, in the lungs of vaccinated mice at this time point. Although the vaccine-related immune responses evoked in mice immunized with the DNA vaccine were relatively limited at 10 days postinfection, upregulation of IFN-γ RNA synthesis as well as increased expression levels of CXCL9, CXCL10, and CXCL11 chemokines were detected.
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Pradhananga, Sarbendra, and Jon R. Sayers. "Natural synthesis: Biologics, biosimilars and biobetters in protein hormone therapy." Biochemist 34, no. 1 (February 1, 2012): 10–15. http://dx.doi.org/10.1042/bio03401010.
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Hormone therapies have been used since the early 20th Century and belong to a group of drugs that has recently become known as ‘biologics’. Biologics are medicinal products that have been produced by biological processes as opposed to chemically synthesized drugs. The term biologics spans a wide range of products that include therapeutics such as organs, tissue, cells, blood or blood components, vaccines and proteins. This ‘proteins’ subgroup can be further subdivided into therapeutics such as antibodies, enzymes and hormones. The first hormone therapeutics were extracted from human or animal sources; however, with the advent and development of cloning and protein production technologies from the late-20th Century onwards, protein hormone therapeutics are now produced by recombinant DNA technology.
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Miskovsky, E. P., A. Y. Liu, W. Pavlat, R. Viveen, P. E. Stanhope, D. Finzi, W. M. Fox, R. H. Hruban, E. R. Podack, and R. F. Siliciano. "Studies of the mechanism of cytolysis by HIV-1-specific CD4+ human CTL clones induced by candidate AIDS vaccines." Journal of Immunology 153, no. 6 (September 15, 1994): 2787–99. http://dx.doi.org/10.4049/jimmunol.153.6.2787.
Full textAbstract:
Abstract Vaccine-induced, virus-specific CTLs may rapidly eliminate the host cells that first become infected after virus exposure, thereby preventing disseminated infection. Thus, there is much interest in the ability of candidate AIDS vaccines to elicit CTLs. All HIV-1 envelope (env) protein-based vaccines tested to date in seronegative humans induce CTLs from the CD4+ subset. Because the mechanism of cytolysis by CD4+ CTLs is controversial, a detailed study of the cytolytic reactions mediated by vaccine-induced, HIV-1-specific human CD4+ CTL clones was conducted. CD4+ CTL clones induced rapid destruction of Ag-pulsed target cells. Lysis was readily detectable within 15 min. Lysis was not a result of syncytium formation between CD4+ effector cells and env-expressing targets. Target cell destruction was not dependent upon de novo RNA or protein synthesis in either the effector or the target cell. Expression of perforin mRNA was detected by Northern blotting and reverse-transcriptase-PCR in CD4+ CTL clones but not in autologous B lymphoblastoid cell lines. Immunohistochemical studies demonstrated perforin protein in cytoplasmic granules in CD4+ CTL clones. Lysis by CD4+ CTLs was strictly dependent upon extracellular Ca2+ and was highly specific, with no lysis of innocent bystander cells. DNA fragmentation was detectable in target cells, but did not precede 51Cr release. Taken together, these results provide a dramatically different view of cytolysis by human CD4+ CTLs. Target cells are lysed by a rapid and efficient mechanism that involves a preformed mediator and that is functionally similar to the mechanism used by CD8+ CTLs.
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Cai, Wenlong, and Covadonga R. Arias. "Deciphering the Molecular Basis for Attenuation of Flavobacterium columnare Strain Fc1723 Used as Modified Live Vaccine against Columnaris Disease." Vaccines 9, no. 11 (November 22, 2021): 1370. http://dx.doi.org/10.3390/vaccines9111370.
Full textAbstract:
Vaccines are widely employed in aquaculture to prevent bacterial infections, but their use by the U.S. catfish industry is very limited. One of the main diseases affecting catfish aquaculture is columnaris disease, caused by the bacterial pathogen Flavobacterium columnare. In 2011, a modified-live vaccine against columnaris disease was developed by selecting mutants that were resistant to rifampin. The previous study has suggested that this vaccine is stable, safe, and effective, but the mechanisms that resulted in attenuation remained uncharacterized. To understand the molecular basis for attenuation, a comparative genomic analysis was conducted to identify specific point mutations. The PacBio RS long-read sequencing platform was used to obtain draft genomes of the mutant attenuated strain (Fc1723) and the parent virulent strain (FcB27). Sequence-based genome comparison identified 16 single nucleotide polymorphisms (SNP) unique to the mutant. Genes that contained mutations were involved in rifampin resistance, gliding motility, DNA transcription, toxin secretion, and extracellular protease synthesis. The results also found that the vaccine strain formed biofilm at a significantly lower rate than the parent strain. These observations suggested that the rifampin-resistant phenotype and the associated attenuation of the vaccine strain result from the altered activity of RNA polymerase (RpoB) and possible disrupted protein secretion systems.
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Rumyantsev, Andrey M., Mikhail A. Tsygankov, Vladislav V. Veretennikov, Elena V. Sambuk, and Marina V. Padkina. "Heterologous synthesis of N and M fragments of capsid protein VP2 of avian infectious bursal disease virus in yeast Pichia pastoris." Ecological genetics 20, no. 1 (May 20, 2022): 49–59. http://dx.doi.org/10.17816/ecogen83441.
Full textAbstract:
BACKGROUND: Infectious bursal disease is one of the most common and economically important viral diseases of birds. Vaccination is currently the most effective way to control IBD. Subunit vaccines contain only the immunogenic protein of the pathogen or its fragments, but do not contain other proteins, lipopolysaccharides, toxins, which avoids vaccination side effects.
AIM: The aim of the work was to obtain yeast Pichia pastoris strains that synthesize and secrete the fragments of major coat protein VP2 of the infectious bursal disease virus.
MATERIALS AND METHODS: The DNA sequences encoding the N and M fragments of VP2 protein, were cloned under the control of the AOX1 gene promoter and integrated into the genome of P. pastoris strains X-33 (mut+) and GS115 (his4).
RESULTS: The analysis of proteins secreted by the obtained strains revealed the presence of additional proteins with a molecular weights corresponding to the target proteins.
CONCLUSIONS: Thus, the obtained strains of P. pastoris producers of N and M fragments of VP2 protein can be used for antigen production to create a subunit vaccine against avian IBD.
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Hahm, Heung Sik, Mark K. Schlegel, Mattan Hurevich, Steffen Eller, Frank Schuhmacher, Johanna Hofmann, Kevin Pagel, and Peter H. Seeberger. "Automated glycan assembly using the Glyconeer 2.1 synthesizer." Proceedings of the National Academy of Sciences 114, no. 17 (April 10, 2017): E3385—E3389. http://dx.doi.org/10.1073/pnas.1700141114.
Full textAbstract:
Reliable and rapid access to defined biopolymers by automated DNA and peptide synthesis has fundamentally altered biological research and medical practice. Similarly, the procurement of defined glycans is key to establishing structure–activity relationships and thereby progress in the glycosciences. Here, we describe the rapid assembly of oligosaccharides using the commercially available Glyconeer 2.1 automated glycan synthesizer, monosaccharide building blocks, and a linker-functionalized polystyrene solid support. Purification and quality-control protocols for the oligosaccharide products have been standardized. Synthetic glycans prepared in this way are useful reagents as the basis for glycan arrays, diagnostics, and carbohydrate-based vaccines.
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Nersesyan, Armen, Hairapet Galstyan, and Robert Adamyan. "Tularemia live vaccine as a modifier of genotoxic and carcinogenic action of environmental factors." Archive of Oncology 10, no. 4 (2002): 273–77. http://dx.doi.org/10.2298/aoo0204273n.
Full textAbstract:
In this review we presented the data concerning the influence of tularemia live vaccine (TLV, produced in the USSR and Russia) on mutagenesis and carcinogenesis. The data show that rodents immunized with TLV are resistant to mutagen of any origin (chemical, physical, biological) independently of species (rats, mice, gray hamsters, guinea pigs). Exposure of immunized rodents to carcinogens decreased the incidence of tumors, prolonged the mean latency period of tumor development, and decreased the mean tumor weight. The most pronounced effect was observed when rodents were exposed to mutagens/carcinogens on day 15 after immunization. This universal antimutagenic/anticarcinogenic effect is associated with increased resistance of DNA of immunized rodents to genotoxic action of environmental agents, the influence on the process of metabolic activation of xenobiotics, and increased synthesis of interferon, catalase and superoxide dismutase. The problem of influence of vaccines against dangerous infections on mutagenesis and carcinogenesis is a topical issue because the best way of protection against dangerous infections in case of epidemic or terrorist act is vaccination.
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Giannios, J., J. Peristeris, N. Alexandropoulos, T. Kononas, and P. Ginopoulos. "Effect on humoral and cellular immunity and on apoptosis in CIN2, CIN3, and HPV16+ cervical cancer of therapeutic divalent genetic vaccination with CMV replicon system (CRS) delivering HPV16 recombinantly mutated E6 and E7 viral oncogenes targeting p53 and Rb, respectively." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): 3062. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.3062.
Full textAbstract:
3062 Background: Prophylactic vaccines have no therapeutic capacity for all the women who are already infected with HPV16 and have developed cervical intraepithelial neoplasia (CIN) or cervical cancer. Approximately 300 million women with CIN and cervical cancer will require therapy in the next decades. Thus, there is a great demand for a therapeutic HPV vaccine. Methods: We developed a cytomegalovirus (CMV) replicon system (CRS) for delivery of the HPV16 recombinantly mutated E6 and E7 genes replacing part of the CMV genome for the HPV genes, which were genetically altered to block binding sites for p53 and Rb.The replicon-vectors infected and co-transfected CIN and cervical cancer cells in animal models derived from HPV16(+) CIN, and cervical Ca cells obtained from patients. The genetic vaccine was administered subcutaneously (SC) with a needless injection system. Results: After vaccination,the viral E6 oncogene did not degrade apoptotic p53, and it blocked activation of telomerase. This induced apoptosis and DNA repair in CIN and cervical cancer cells. Furthermore, the E7 viral oncogene did not degrade the retinoblastoma oncogene (Rb) protein releasing transcription factor E2F. This vaccination led to scheduled cell cycle entry, genetic stability, and mortalization of tumor cells. Humoral and cellular immune responses were exhibited, which led to irreversible D2 apoptotic stage of PCD type I leading to a bystander killing effect of CIN, and cervical cancer cells. BrdU and MTT analysis exhibited inhibition of DNA synthesis and metabolic activity of vaccinated tumor cells compared to controls. Conclusions: This genetic divalent vaccine coding for E6 and E7 mutations designed to prevent p53 and Rb binding sites activated humoral and cellular immune responses leading to apoptosis of CIN and cervical cancer cells. It is up to translational medicine to bring this therapeutic vaccine into the clinic for patients with CIN2, CIN3, and cervical cancer patients. No significant financial relationships to disclose.
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Febriyana, Dwi, Sunarno Sunarno, Yudi Hartoyo, Sundari Nursofiah, Tati Febrianti, Ratih Dian Saraswati, Nelly Puspandari, et al. "Analisis Gen Tox Corynebacterium Diphtheriae Penyebab Difteri di Beberapa Wilayah Indonesia." Buletin Penelitian Kesehatan 49, no. 1 (May 25, 2021): 63–70. http://dx.doi.org/10.22435/bpk.v0i0.3844.
Full textAbstract:
Diphtheria is a vaccine-preventable disease. The clinical features and complications of diphtheria are associated with toxins produced by the causative bacteria. Diphtheria toxin synthesis is encoded by tox gene. This study aimed to provide an overview of the DNA sequences of the tox gene of Corynebacterium diphtheriae causing diphtheria in several region of Indonesia. A total of 65 Corynebacterium diphtheriae isolated from several provinces in Indonesia (2010-2017) were used as samples. Isolates recultured on blood agar medium (BA), incubated at 37 0 C overnight. DNA extraction conducted using the QiaAmp DNA Mini Kit. The DNA sequencing was carried out using the Whole Genome Sequencing (WGS) approach. The data conversion and analysis conducted using U-gene and BioEdit programs. Examination of 65 isolate C. diphtheriae with 1683 bp of tox gene sequences showed that there are 3 patterns of gene sequences with 3 mutation site. All mutations were silent mutation. The mutation sites were also not commonly used as 3’end binding site of the PCR primer. We concluded that tox gene of C. diphtheriae that causes diphtheria in some provinces in Indonesia have limited variations and these variations do not encode amino acid changes. This indicates that the vaccines used in Indonesia are still in accordance with the variations in circulating bacteria and PCR can be used for screening and predicting the toxigenicity of diphtheria-causing bacteria. Keywords: C. diphtheriae, gene tox, diphtheria, Indonesia
Abstrak
Difteri merupakan salah satu penyakit yang dapat dicegah dengan imunisasi (PD3I). Gambaran klinis dan komplikasi difteri dikaitkan dengan toksin yang diproduksi oleh bakteri penyebab. Sintesis toksin difteri dikode oleh gen tox. Penelitian ini bertujuan untuk memberikan gambaran sekuens DNA gen tox Corynebacterium diphtheriae penyebab difteri di beberapa wilayah Indonesia. Sebanyak 65 isolat C. diphtheriae tersimpan milik Badan Litbangkes yang diisolasi dari beberapa wilayah Indonesia tahun 2010- 2017 dijadikan sebagai sampel. Rekultur dilakukan pada medium agar darah (BA), diinkubasi pada suhu 37 o C selama sehari semalam. Ekstraksi DNA menggunakan kit QiaAmp DNA Minikit. Sekuensing DNA dilakukan dengan pendekatan Whole Genome Sequencing (WGS). Konversi dan analisis data menggunakan program U-gene dan BioEdit.Pemeriksaan 65 isolat C. diphtheriae dengan 1683 bp sekuens gen tox menunjukkan ada 3 pola sekuens gen dengan 3 lokasi mutasi. Seluruh mutasi bersifat silent mutation. Lokasi mutasi juga bukan merupakan tempat penempelan ujung 3’ primer PCR yang umum digunakan. Berdasarkan hasil penelitian dapat disimpulkan bahwa variasi gen tox yang ditemukan pada C. diphtheriae penyebab difteri di Indonesia memiliki variasi yang terbatas dan mutasi yang ada tidak mengkode perubahan asam amino. Hal ini mengindikasikan bahwa vaksin yang digunakan di Indonesia masih sesuai dengan variasi bakteri yang bersirkulasi. Hasil penelitian juga mengindikasikan bahwa PCR dapat digunakan untuk skrining dan memprediksi toksigenisitas bakteri penyebab difteri.
Kata kunci : C. diphtheriae, gen tox, difteri, Indonesia
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Febriyana, Dwi, Sunarno Sunarno, Yudi Hartoyo, Sundari Nursofiah, Tati Febrianti, Ratih Dian Saraswati, Nelly Puspandari, et al. "Analisis Gen Tox Corynebacterium Diphtheriae Penyebab Difteri di Beberapa Wilayah Indonesia." Buletin Penelitian Kesehatan 49, no. 1 (May 25, 2021): 63–70. http://dx.doi.org/10.22435/bpk.v49i1.3844.
Full textAbstract:
Diphtheria is a vaccine-preventable disease. The clinical features and complications of diphtheria are associated with toxins produced by the causative bacteria. Diphtheria toxin synthesis is encoded by tox gene. This study aimed to provide an overview of the DNA sequences of the tox gene of Corynebacterium diphtheriae causing diphtheria in several region of Indonesia. A total of 65 Corynebacterium diphtheriae isolated from several provinces in Indonesia (2010-2017) were used as samples. Isolates recultured on blood agar medium (BA), incubated at 37 0 C overnight. DNA extraction conducted using the QiaAmp DNA Mini Kit. The DNA sequencing was carried out using the Whole Genome Sequencing (WGS) approach. The data conversion and analysis conducted using U-gene and BioEdit programs. Examination of 65 isolate C. diphtheriae with 1683 bp of tox gene sequences showed that there are 3 patterns of gene sequences with 3 mutation site. All mutations were silent mutation. The mutation sites were also not commonly used as 3’end binding site of the PCR primer. We concluded that tox gene of C. diphtheriae that causes diphtheria in some provinces in Indonesia have limited variations and these variations do not encode amino acid changes. This indicates that the vaccines used in Indonesia are still in accordance with the variations in circulating bacteria and PCR can be used for screening and predicting the toxigenicity of diphtheria-causing bacteria. Keywords: C. diphtheriae, gene tox, diphtheria, Indonesia
Abstrak
Difteri merupakan salah satu penyakit yang dapat dicegah dengan imunisasi (PD3I). Gambaran klinis dan komplikasi difteri dikaitkan dengan toksin yang diproduksi oleh bakteri penyebab. Sintesis toksin difteri dikode oleh gen tox. Penelitian ini bertujuan untuk memberikan gambaran sekuens DNA gen tox Corynebacterium diphtheriae penyebab difteri di beberapa wilayah Indonesia. Sebanyak 65 isolat C. diphtheriae tersimpan milik Badan Litbangkes yang diisolasi dari beberapa wilayah Indonesia tahun 2010- 2017 dijadikan sebagai sampel. Rekultur dilakukan pada medium agar darah (BA), diinkubasi pada suhu 37 o C selama sehari semalam. Ekstraksi DNA menggunakan kit QiaAmp DNA Minikit. Sekuensing DNA dilakukan dengan pendekatan Whole Genome Sequencing (WGS). Konversi dan analisis data menggunakan program U-gene dan BioEdit.Pemeriksaan 65 isolat C. diphtheriae dengan 1683 bp sekuens gen tox menunjukkan ada 3 pola sekuens gen dengan 3 lokasi mutasi. Seluruh mutasi bersifat silent mutation. Lokasi mutasi juga bukan merupakan tempat penempelan ujung 3’ primer PCR yang umum digunakan. Berdasarkan hasil penelitian dapat disimpulkan bahwa variasi gen tox yang ditemukan pada C. diphtheriae penyebab difteri di Indonesia memiliki variasi yang terbatas dan mutasi yang ada tidak mengkode perubahan asam amino. Hal ini mengindikasikan bahwa vaksin yang digunakan di Indonesia masih sesuai dengan variasi bakteri yang bersirkulasi. Hasil penelitian juga mengindikasikan bahwa PCR dapat digunakan untuk skrining dan memprediksi toksigenisitas bakteri penyebab difteri.
Kata kunci : C. diphtheriae, gen tox, difteri, Indonesia
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Ramachandran, Srividya, Katherine A. Davoli, Michael B. Yee, Robert L. Hendricks, and Paul R. Kinchington. "Delaying the Expression of Herpes Simplex Virus Type 1 Glycoprotein B (gB) to a True Late Gene Alters Neurovirulence and Inhibits the gB-CD8+ T-Cell Response in the Trigeminal Ganglion." Journal of Virology 84, no. 17 (June 23, 2010): 8811–20. http://dx.doi.org/10.1128/jvi.00496-10.
Full textAbstract:
ABSTRACT Following herpes simplex virus type 1 (HSV-1) ocular infection of C57BL/6 mice, activated CD8+ T cells specific for an immunodominant epitope on HSV-1 glycoprotein B (gB-CD8 cells) establish a stable memory population in HSV-1 latently infected trigeminal ganglia (TG), whereas non-HSV-specific CD8+ T cells are lost over time. The retention and activation of gB-CD8 cells appear to be influenced by persistent viral antigenic exposure within the latently infected TG. We hypothesized that the low-level expression of gB from its native promoter before viral DNA synthesis is critical for the retention and activation of gB-CD8 cells in the TG during HSV-1 latency and for their ability to block HSV-1 reactivation from latency. To test this, we created a recombinant HSV-1 in which gB is expressed only after viral DNA synthesis from the true late gC promoter (gCp-gB). Despite minor growth differences compared to its rescuant in infected corneas, gCp-gB was significantly growth impaired in the TG and produced a reduced latent genome load. The gCp-gB- and rescuant-infected mice mounted similar gB-CD8 effector responses, but the size and activation phenotypes of the memory gB-CD8 cells were diminished in gCp-gB latently infected TG, suggesting that the stimulation of gB-CD8 cells requires gB expression before viral DNA synthesis. Surprisingly, late gB expression did not compromise the capacity of gB-CD8 cells to inhibit HSV-1 reactivation from latency in ex vivo TG cultures, suggesting that gB-CD8 cells can block HSV-1 reactivation at a very late stage in the viral life cycle. These data have implications for designing better immunogens for vaccines to prevent HSV-1 reactivation.
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Rice, Alan M., Atahualpa Castillo Morales, Alexander T. Ho, Christine Mordstein, Stefanie Mühlhausen, Samir Watson, Laura Cano, Bethan Young, Grzegorz Kudla, and Laurence D. Hurst. "Evidence for Strong Mutation Bias toward, and Selection against, U Content in SARS-CoV-2: Implications for Vaccine Design." Molecular Biology and Evolution 38, no. 1 (July 20, 2020): 67–83. http://dx.doi.org/10.1093/molbev/msaa188.
Full textAbstract:
Abstract Large-scale re-engineering of synonymous sites is a promising strategy to generate vaccines either through synthesis of attenuated viruses or via codon-optimized genes in DNA vaccines. Attenuation typically relies on deoptimization of codon pairs and maximization of CpG dinucleotide frequencies. So as to formulate evolutionarily informed attenuation strategies that aim to force nucleotide usage against the direction favored by selection, here, we examine available whole-genome sequences of SARS-CoV-2 to infer patterns of mutation and selection on synonymous sites. Analysis of mutational profiles indicates a strong mutation bias toward U. In turn, analysis of observed synonymous site composition implicates selection against U. Accounting for dinucleotide effects reinforces this conclusion, observed UU content being a quarter of that expected under neutrality. Possible mechanisms of selection against U mutations include selection for higher expression, for high mRNA stability or lower immunogenicity of viral genes. Consistent with gene-specific selection against CpG dinucleotides, we observe systematic differences of CpG content between SARS-CoV-2 genes. We propose an evolutionarily informed approach to attenuation that, unusually, seeks to increase usage of the already most common synonymous codons. Comparable analysis of H1N1 and Ebola finds that GC3 deviated from neutral equilibrium is not a universal feature, cautioning against generalization of results.
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Yang, Lei, Liming Gong, Ping Wang, Xinghui Zhao, Feng Zhao, Zhijie Zhang, Yunfei Li, and Wei Huang. "Recent Advances in Lipid Nanoparticles for Delivery of mRNA." Pharmaceutics 14, no. 12 (December 1, 2022): 2682. http://dx.doi.org/10.3390/pharmaceutics14122682.
Full textAbstract:
Messenger RNA (mRNA), which is composed of ribonucleotides that carry genetic information and direct protein synthesis, is transcribed from a strand of DNA as a template. On this basis, mRNA technology can take advantage of the body’s own translation system to express proteins with multiple functions for the treatment of various diseases. Due to the advancement of mRNA synthesis and purification, modification and sequence optimization technologies, and the emerging lipid nanomaterials and other delivery systems, mRNA therapeutic regimens are becoming clinically feasible and exhibit significant reliability in mRNA stability, translation efficiency, and controlled immunogenicity. Lipid nanoparticles (LNPs), currently the leading non-viral delivery vehicles, have made many exciting advances in clinical translation as part of the COVID-19 vaccines and therefore have the potential to accelerate the clinical translation of gene drugs. Additionally, due to their small size, biocompatibility, and biodegradability, LNPs can effectively deliver nucleic acids into cells, which is particularly important for the current mRNA regimens. Therefore, the cutting-edge LNP@mRNA regimens hold great promise for cancer vaccines, infectious disease prevention, protein replacement therapy, gene editing, and rare disease treatment. To shed more lights on LNP@mRNA, this paper mainly discusses the rational of choosing LNPs as the non-viral vectors to deliver mRNA, the general rules for mRNA optimization and LNP preparation, and the various parameters affecting the delivery efficiency of LNP@mRNA, and finally summarizes the current research status as well as the current challenges. The latest research progress of LNPs in the treatment of other diseases such as oncological, cardiovascular, and infectious diseases is also given. Finally, the future applications and perspectives for LNP@mRNA are generally introduced.
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Oca, Saúl Aguilar-Montes de, Roberto Montes-de-Oca-Jiménez, Juan Carlos Vázquez-Chagoyán, Alberto Barbabosa-Pliego, Pilar Eliana Rivadeneira-Barreiro, and Pablo C. Zambrano-Rodríguez. "The Use of Peptides in Veterinary Serodiagnosis of Infectious Diseases: A Review." Veterinary Sciences 9, no. 10 (October 12, 2022): 561. http://dx.doi.org/10.3390/vetsci9100561.
Full textAbstract:
Peptides constitute an alternative and interesting option to develop treatments, vaccines, and diagnostic tools as they demonstrate their scope in several health aspects; as proof of this, commercial peptides for humans and animals are available on the market and used daily. This review aimed to know the role of peptides in the field of veterinary diagnosis, and include peptide-based enzyme-linked immunosorbent assay (pELISA), lateral flow devices, and peptide latex agglutination tests that have been developed to detect several pathogens including viruses and bacteria of health and production relevance in domestic animals. Studies in cattle, small ruminants, dogs, cats, poultry, horses, and even aquatic organisms were reviewed. Different studies showed good levels of sensitivity and specificity against their target, moreover, comparisons with commercial kits and official tests were performed which allowed appraising their performance. Chemical synthesis, recombinant DNA technology, and enzymatic synthesis were reviewed as well as their advantages and drawbacks. In addition, we discussed the intrinsic limitations such as the small size or affinity to polystyrene membrane and mention several strategies to overcome these problems. The use of peptides will increase in the coming years and their utility for diagnostic purposes in animals must be evaluated.
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Hornemann, Simone, Olof Harlin, Caroline Staib, Sigrid Kisling, Volker Erfle, Bernd Kaspers, Georg Häcker, and Gerd Sutter. "Replication of Modified Vaccinia Virus Ankara in Primary Chicken Embryo Fibroblasts Requires Expression of the Interferon Resistance Gene E3L." Journal of Virology 77, no. 15 (August 1, 2003): 8394–407. http://dx.doi.org/10.1128/jvi.77.15.8394-8407.2003.
Full textAbstract:
ABSTRACT Highly attenuated modified vaccinia virus Ankara (MVA) serves as a candidate vaccine to immunize against infectious diseases and cancer. MVA was randomly obtained by serial growth in cultures of chicken embryo fibroblasts (CEF), resulting in the loss of substantial genomic information including many genes regulating virus-host interactions. The vaccinia virus interferon (IFN) resistance gene E3L is among the few conserved open reading frames encoding viral immune defense proteins. To investigate the relevance of E3L in the MVA life cycle, we generated the deletion mutant MVA-ΔE3L. Surprisingly, we found that MVA-ΔE3L had lost the ability to grow in CEF, which is the first finding of a vaccinia virus host range phenotype in this otherwise highly permissive cell culture. Reinsertion of E3L led to the generation of revertant virus MVA-E3rev and rescued productive replication in CEF. Nonproductive infection of CEF with MVA-ΔE3L allowed viral DNA replication to occur but resulted in an abrupt inhibition of viral protein synthesis at late times. Under these nonpermissive conditions, CEF underwent apoptosis starting as early as 6 h after infection, as shown by DNA fragmentation, Hoechst staining, and caspase activation. Moreover, we detected high levels of active chicken alpha/beta IFN (IFN-α/β) in supernatants of MVA-ΔE3L-infected CEF, while moderate IFN quantities were found after MVA or MVA-E3rev infection and no IFN activity was present upon infection with wild-type vaccinia viruses. Interestingly, pretreatment of CEF with similar amounts of recombinant chicken IFN-α inhibited growth of vaccinia viruses, including MVA. We conclude that efficient propagation of MVA in CEF, the tissue culture system used for production of MVA-based vaccines, essentially requires conserved E3L gene function as an inhibitor of apoptosis and/or IFN induction.
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BATISTA, D. G. J., M. G. O. PACHECO, A. KUMAR, D. BRANOWSKA, M. A. ISMAIL, L. HU, D. W. BOYKIN, and M. N. C. SOEIRO. "Biological, ultrastructural effect and subcellular localization of aromatic diamidines in Trypanosoma cruzi." Parasitology 137, no. 2 (September 21, 2009): 251–59. http://dx.doi.org/10.1017/s0031182009991223.
Full textAbstract:
SUMMARYNo vaccines or safe chemotherapy are available for Chagas disease. Pentamidine and related di-cations are DNA minor groove-binders with broad-spectrum anti-protozoal activity. Therefore our aim was to evaluate the in vitro efficacy of di-cationic compounds – DB1645, DB1582, DB1651, DB1646, DB1670 and DB1627 – against bloodstream trypomastigotes (BT) and intracellular forms of Trypanosoma cruzi. Cellular targets of these compounds in treated parasites were also analysed by fluorescence and transmission electron microscopy (TEM). DB1645, DB1582 and DB1651 were the most active against BT showing IC50 values ranging between 0·15 and 6·9 μm. All compounds displayed low toxicity towards mammalian cells and DB1645, DB1582 and DB1651 were also the most effective against intracellular parasites, with IC50 values ranging between 7·3 and 13·3 μm. All compounds localized in parasite nuclei and kDNA (with greater intensity in the latter structure), and DB1582 and DB1651 also concentrated in non-DNA-containing cytoplasmic organelles possibly acidocalcisomes. TEM revealed alterations in mitochondria and kinetoplasts, as well as important disorganization of microtubules. Our data provide further information regarding the activity of this class of compounds upon T. cruzi which should aid future design and synthesis of agents that could be used for Chagas disease therapy.
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Márquez-Jurado, Silvia, Aitor Nogales, Ginés Ávila-Pérez, Francisco Iborra, Luis Martínez-Sobrido, and Fernando Almazán. "An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo." Viruses 10, no. 10 (October 7, 2018): 547. http://dx.doi.org/10.3390/v10100547.
Full textAbstract:
The recent outbreaks of Zika virus (ZIKV), its association with Guillain–Barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat ZIKV disease. In this respect, infectious clones constitute excellent tools to accomplish these goals. However, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. To bypass this problem, several alternative approaches have been applied for the generation of ZIKV clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. Here, we report a simple and novel DNA-launched approach based on the use of a bacterial artificial chromosome (BAC) to generate a cDNA clone of Rio Grande do Norte Natal ZIKV strain. The sequence was identified from the brain tissue of an aborted fetus with microcephaly. The BAC clone was fully stable in bacteria and the infectious virus was efficiently recovered in Vero cells through direct delivery of the cDNA clone. The rescued virus yielded high titers in Vero cells and was pathogenic in a validated mouse model (A129 mice) of ZIKV infection. Furthermore, using this infectious clone we have generated a mutant ZIKV containing a single amino acid substitution (A175V) in the NS2A protein that presented reduced viral RNA synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with ZIKV wild-type. This BAC approach provides a stable and reliable reverse genetic system for ZIKV that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies.
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Markin, Vladimir A. "Marburg virus and the disease it causes." Journal of microbiology, epidemiology and immunobiology 99, no. 5 (December 7, 2022): 605–18. http://dx.doi.org/10.36233/0372-9311-273.
Full textAbstract:
Over the 50 years since its discovery, many properties of the Marburg virus have been studied, but no reliable medical remedies of preventing and treating the infection it causes have been developed, although it can potentially cause large-scale epidemics.
Marburg fever is relevant due to the risk of importation to other countries. The source of infection in nature is bats (reservoir) and monkeys (intermediate host), and the routes of transmission are aerosol, contact and alimentary. The mortality rate in recent outbreaks has reached 90%. In convalescents the causative agent was identified in tears, semen, and liver biopsies weeks and months after recovery.
The lack of therapeutic and prophylactic antiviral drugs, high rates of mortality, infectivity, the ability of aerosol contamination, and a high epidemic potential all together define Marburg fever as a serious global threat to international health. The development of medical protection against this infection should be an urgent task of ensuring the biological safety of the population of the Russian Federation.
The most promising ways to develop vaccines against Marburg fever are the construction of recombinants based on adenovirus, vesicular stomatitis virus or alphavirus replicon, DNA vaccines. A reliable protective effect of the chemotherapy drug remdesivir in combination with human antibodies, as well as an etiotropic drug with an antisense mechanism of action and an interferon inducer has been shown. In model experiments with pseudovirus, fundamentally new ways of developing pathogen inhibitors were found preventing its exit from cells, as well as the construction of anti-gene-binding Fab fragments that inhibit the synthesis of viral RNA.
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Santiago, Daniel. "A Partial Answer to the Question Posed by David A. Hughes, PhD, in the Article: “What is in the so-called COVID-19 ‘Vaccines’? Part 1: Evidence of a Global Crime Against Humanity”." International Journal of Vaccine Theory, Practice, and Research 2, no. 2 (September 7, 2022): 587–94. http://dx.doi.org/10.56098/ijvtpr.v2i2.56.
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In this comment, originally thought of as a “Letter to the Editor”, I want to address the opening question posed by David A. Hughes in the immediately preceding entry in this journal: “What is in the so-called COVID-19 ‘Vaccines’?” The views from under the microscope, ordinary light or electron scanning, all show undisclosed foreign objects that seem to activate themselves and aggregate into complexes that disrupt blood flow in all organ systems. With the spectral analysis using electron microscopy it is possible to determine the specific elements and relative quantities of the elements in those foreign entities. In this comment, I want to focus on the absence of certain elements that are universally present in the proteins of naturally occurring life forms from humans right down to bacteria and even the proteins formed from viruses. What is missing from the spectral analyses of the foreign elements in the main COVID-19 vaccines, Pfizer and Moderna for certain, and probably also missing from the other experimental products being widely distributed that are known to contain foreign aggregates of strange materials similar to those found in the Moderna and Pfizer injections, are the elements nitrogen and phosphorous. This is revealing because all natural DNA, RNA, and their protein products contain those missing elements. Nitrogen for protein synthesis and phosphorus for DNA, RNA, and energy transfer. Therefore, their absence from the foreign structures seen under many different microscopes in all of the COVID-19 so-called “vaccines” that have been examined, and also found in blood samples of persons injected with the Moderna and Pfizer concoctions, proves that these intentionally manufactured self-assembling components, built mainly from carbon-based materials used in computing and super-conductors, are connected with the avant-guard evolutionary theory and experimentation with what is known as XNA, Xeno (Greek for “foreign”), Nucleic Acid. Most of the relevant information is behind significant paywalls in esoteric journals specializing in this peculiar branch of highly theoretical and experimental chemistry. To leap to the bottom-line of my urgent comment on the Hughes’ paper, the edgy modified mRNA with N1-methylpseudouridine (Ψ) replacing the naturally occurring RNA nucleotide uridine (U) at least 728 times in each one of the 30 billion mRNA molecules in each of the Pfizer injections is an exmplary XNA. In this comment I want to explain why the inclusion of such an XNA may be the clue that leads to the unraveling of the already devastating and potentially exterminating impact of the ongoing COVID-19 experiment on the human race.
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Zhang, Yana, Jian Zhang, Masum Shahriar, and Seah H. Lim. "Clofarabine Induces Hypomethylation of DNA and Expression of Cancer-Testis Antigens." Blood 112, no. 11 (November 16, 2008): 379. http://dx.doi.org/10.1182/blood.v112.11.379.379.
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Abstract Clofarabine is a second generation purine nucleoside analogue, active in non-dividing cells and in cells with a low proliferation rate. Although clofarabine has been used in patients with refractory AML, ALL and MDS, the precise mechanism of action of clofarabine is unclear. In vitro data suggests that clofarabine induce cytotoxicity through inhibition of DNA synthesis. Clofarabine 5′-triphosphate inhibits both DNA polymerase a and ribonucleotide reductase and causes a depletion of the intracellular deoxynucleotide triphosphate pools and inhibition of elongation of DNA strands during synthesis. Clofarabine also induces apoptosis and disrupts the integrity of mitochondria. Cancer-Testis (CT) antigens are a group of normal testicular proteins aberrantly expressed in tumor cells. They are potential targets for tumor vaccine development because of their limited expression in normal tissues and their in vivo immunogenicity. Changes in genomic methylation associated with malignant transformation of normal cells are thought to be responsible for the aberrant expression of the genes encoding some CT antigens. Using the LINE-1 PCR approach to amplify the Alu repeats of the long interspersed nucleotide elements of genomic DNA, there were significant decreases in the LINE-1 hypomethylation of the DNA in tumor cell lines following treatment with clofarabine when compared to cells cultured in medium without clofarabine (p < 0.05). With clofarabine concentrations between 1 × 10−10 M to 1 × 10−7 M, there was a maximum decrease in the LINE-1 hypomethylation of 13% in JVM-2 cells, 14% in RL cells and 21% in Granta 519 cells. DNA hypomethylation was observed in all three cell lines with a clofarabine concentration as low as 1 × 10−10 M. These results, therefore, support the DNA hypomethylating property of low concentration clofarabine. Maximum hypomethylating effects of clofarabine was observed at concentrations of 1 × 10−7 M for JVM-2 cells, between 1 × 10−10 and 1 × 10−9 M for RL cells and 1 × 10−8 M for Granta 519 cells. Above these concentrations, the % of LINE-1 hypomethylation of the DNA declined as the degree of apoptosis, as demonstrated by Annexin V staining, increased. Clofarabine treatment of these tumor cells resulted in the upregulation of the CT antigens. Before treatment with clofarabine, JVM-2 and Granta 519 cells expressed a very low level of Sp17 gene and undetectable Sp17 protein, and RL cells did not express any Sp17 gene or protein. In all three tumor cells, Sp17 expression was either induced or upregulated at the transcript and protein levels following treatment of the tumor cells with clofarabine. Using real time PCR, we also found a dose response of the mRNA levels encoding Sp17 with increasing concentrations of clofarabine used in the cell culture, up to a clofarabine concentration of 1 × 10−8 M. However, above this concentration, the levels of mRNA encoding Sp17 declined progressively as apoptosis increased. Similar results were seen with SPAN-Xb expression. Before treatment, transcripts encoding SPAN-Xb were detected in all three cell lines by RT-PCR. At the protein level, RL cells and Granta 519 cells expressed high level and JVM-2 cells low level of SPAN-Xb protein by immunocytochemistry. Following treatment with clofarabine, SPAN-Xb protein expression was greatly upregulated in all three tumor cell lines. These results, therefore, provide the first evidence showing the ability of clofarabine to induce the expression of Sp17 and SPAN-Xb and support the notion that low concentration clofarabine induces DNA hypomethylation, as suggested by the LINE-1 PCR analysis. However, not unlike in Sp17, we also found a dose response of the mRNA levels encoding SPAN-Xb with increasing concentrations of clofarabine used in the cell culture, up to concentrations between 1 × 10−9 and 1 × 10−8 M. Above this optimal concentration, SPAN-Xb gene expression declined as apoptosis increased. In conclusion, clofarabine has a DNA hypomethylating property and is capable of inducing CT antigen expression. Since DNA hypomethylation with clofarabine is most optimal at very low clofarabine concentrations, low dose clofarabine should be used if the mechanism needed for response to treatment is that of DNA hypomethylation. Furthermore, there may be opportunity to administer low dose clofarabine to patients to upregulate the expression of CT antigens to increase the susceptibility of the tumor cells to the cytotoxic effect of antigen-specific cytotoxic T cells prior to specific tumor vaccines targeting CT antigens.
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Marciani, Dante J. "Sole Anti-inflammatory Immunomodulators: Innovative Drugs to Prevent and Treat Autoimmune Diseases and Proteopathies." Current Chinese Science 1, no. 2 (April 19, 2021): 273–85. http://dx.doi.org/10.2174/2210298101666210108110556.
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Objective: To review the available sole anti-inflammatory immunomodulators or adjuvants, different from pro-inflammatory ones, which elicit a Th2 immunity while inhibiting but without abrogating Th1/Th17 immunities. Adjuvants that are useful to develop vaccines for T-cell mediated autoimmune conditions. Methods: A literature search using PubMed and Google Scholar databases was made to identify reports regarding adjuvants, mechanisms of action, pro-inflammatory autoimmunity and vaccines to treat it, immunosuppressive agents, dendritic cells, helminths, immunotolerance, and infectious diseases causing autoimmunity. Results: Some anti-inflammatory drugs to treat autoimmune diseases inhibit DNA or protein synthesis causing global immunosuppression, which is reduced by using biologics to block key steps in the inflammatory cascade. Fucosylated glycans from helminths, which are anti-inflammatory but not immune-suppressive, offer an avenue to develop better drugs. Fucosylated glycans bind to DC-SIGN, a receptor on dendritic cells, entering the cells via receptor-mediated endocytosis, biasing their immunoresponse to a sole Th2 anti-inflammatory immunity, while inhibiting the proinflammatory Th1/Th17 immunities. New anti-inflammatory drugs are particular plant-derived fucosylated glycosides with immunological properties like those of helminth-derived glycans. Another class of anti-inflammatory immunomodulators is ligands of the aromatic-hydrocarbon receptor, which by activating this intracellular receptor, boosts the differentiation of regulatory Tcells, inducing an anti-inflammatory immunity. However, aromatic ligands can also stimulate a pro-inflammatory response. Exogenous aromatic ligands are usually delivered intracellularly using carriers like nanoparticles, which upon translocation to the nucleus, activate this receptor. Conclusions: Autoimmune conditions and some infectious diseases, characterized by organ damage due to pro-inflammatory autoimmune immunoresponses, could benefit from nonimmunosuppressive agents to modulate immunity; this way, averting a damaging inflammation.
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Glushkov, Andrew N. "Immune-hormonal imbalance in chemical cancerogenesis." Russian Journal of Immunology 25, no. 1 (August 3, 2022): 23–36. http://dx.doi.org/10.46235/1028-7221-1091-ihi.
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The present article deals with experimental and clinical aspects of immuno-hormonal interactions in chemical carcinogenesis i.e., formation of DNA-adducts with chemical carcinogens as a trigger of tumor initiation; synthesis of specific antibodies as markers of human exposure to environmental carcinogens; immunomodulation of chemical carcinogenesis by the specific antibodies in experimental studies; interactions of antibodies against environmental carcinogens with endogenous steroid hormones in human carcinogenesis; immunological interference and inversion of immuno-hormonal interactions by the action of antibodies against environmental carcinogens; immune stimulation of tumor progression in cancer patients. It is shown that antibodies specific to estradiol and progesterone participate in regulation of serum estradiol and progesterone levels in healthy women. Excessive production of antibodies against benzo[a]pyrene is associated with impaired physiological balance between the levels of antibodies to estradiol and progesterone, thus causing disturbed physiological balance between serum estradiol and progesterone. Immuno-hormonal imbalance promotes tumor initiation, its growth and progression. The new approaches to the personalized cancer immunoprediction and immune prevention are discussed. Coordinated synthesis of antibodies against benzo[a]pyrene and estradiol seems to reflect production of DNA-adducts with genotoxic metabolic effects of these compounds manifesting as synergistic carcinogenic effects upon the target cells. Hence, simultaneously increased levels of serum antibodies against benzo[a]pyrene and estradiol in healthy people may be considered an immunological marker of high oncological risk and an reason to use of new immunoprotective tools against polycyclic aromatic hydrocarbons and phytoestrogens. However, ability of these antibodies to raise the blood serum levels of environmental carcinogens and endogenous estradiol, as shown in vitro and in vivo, excludes the opportunity for active cancer immune prevention. Usage of anticarcinogen vaccines aimed for induction of protective secretory antibodies is likely to further increase high levels of procarcinogenic serum antibodies against benzo[a]pyrene and estradiol, followed by additional enhancement of immuno-hormonal imbalance and promotion of carcinogenesis. Development of probiotics transduced with genes encoding human antibodies against environmental carcinogens may present an alternative approach to cancer immune prevention. The antibodies produced by such probiotics would bind appropriate carcinogens and prevent their invasion into the organism, thus inhibiting emergence of DNA-adducts and suppressing synthesis of specific autoantibodies that may promote carcinogenesis. The aim is to substantiate the concept of immuno-hormonal imbalance for the carcinogen-induced hormone-dependent tumors.
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Kopteva, Olga S., Yulia A. Desheva, Alexandra N. Ivanova, Maxim G. Vorobyov, Galina F. Leontieva, Tatiana V. Gupalova, Elena A. Bormotova, and Alexander N. Suvorov. "Surface expression of SARS-CoV-2 epitopes in <i>Enterococcus faecium</i> L3 for live oral vaccine against new coronavirus infection." Medical academic journal 2, no. 2 (November 6, 2022): 197–202. http://dx.doi.org/10.17816/maj108671.
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BACKGROUND: Probiotic microorganisms are currently considered as a promising platform for the development of recombinant vaccines expressing viral or bacterial antigens. Probiotic-based mucosal vaccines are easy to produce in large quantities, they have a low cost, provide a fairly long T-cell memory.
AIM: The aim was to study expression of mRNA fragment of S1 SARS-CoV-2 gene in Enterococcus faecium L3 culture and to confirm the insertion of S1 SARS-CoV-2 protein fragment into the pili of this bacterial strain by immunoelectron microscopy of original (E. faecium L3) and genetically modified strain (L3-SARS) with human sera obtained from patients with SARS-CoV-2.
MATERIALS AND METHODS: mRNA expression was studied by real-time PCR with reverse transcription using primers specific to S1 protein. Immunoelectron microscopy was aimed to study the structure of E. faecium L3 pili with the expression of viral protein SARS-CoV-2. Bacteria were washed three times in PBS by centrifugation at 3500 rpm for 20 min and suspended in 0.1 M NaCl. A 10-fold bacterial concentrate was used. The source of the primary antibodies was a set of polyclonal human sera containing IgG. Labeling was performed using goat IgG conjugated with 18 nm gold particles.
RESULTS: A sharp increase in mRNA amplification of inserted genetic sequence of S1 SARS-CoV-2 gene fragment relatively to the control was demonstrated. These results confirmed that DNA of S1 gene in E. faecium L3 genome is transcribed together with the target pili gene in E. faecium genome. Specific antigens of SARS-CoV-2 on the surface of L3-SARS were determined using electron microscopy, which demonstrated the correct assembly of chimeric molecules of pili on the surface of bacteria.
CONCLUSIONS: Evaluation in expression of SARS-CoV-2 S1 protein after introduction of the corresponding genetic elements into genome of probiotic strain E. faecium L3 allows us to conclude that selected DNA fragments of SARS-CoV-2 were able to direct the synthesis of immunogenic protein S1 that was expressed by the strain E. faecium L3-SARS.
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Rajpoot, Kuldeep. "Solid Lipid Nanoparticles: A Promising Nanomaterial in Drug Delivery." Current Pharmaceutical Design 25, no. 37 (December 17, 2019): 3943–59. http://dx.doi.org/10.2174/1381612825666190903155321.
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The solid lipid nanoparticles (SLNs) usually consists of active drug molecules along with solid lipids, surfactants, and/or co-surfactants. They possess some potential features such as nano-size, surface with a free functional group to attach ligands, and as well they prove safe homing for both lipophilic as well as hydrophilic molecules. As far as synthesis is concerned, SLNs can be prepared by employing various techniques viz., homogenization techniques (e.g., high-pressure, high-speed, cold, or hot homogenization), spray drying technique, ultrasonication, solvent emulsification, double emulsion technique, etc. Apart from this, they are characterized by different methods for determining various parameters like particle-size, polydispersity-index, surface morphology, DSC, XRD, etc. SLNs show good stability as well as the ability for surface tailoring with the specific ligand, which makes them a suitable candidate in the therapy of numerous illnesses, especially in the targeting of the cancers. In spite of this, SLNs have witnessed their application via various routes e.g., oral, parenteral, topical, pulmonary, rectal routes, etc. Eventually, SLNs have also shown great potential for delivery of gene/DNA, vaccines, as well as in cosmeceuticals. Hence, SLNs have emerged as a promising nanomaterial for efficient delivery of various Active Pharmaceutical Ingredients (APIs).
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Casais, Rosa, Volker Thiel, Stuart G. Siddell, David Cavanagh, and Paul Britton. "Reverse Genetics System for the Avian Coronavirus Infectious Bronchitis Virus." Journal of Virology 75, no. 24 (December 15, 2001): 12359–69. http://dx.doi.org/10.1128/jvi.75.24.12359-12369.2001.
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ABSTRACT Major advances in the study of the molecular biology of RNA viruses have resulted from the ability to generate and manipulate full-length genomic cDNAs of the viral genomes with the subsequent synthesis of infectious RNA for the generation of recombinant viruses. Coronaviruses have the largest RNA virus genomes and, together with genetic instability of some cDNA sequences in Escherichia coli, this has hampered the generation of a reverse-genetics system for this group of viruses. In this report, we describe the assembly of a full-length cDNA from the positive-sense genomic RNA of the avian coronavirus, infectious bronchitis virus (IBV), an important poultry pathogen. The IBV genomic cDNA was assembled immediately downstream of a T7 RNA polymerase promoter by in vitro ligation and cloned directly into the vaccinia virus genome. Infectious IBV RNA was generated in situ after the transfection of restricted recombinant vaccinia virus DNA into primary chick kidney cells previously infected with a recombinant fowlpox virus expressing T7 RNA polymerase. Recombinant IBV, containing two marker mutations, was recovered from the transfected cells. These results describe a reverse-genetics system for studying the molecular biology of IBV and establish a paradigm for generating genetically defined vaccines for IBV.
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Sheth, Alpa C., and Paresh U. Patel. "Review of Elemental Impurities in Pharmaceuticals Arena." INTERNATIONAL JOURNAL OF PHARMACEUTICAL QUALITY ASSURANCE 11, no. 02 (June 25, 2020): 214–18. http://dx.doi.org/10.25258/ijpqa.11.2.3.
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Elemental impurities in drug products may arise from several sources; they may be residual catalysts that were added intentionally in the synthesis or may be present as impurities (e.g., through interactions with processing equipment or container/closure systems or by being present in components of the drug product). Because elemental impurities do not provide any therapeutic benefit to the patient, their levels in the drug product should be controlled within acceptable limits. The main objective of the International Conference on Harmonization (ICH) (Q3D) guideline applies to new finished drug products and new drug products containing existing drug substances. The drug products containing purified proteins and polypeptides. This guideline does not apply to herbal products, radiopharmaceuticals, vaccines, cell metabolites, DNA products, allergenic extracts, cells, whole blood, cellular blood components, or blood derivatives, including plasma and plasma derivatives. The evaluation of the toxicity data for potential elemental impurities; the establishment of a permitted daily exposure (PDE) for each element of toxicological concern; application of a risk-based approach to control elemental impurities in drug products. Different analytical techniques for elemental impurities detection: flame atomic absorption spectrometry (FAAS), graphite furnace atomic absorption spectrometry (GFAAS), atomic fluorescence spectrometry, X-ray fluorescence spectrometry (XRF), instrumental neutron activation analysis (INAA), inductively coupled plasma-atomic emission spectroscopy (optical emission spectroscopy, ICP-OES), inductively coupled plasma mass spectrometry (ICP-MS), and microwave plasma atomic emission spectrometry (MP-AES).
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SOEIRO, M. N. C., K. WERBOVETZ, D. W. BOYKIN, W. D. WILSON, M. Z. WANG, and A. HEMPHILL. "Novel amidines and analogues as promising agents against intracellular parasites: a systematic review." Parasitology 140, no. 8 (April 8, 2013): 929–51. http://dx.doi.org/10.1017/s0031182013000292.
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SUMMARYParasitic protozoa comprise diverse aetiological agents responsible for important diseases in humans and animals including sleeping sickness, Chagas disease, leishmaniasis, malaria, toxoplasmosis and others. They are major causes of mortality and morbidity in tropical and subtropical countries, and are also responsible for important economic losses. However, up to now, for most of these parasitic diseases, effective vaccines are lacking and the approved chemotherapeutic compounds present high toxicity, increasing resistance, limited efficacy and require long periods of treatment. Many of these parasitic illnesses predominantly affect low-income populations of developing countries for which new pharmaceutical alternatives are urgently needed. Thus, very low research funding is available. Amidine-containing compounds such as pentamidine are DNA minor groove binders with a broad spectrum of activities against human and veterinary pathogens. Due to their promising microbicidal activity but their rather poor bioavailability and high toxicity, many analogues and derivatives, including pro-drugs, have been synthesized and screened in vitro and in vivo in order to improve their selectivity and pharmacological properties. This review summarizes the knowledge on amidines and analogues with respect to their synthesis, pharmacological profile, mechanistic and biological effects upon a range of intracellular protozoan parasites. The bulk of these data may contribute to the future design and structure optimization of new aromatic dicationic compounds as novel antiparasitic drug candidates.
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Meuleman, Theodorus J., Vanessa M. Cowton, Arvind H. Patel, and Rob M. J. Liskamp. "Design and Synthesis of HCV-E2 Glycoprotein Epitope Mimics in Molecular Construction of Potential Synthetic Vaccines." Viruses 13, no. 2 (February 20, 2021): 326. http://dx.doi.org/10.3390/v13020326.
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Hepatitis C virus remains a global threat, despite the availability of highly effective direct-acting antiviral (DAA) drugs. With thousands of new infections annually, the need for a prophylactic vaccine is evident. However, traditional vaccine design has been unable to provide effective vaccines so far. Therefore, alternative strategies need to be investigated. In this work, a chemistry-based approach is explored towards fully synthetic peptide-based vaccines using epitope mimicry, by focusing on highly effective and conserved amino acid sequences in HCV, which, upon antibody binding, inhibit its bio-activity. Continuous and discontinuous epitope mimics were both chemically synthesized based on the HCV-E2 glycoprotein while using designed fully synthetic cyclic peptides. These cyclic epitope mimics were assembled on an orthogonally protected scaffold. The scaffolded epitope mimics have been assessed in immunization experiments to investigate the elicitation of anti-HCV-E2 glycoprotein antibodies. The neutralizing potential of the elicited antibodies was investigated, representing a first step in employing chemically synthesized epitope mimics as a novel strategy towards vaccine design.
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Lee, Jae-Yeol, Se-Jeong Ahn, Chanseop Park, Hee-Won Bae, Eun Sook Kim, and You-Hee Cho. "Reverse Genetic Systems for Pseudomonas aeruginosa Leviphages." Methods and Protocols 2, no. 1 (March 5, 2019): 22. http://dx.doi.org/10.3390/mps2010022.
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Reverse genetic systems for RNA viruses are the platforms to introduce mutations into the RNA genomes and thus have helped understand their life cycle and harness them for human purposes to develop vaccines and delivery systems. These systems are based on the complementary DNA (cDNA) of the RNA viruses, whose transcripts derived from bacterial RNA polymerases act not only as the primary mRNA for phage protein synthesis, but also as the template for phage RNA replicases (aka. RNA-dependent RNA polymerases). Here, we present a protocol optimized for the small RNA phages of Leviviridae (i.e., leviphages) infecting Pseudomonas aeruginosa. This protocol includes three fundamental steps: (i) Creation of a promoter-fused cDNA, (ii) generation of a clone into mini-Tn7-based vector, and (iii) introduction of the clone into non-susceptible hosts. As the representative example, we describe the reverse genetic system for PP7, which infects a set of P. aeruginosa strains such as PAO1. The cDNA was fused to the T7 promoter, which was cloned in mini-Tn7-Gm. This construct was introduced into P. aeruginosa PAK and E. coli HB101. Functional assembly of PP7 phages from the culture supernatants were assessed by plaque formation on PAO1 and the phage particles were observed under transmission microscope. We found that the host cells should be cultured at 30 °C for the maximal phage production (~1012 pfu/mL). The reverse genetic systems will provide a new insight into the life cycle of the RNA phages and help develop engineered variants with new traits for phage applications regarding selective diagnosis and efficient therapy.
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Poly, Frédéric, Timothy D. Read, Yu-Han Chen, Mario A. Monteiro, Oralak Serichantalergs, Piyarat Pootong, Ladaporn Bodhidatta, et al. "Characterization of Two Campylobacter jejuni Strains for Use in Volunteer Experimental-Infection Studies." Infection and Immunity 76, no. 12 (September 22, 2008): 5655–67. http://dx.doi.org/10.1128/iai.00780-08.
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ABSTRACT The development of vaccines against Campylobacter jejuni would be facilitated by the ability to perform phase II challenge studies. However, molecular mimicry of the lipooligosaccharide (LOS) of most C. jejuni strains with human gangliosides presents safety concerns about the development of Guillain-Barré syndrome. Clinical isolates of C. jejuni that appeared to lack genes for the synthesis of ganglioside mimics were identified by DNA probe analyses. Two clinical isolates from Southeast Asia (strains BH-01-0142 and CG8421) were determined to express the LOS type containing N-acetyl quinovosamine. No ganglioside structures were observed to be present in the LOSs of these strains, and pyrosequence analyses of the genomes of both strains confirmed the absence of genes involved in ganglioside mimicry. The capsule polysaccharide (CPS) of BH-01-0142 was determined to be composed of galactose (Gal), 6-deoxy-ido-heptose, and, in smaller amounts, d-glycero-d-ido-heptose, and the CPS of CG8421 was observed to contain Gal, 6-deoxy-altro-heptose, N-acetyl-glucosamine, and minor amounts of 6-deoxy-3-O-Me-altro-heptose. Both CPSs were shown to carry O-methyl-phosphoramidate. The two genomes contained strain-specific zones, some of which could be traced to a plasmid origin, and both contained a large chromosomal insertion related to the CJEI3 element of C. jejuni RM1221. The genomes of both strains shared a high degree of similarity to each other and, with the exception of the capsule locus of CG8421, to the type strain of the HS3 serotype, TGH9011.
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Lewis, T., L. Zsak, T. G. Burrage, Z. Lu, G. F. Kutish, J. G. Neilan, and D. L. Rock. "An African Swine Fever Virus ERV1-ALRHomologue, 9GL, Affects Virion Maturation and Viral Growth in Macrophages and Viral Virulence in Swine." Journal of Virology 74, no. 3 (February 1, 2000): 1275–85. http://dx.doi.org/10.1128/jvi.74.3.1275-1285.2000.
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ABSTRACT The African swine fever virus (ASFV) genome contains a gene,9GL, with similarity to yeast ERV1 andALR genes. ERV1 has been shown to function in oxidative phosphorylation and in cell growth, while ALR has hepatotrophic activity. 9GL encodes a protein of 119 amino acids and was highly conserved at both nucleotide and amino acid levels among all ASFV field isolates examined. Monospecific rabbit polyclonal antibody produced to a glutathione S-transferase–9GL fusion protein specifically immunoprecipitated a 14-kDa protein from macrophage cell cultures infected with the ASFV isolate Malawi Lil-20/1 (MAL). Time course analysis and viral DNA synthesis inhibitor experiments indicated that p14 was a late viral protein. A9GL gene deletion mutant of MAL (Δ9GL), exhibited a growth defect in macrophages of approximately 2 log10 units and had a small-plaque phenotype compared to either a revertant (9GL-R) or the parental virus. 9GL affected normal virion maturation; virions containing acentric nucleoid structures comprised 90 to 99% of all virions observed in Δ9GL-infected macrophages. The Δ9GL virus was markedly attenuated in swine. In contrast to 9GL-R infection, where mortality was 100%, all Δ9GL-infected animals survived infection. With the exception of a transient fever response in some animals, Δ9GL-infected animals remained clinically normal and exhibited significant 100- to 10,000-fold reductions in viremia titers. All pigs previously infected with Δ9GL survived infection when subsequently challenged with a lethal dose of virulent parental MAL. Thus, ASFV9GL gene deletion mutants may prove useful as live-attenuated ASF vaccines.
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Grethe, Stefanie, Masyar Monazahian, Ingo Böhme, and Reiner Thomssen. "Characterization of Unusual Escape Variants of Hepatitis B Virus Isolated from a Hepatitis B Surface Antigen-Negative Subject." Journal of Virology 72, no. 9 (September 1, 1998): 7692–96. http://dx.doi.org/10.1128/jvi.72.9.7692-7696.1998.
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ABSTRACT Hepatitis B virus DNA was extracted from serial serum samples of a hepatitis B surface antigen-negative patient with antibodies to the core protein as the only marker of an infection with hepatitis B virus. This patient showed no symptoms of hepatic injury. Sequencing of the amplified viral DNA demonstrated multiple amino acid changes clustering in surface-exposed regions of the surface protein. Synthesis and association of the middle (M) and small (S) surface proteins could be shown in vitro. The variant surface antigens were recognized neither by monoclonal antibodies to the surface antigen nor by the vaccinee’s sera. Consequences for hepatitis B surface antigen testing and vaccine development are discussed.
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Niu, Y., J. Sun, M. Fan, Q. A. Xu, J. Guo, R. Jia, and Y. Li. "Construction of a New Fusion Anti-caries DNA Vaccine." Journal of Dental Research 88, no. 5 (May 2009): 455–60. http://dx.doi.org/10.1177/0022034509336727.
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Mutans streptococci (MS) are generally considered to be the principal etiological agent of dental caries. MS have two important virulence factors: cell- surface protein PAc and glucosyltransferases (GTFs). GTFs have two functional domains: an N-terminal catalytic sucrose-binding domain (CAT) and a C-terminal glucan-binding domain (GLU). A fusion anti-caries DNA vaccine, pGJA-P/VAX, encoding two important antigenic domains, PAc and GLU, of S. mutans, was successful in reducing the levels of dental caries caused by S. mutans in gnotobiotic animals. However, its protective effect against S. sobrinus infection proved to be weak. Does the DNA vaccine need an antigen of S. sobrinus to enhance its ability to inhibit infection? To answer this question, in this study, we cloned the catalytic ( cat) fragment of S. sobrinus gtf-I, which demonstrated its ability to inhibit water-insoluble glucan synthesis by S. sobrinus, into pGJA-P/VAX to produce a new anti-caries DNA vaccine.