scholarly journals Human Adenovirus Serotypes Efficiently Transducing HEK293 Cells: An In Vitro Propagation of HAdv

2021 ◽  
Vol 8 (5) ◽  
Author(s):  
Mr. Utkalendu Suvendusekhar Samantaray ◽  
Ms. Piyanki Santra
Keyword(s):  
Vaccine Development ◽  
Recombinant Adenovirus ◽  
Scale Up ◽  
Human Adenovirus ◽  
Adenoviral Vectors ◽  
Hek293 Cells ◽  
Target Cells ◽  
Hek 293 ◽  
Mammalian Cell Lines

Generally, a gene which is inserted directly into a cell does not operate independently. Instead, the transmission of the gene is genetically modified by a biological messenger called a vector, consists of a transgene and a large DNA sequence as a backbone. Since they can deliver the new gene by infecting the cell, such viruses are also used as vectors. The adenovirus is a non-enveloped virus that can be tailored to transfer DNA to target cells, and it has sparked a lot of interest in the field, particularly in clinical trial therapy techniques. For the new age production of COVID-19 vaccine, development of different mammalian cell lines like HEK293 (most reliable growth and prosperity for transfection) and recombinant adenoviral vectors have become the first priority for biopharmaceutical giants and globally approved vaccine manufacturers to scale up their vaccine production. Adenoviruses have an icosahedral shape, with a protein coat encasing the viral double-stranded DNA genome. Because the adenovirus genome is relatively small, it's a good candidate for insertion of foreign DNA. The adenovirus E1A gene is deleted, and the virus loses its capacity to replicate. This ability can be restored during cell culture propagation by employing cells that produce the E1A protein, for example. Hence, in this mini research, I have shared an overview of the propagation of adenoviral vectors, i.e. recombinant adenovirus SARS CoV-2 vector in HEK-293 cell suspension culture.

scholarly journals Construction and Immunogenicity of Recombinant Adenovirus Vaccines Expressing the HMW1, HMW2, or Hia Adhesion Protein of Nontypeable Haemophilus influenzae

2010 ◽  
Vol 17 (10) ◽  
pp. 1567-1575 ◽  
Author(s):  
Linda E. Winter ◽  
Stephen J. Barenkamp
Keyword(s):  
Haemophilus Influenzae ◽  
Vaccine Development ◽  
Recombinant Adenovirus ◽  
Serum Antibody ◽  
Nontypeable Haemophilus Influenzae ◽  
Hek 293 ◽  
Adhesion Protein ◽  
Protective Antigens ◽  
Antibody Titers ◽  
Adenoviral Construct

ABSTRACT The objective of the present study was to construct and assess the immunogenicity of recombinant adenovirus vectors expressing the HMW1, HMW2, or Hia protein of nontypeable Haemophilus influenzae (NTHi). These proteins are critical adhesins and potential protective antigens expressed by NTHi. Segments of the hmw1A and hmw2A structural genes that encode the distal one-half of mature HMW1 or HMW2 were cloned into the T7 expression vector pGEMEX-2. These constructs encoded stable HMW1 or HMW2 recombinant fusion protein that expresses B-cell epitopes common to most NTHi strains. A segment of the hia gene that encodes the surface-exposed portion of mature Hia was also cloned into pGEMEX-2. The resulting T7 gene 10 translational fusions were excised from the parent plasmids and cloned into the shuttle plasmid pDC316. Cotransfection of HEK 293 cells with the pDC316 derivatives and pBHGloxΔE1,3Cre resulted in the production of viral plaques from which recombinant adenoviruses expressing fusion proteins were recovered. Chinchillas immunized intraperitoneally with a single 108-PFU dose of either the HMW2 or Hia adenoviral construct developed high anti-HMW2 or anti-Hia serum antibody titers within 4 weeks of immunization. Chinchillas immunized intranasally with a single 107- to 109-PFU dose of the Hia adenoviral construct also developed high anti-Hia serum antibody titers within 8 weeks of immunization. Recombinant adenoviruses represent a promising system to induce mucosal and systemic immunity and protection against mucosal diseases such as otitis media. Recombinant adenoviruses expressing recombinant HMW1, HMW2, or Hia protein will be important new tools in NTHi vaccine development efforts.

scholarly journals Exosome-Mediated mRNA Delivery For SARS-CoV-2 Vaccination

2020 ◽  
Author(s):  
Shang-Jui Tsai ◽  
Chenxu Guo ◽  
Nadia A. Atai ◽  
Stephen J. Gould
Keyword(s):  
Viral Vectors ◽  
Vaccine Development ◽  
Spike Protein ◽  
Interleukin 4 ◽  
Target Cells ◽  
Durable Response ◽  
Elevated Expression ◽  
Cellular Immune

AbstractBackgroundIn less than a year from its zoonotic entry into the human population, SARS-CoV-2 has infected more than 45 million people, caused 1.2 million deaths, and induced widespread societal disruption. Leading SARS-CoV-2 vaccine candidates immunize with the viral spike protein delivered on viral vectors, encoded by injected mRNAs, or as purified protein. Here we describe a different approach to SARS-CoV-2 vaccine development that uses exosomes to deliver mRNAs that encode antigens from multiple SARS-CoV-2 structural proteins.ApproachExosomes were purified and loaded with mRNAs designed to express (i) an artificial fusion protein, LSNME, that contains portions of the viral spike, nucleocapsid, membrane, and envelope proteins, and (ii) a functional form of spike. The resulting combinatorial vaccine, LSNME/SW1, was injected into thirteen weeks-old, male C57BL/6J mice, followed by interrogation of humoral and cellular immune responses to the SARS-CoV-2 nucleocapsid and spike proteins, as well as hematological and histological analysis to interrogate animals for possible adverse effects.ResultsImmunized mice developed CD4+, and CD8+ T-cell reactivities that respond to both the SARS-CoV-2 nucelocapsid protein and the SARS-CoV-2 spike protein. These responses were apparent nearly two months after the conclusion of vaccination, as expected for a durable response to vaccination. In addition, the spike-reactive CD4+ T-cells response was associated with elevated expression of interferon gamma, indicative of a Th1 response, and a lesser induction of interleukin 4, a Th2-associated cytokine. Vaccinated mice showed no sign of altered growth, injection-site hypersensitivity, change in white blood cell profiles, or alterations in organ morphology. Consistent with these results, we also detected moderate but sustained anti-nucleocapsid and anti-spike antibodies in the plasma of vaccinated animals.ConclusionTaken together, these results validate the use of exosomes for delivering functional mRNAs into target cells in vitro and in vivo, and more specifically, establish that the LSNME/SW1 vaccine induced broad immunity to multiple SARS-CoV-2 proteins.

scholarly journals Rapid development of SARS-CoV-2 receptor binding domain-conjugated nanoparticle vaccine candidate

2020 ◽  
Author(s):  
Yin-Feng Kang ◽  
Cong Sun ◽  
Zhen Zhuang ◽  
Run-Yu Yuan ◽  
Qing-Bing Zheng ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Vaccine Development ◽  
Vaccine Candidate ◽  
Rapid Development ◽  
Scale Up ◽  
Physical Stability ◽  
Covalent Bond ◽  
Neutralizing Antibody ◽  
Economic Losses

AbstractThe ongoing of coronavirus disease 2019 (COVID-19) pandemic caused by novel SARS-CoV-2 coronavirus, resulting in economic losses and seriously threating the human health in worldwide, highlighting the urgent need of a stabilized, easily produced and effective preventive vaccine. The SARS-COV-2 spike protein receptor binding region (RBD) plays an important role in the process of viral binding receptor angiotensin-converting enzyme 2 (ACE2) and membrane fusion, making it an ideal target for vaccine development. In this study, we designed three different RBD-conjugated nanoparticles vaccine candidates, RBD-Ferritin (24-mer), RBD-mi3 (60-mer) and RBD-I53-50 (120-mer), with the application of covalent bond linking by SpyTag-SpyCatcher system. It was demonstrated that the neutralizing capability of sera from mice immunized with three RBD-conjugated nanoparticles adjuvanted with AddaVax or Sigma Systerm Adjuvant (SAS) after each immunization was ~8-to 120-fold greater than monomeric RBD group in SARS-CoV-2 pseudovirus and authentic virus neutralization assay. Most importantly, sera from RBD-conjugated NPs groups more efficiently blocked the binding of RBD to ACE2 or neutralizing antibody in vitro, a further proof of promising immunization effect. Besides, high physical stability and flexibility in assembly consolidated the benefit for rapid scale-up production of vaccine. These results supported that our designed SARS-CoV-2 RBD-conjugated nanoparticle was competitive vaccine candidate and the carrier nanoparticles could be adopted as universal platform for future vaccine development.

scholarly journals Improvement of Immunogenicity of Meningococcal Lipooligosaccharide by Coformulation with Lipidated Transferrin-Binding Protein B in Liposomes: Implications for Vaccine Development

2012 ◽  
Vol 19 (5) ◽  
pp. 711-722 ◽  
Author(s):  
Noëlle Mistretta ◽  
Bruno Guy ◽  
Yves Bérard ◽  
François Dalençon ◽  
Olivia Fratantonio ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Binding Protein ◽  
Hek293 Cells ◽  
Molar Ratio ◽  
Adjuvant Effect ◽  
Content Type ◽  
Endotoxic Activity ◽  
Protein B

ABSTRACTAmong various meningococcal antigens, lipooligosaccharide (LOS) and recombinant lipidated transferrin-binding protein B (rlip-TbpB) are considered to be putative vaccine candidates against group BNeisseria meningitidis. In the present work, we report the development of a new liposome-based vaccine formulation containing both rlip-TbpB and L8 LOS. The endotoxic activity of the liposomal LOS was evaluatedin vitrousing theLimulusAmebocyte Lysate assay and compared to the endotoxic activity of free LOS. Above a 250:1 lipid/LOS molar ratio, liposomes were shown to effectively detoxify the LOS as the endotoxic activity of the LOS was reduced by more than 99%. Immunogenicity studies in rabbits showed that the presence of rlip-TbpB dramatically increased the immunogenicity of the LOS. While the formulation raised a strong anti-TbpB response, it elicited a higher anti-LOS IgG level than the liposomal LOS alone. Sera from rabbits immunized with rlip-TbpB/liposomal LOS displayed increased ability to recognize LOS on live bacteria expressing the L8 immunotype and increased anti-LOS-specific bactericidal activity compared to sera from rabbits immunized with liposomal LOS alone. Measurement of interleukin-8 (IL-8) produced by HEK293 cells transfected with Toll-like receptor (TLR) after stimulation with rlip-TbpB showed that the protein is a TLR2 agonist, which is in accordance with the structure of its lipid. Furthermore, anin vivostudy demonstrated that the lipid moiety is not only required for its adjuvant effect but also has to be linked to the protein. Overall, the rlip-TbpB/LOS liposomal formulation was demonstrated to induce an effective anti-LOS response due to the adjuvant effect of rlip-TbpB on LOS.

scholarly journals Allogeneic Antigen Composition for Preparing Universal Cancer Vaccines

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Petr G. Lokhov ◽  
Elena E. Balashova
Keyword(s):  
Endothelial Cells ◽  
Vaccine Development ◽  
Present Report ◽  
Tumor Vasculature ◽  
Target Cells ◽  
Human Endothelial Cells ◽  
Cell Surface Antigens ◽  
Tumor Endothelium ◽  
Natural Target

Recently it was demonstrated that tumors induce specific changes to the surface of human endothelial cells thereby providing the basis for designing endothelial cell-based vaccines that directly target antigens expressed by the tumor endothelium. The present report extends these studiesin vitroby investigating the efficacy of allogeneic antigens with regard to their ability to target immune responses against the tumor vasculature since alloantigens simplify vaccine development and implementation in clinical practice. We demonstrated that allogeneic SANTAVAC (Set of All Natural Target Antigens for Vaccination Against Cancer), which presents a specifically prepared composition of cell surface antigens from tumor-stimulated endothelial cells, allows targeting of the tumor vasculature with efficacy of 17, where efficacy represents the killing rate of target cells before normal cells are adversely affected, and efficacy of 60, where efficacy represents the fold decrease in the number of target cells and directly relates to tumor growth arrest. These data suggest that allogeneic SANTAVAC may be considered an antigenic composition that following administration in the presence of respective adjuvants may be clinically tested as a therapeutic or prophylactic universal cancer vaccine without adverse side effects to the normal vasculature.

scholarly journals Targeting Adenoviral Vectors by Using the Extracellular Domain of the Coxsackie-Adenovirus Receptor: Improved Potency via Trimerization

2002 ◽  
Vol 76 (4) ◽  
pp. 1892-1903 ◽  
Author(s):  
Jin Kim ◽  
Theodore Smith ◽  
Neeraja Idamakanti ◽  
Kathy Mulgrew ◽  
Michele Kaloss ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Extracellular Domain ◽  
Adenoviral Vectors ◽  
Target Cells ◽  
Coxsackie Adenovirus Receptor ◽  
Liver Gene ◽  
Peptide Targeting ◽  
Adenovirus Receptor

ABSTRACT Adenovirus binds to mammalian cells via interaction of fiber with the coxsackie-adenovirus receptor (CAR). Redirecting adenoviral vectors to enter target cells via new receptors has the advantage of increasing the efficiency of gene delivery and reducing nonspecific transduction of untargeted tissues. In an attempt to reach this goal, we have produced bifunctional molecules with soluble CAR (sCAR), which is the extracellular domain of CAR fused to peptide-targeting ligands. Two peptide-targeting ligands have been evaluated: a cyclic RGD peptide (cRGD) and the receptor-binding domain of apolipoprotein E (ApoE). Human diploid fibroblasts (HDF) are poorly transduced by adenovirus due to a lack of CAR on the surface. Addition of the sCAR-cRGD or sCAR-ApoE targeting protein to adenovirus redirected binding to the appropriate receptor on HDF. However, a large excess of the monomeric protein was needed for maximal transduction, indicating a suboptimal interaction. To improve interaction of sCAR with the fiber knob, an isoleucine GCN4 trimerization domain was introduced, and trimerization was verified by cross-linking analysis. Trimerized sCAR proteins were significantly better at interacting with fiber and inhibiting binding to HeLa cells. Trimeric sCAR proteins containing cRGD and ApoE were more efficient at transducing HDF in vitro than the monomeric proteins. In addition, the trimerized sCAR protein without targeting ligands efficiently blocked liver gene transfer in normal C57BL/6 mice. However, addition of either ligand failed to retarget the liver in vivo. One explanation may be the large complex size, which serves to decrease the bioavailability of the trimeric sCAR-adenovirus complexes. In summary, we have demonstrated that trimerization of sCAR proteins can significantly improve the potency of this targeting approach in altering vector tropism in vitro and allow the efficient blocking of liver gene transfer in vivo.

scholarly journals Review on Adeno Virus; As a Vaccine Vehicle

2017 ◽  
Vol 2 (3) ◽  
pp. 1-12
Author(s):  
Tadesse B
Keyword(s):  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Human Adenovirus ◽  
Antigen Expression ◽  
Adenovirus Type ◽  
Adenoviral Vectors ◽  
Vaccine Antigen ◽  
Mammalian Host ◽  
Antigen Delivery ◽  
Vaccine Vectors

Adenoviruses have moved to the forefront of vaccinology and are showing substantial prom ise as vehicles for antigen delivery for a number of vaccines currently being developed. Most studies to date have focused on human serotype adenoviruses, particularly human adenovirus type 5. Human serotype adenovirus vaccine vectors are particularly usef ul for development of veterinary vaccines as neutralizing antibodies to the vector will not usually be present in the vaccinates. Most vectors currently used as vaccine carriers are deleted in E1 gene. The original E1 deleted adenoviral vectors were constr ucted by homologous recombination. Replication incompetent vectors contain an antigen expression cassette substituted for the deleted E1A – E1B region. These replication incompitant adenoviruses can not replicate because of the deletion of the essential vir al E1 gene region containing two genes. Replication competent adenoviral vectors encode all of the remaining adenoviral antigens in addition to the transgene product, i.e., the vaccine antigen. The potential for adenoviruses to elicit powerful B cell and T cell responses in the mammalian host are the main reason for the use of these vectors in vaccine development. For effective veterinary use, extensive research on adenoviral vaccine vectors should be undertaken.

scholarly journals An alternative to the adenovirus inverted terminal repeat sequence increases the viral genome replication rate and provides a selective advantage in vitro

2014 ◽  
Vol 95 (7) ◽  
pp. 1574-1584 ◽  
Author(s):  
Kerstin Wunderlich ◽  
Esmeralda van der Helm ◽  
Dirk Spek ◽  
Mark Vermeulen ◽  
Adile Gecgel ◽  
...  
Keyword(s):  
Human Adenovirus ◽  
Selective Advantage ◽  
Adenoviral Vectors ◽  
Repeat Sequence ◽  
Inverted Terminal Repeat ◽  
Genome Replication ◽  
Inverted Terminal Repeat Sequence ◽  
Viral Genome Replication ◽  
Terminal Repeat Sequence

During the development of human adenovirus 35-derived replication-incompetent (rAd35) vaccine vectors for prevention of infectious diseases, we detected mutations in the terminal 8 nt of the inverted terminal repeats (ITRs) of rAd35. The switch from the plasmid-encoded sequence 5′-CATCATCA-3′ to the alternative sequence 5′-CTATCTAT-3′ in the ITRs was found to be a general in vitro propagation phenomenon, as shown for several vectors carrying different transgenes or being derived from different adenovirus serotypes. In each tested case, the plasmid-encoded ITR sequence changed to exactly the same alternative ITR sequence, 5′-CTATCTAT-3′. The outgrowth of this alternative ITR version should result from a growth advantage conferred by the alternative ITR sequence. Indeed, replication kinetics studies of rAd35 harbouring either the original or alternative ITR sequence confirmed an increase in replication speed for rAd35 vectors with the alternative ITR sequence. These findings can be applied to generate recombinant adenoviral vectors harbouring the alternative ITR sequence, which will facilitate the generation of genetically homogeneous seed virus batches. Moreover, vector production may be accelerated by taking advantage of the observed improved replication kinetics associated with the alternative ITR sequence.

Synthesis and in vitro Study of Antiviral and Virucidal Activity of Novel 2-[(4-Methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetamide Derivatives

2011 ◽  
Vol 66 (7-8) ◽  
pp. 333-339
Author(s):  
Monika Wujec ◽  
Tomasz Plech ◽  
Agata Siwek ◽  
Barbara Rajtar ◽  
Małgorzata Polz-Dacewicz
Keyword(s):  
Viral Replication ◽  
Elemental Analysis ◽  
In Vitro Study ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Virucidal Activity ◽  
Hek 293 ◽  
H Nmr ◽  
Adenovirus Type 5

2-[(4-Methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetamide derivatives were synthesized and their structures were confirmed by 1H NMR, IR, and elemental analysis. Cytotoxicity of the compounds towards HEK-293 and GMK cells was evaluated. Moreover, the antiviral and virucidal activities of these compounds against human adenovirus type 5 and ECHO-9 virus were assessed. Some of the newly synthesized derivatives have the potential to reduce the viral replication of both tested viruses.

scholarly journals Expression of Thanatin in HEK293 Cells and Investigation of its Antibacterial Effects on Some Human Pathogens

2019 ◽  
Vol 27 (1) ◽  
pp. 41-47
Author(s):  
Abbas Tanhaeian ◽  
Marjan Azghandi ◽  
Zahra Mousavi ◽  
Ali Javadmanesh
Keyword(s):  
Antimicrobial Activity ◽  
Negative Control ◽  
Hek293 Cells ◽  
Cytotoxicity Test ◽  
Human Pathogens ◽  
Hek 293 ◽  
Mammalian Cell Lines ◽  
Beta Hairpin ◽  
Co Precipitation ◽  
Conventional Antibiotics

Background: Thanatin is the smallest member of Beta-hairpin class of cationic peptide derived from insects with vast activities against various pathogens. Objective: n this study, the antimicrobial activity of this peptide against some species of human bacterial pathogens as well as its toxicity on NIH cells were evaluated. Method: Thanatin DNA sequence was cloned into pcDNA3.1+ vector and transformed into a DH5α bacterial strain. Then the recombinant plasmids were transfected into HEK-293 cells by calcium phosphate co-precipitation. After applying antibiotic treatment, the supernatant medium containing thanatin was collected. The peptide quantity was estimated by SDS-PAGE and GelQuant software. The antimicrobial activity of this peptide was performed with Minimum Inhibitory Concentration (MIC) method. In addition, its toxicity on NIH cells were evaluated by MTT assay. Results: The peptide quantity was estimated approximately 164.21 µmolL-1. The antibacterial activity of thanatin was estimated between 0.99 and 31.58 µmolL-1 using MIC method. The result of cytotoxicity test on NIH cell line showed that the peptide toxicity up to the concentration of 394.10 µmolL-1 and for 48 hours, was not statistically significant from negative control cells (P>0.05). The antimicrobial assay demonstrated that thanatin had an antibacterial effect on some tested microorganisms. The results obtained in this study also showed that thanatin had no toxicity on mammalian cell lines including HEK293 and NIH. Conclusion: Antimicrobial peptides such as thanatin are considered to be appropriate alternatives to conventional antibiotics in treating various human pathological diseases bacteria.

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