scholarly journals HHi-FiVe: A high-fidelity genetic engineering pipeline for construction of herpesvirus-based vaccines

2021 ◽  
Author(s):  
Michael Jarvis ◽  
Thekla Mauch ◽  
Eleonore Ostermann ◽  
Yvonne Wezel ◽  
Jenna Nichols ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Genetic Manipulation ◽  
Rhesus Macaques ◽  
High Fidelity ◽  
Bacterial Artificial Chromosomes ◽  
Virus Challenge ◽  
Artificial Chromosomes ◽  
Vector Construction ◽  
Animal Populations ◽  
Cellular Tropism

Abstract Herpesvirus-based vectors are attractive for use both as conventional and as transmissible vaccines against emerging zoonoses in hard-to-reach animal populations. However, the threat of off-site mutations during genetic manipulation of vector genomes poses a significant challenge to vaccine construction. Herein, we present the HHi-FiVe (herpesvirus high-fidelity vector) construction pipeline for generating herpesvirus-based vectors by modifying bacterial artificial chromosomes (BACs) and monitoring integrity at each stage by complete genome sequencing. We used this pipeline to repair a highly mutated rhesus cytomegalovirus BAC containing an Ebola virus transgene. The vector derived from this BAC had been shown previously to protect rhesus macaques from lethal Ebola virus challenge by conventional vaccination. Repair of this BAC restored wild-type cellular tropism to the vector, which is essential for transmissible vaccination. Construction of this candidate transmissible vaccine against Ebola virus demonstrates the utility of the HHi-FiVe pipeline for creating precision-made herpesvirus-based vectors.

scholarly journals Targeted Mutagenesis of Guinea Pig Cytomegalovirus Using CRISPR/Cas9-Mediated Gene Editing

2016 ◽  
Vol 90 (15) ◽  
pp. 6989-6998 ◽  
Author(s):  
Craig J. Bierle ◽  
Kaitlyn M. Anderholm ◽  
Jian Ben Wang ◽  
Michael A. McVoy ◽  
Mark R. Schleiss
Keyword(s):  
Guinea Pig ◽  
Viral Genome ◽  
Genetic Manipulation ◽  
Spontaneous Mutation ◽  
Lytic Replication ◽  
Targeted Mutagenesis ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes ◽  
Guinea Pig Cytomegalovirus ◽  
Infected Cells

ABSTRACTThe cytomegaloviruses (CMVs) are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kbp. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes (BACs), which necessitates the maintenance of the viral genome inEscherichia coliand successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection were used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology-driven repair. As CRISPR/Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate genetic manipulation of low-passage or clinical CMV isolates.IMPORTANCEThe cytomegalovirus genome is complex, and viral adaptations to cell culture have complicated the study of infectionin vivo. Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small-animal model of congenital cytomegalovirus disease. CRISPR/Cas9 mutagenesis can introduce the same types of mutations to the viral genome as bacterial artificial chromosome recombineering but does so directly in virus-infected cells. CRISPR/Cas9 mutagenesis is not dependent on a bacterial intermediate, and defined viral mutants can be recovered after a limited number of viral genome replications, minimizing the risk of spontaneous mutation.

scholarly journals Herpesvirus BACs: Past, Present, and Future

2011 ◽  
Vol 2011 ◽  
pp. 1-16 ◽  
Author(s):  
Charles Warden ◽  
Qiyi Tang ◽  
Hua Zhu
Keyword(s):  
Vaccine Development ◽  
Genetic Manipulation ◽  
Large Family ◽  
Site Directed Mutagenesis ◽  
Bacterial Artificial Chromosomes ◽  
Dna Viruses ◽  
Recombinant Viruses ◽  
Artificial Chromosomes ◽  
Viral Genes ◽  
Functional Analyses

The herpesviridae are a large family of DNA viruses with large and complicated genomes. Genetic manipulation and the generation of recombinant viruses have been extremely difficult. However, herpesvirus bacterial artificial chromosomes (BACs) that were developed approximately 10 years ago have become useful and powerful genetic tools for generating recombinant viruses to study the biology and pathogenesis of herpesviruses. For example, BAC-directed deletion mutants are commonly used to determine the function and essentiality of viral genes. In this paper, we discuss the creation of herpesvirus BACs, functional analyses of herpesvirus mutants, and future applications for studies of herpesviruses. We describe commonly used methods to create and mutate herpesvirus BACs (such as site-directed mutagenesis and transposon mutagenesis). We also evaluate the potential future uses of viral BACs, including vaccine development and gene therapy.

scholarly journals Cross-Species RNAi Rescue Platform in Drosophila melanogaster

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 1165-1173 ◽  
Author(s):  
Shu Kondo ◽  
Matthew Booker ◽  
Norbert Perrimon
Keyword(s):  
Cell Culture ◽  
Drosophila Melanogaster ◽  
Large Scale ◽  
Transgenic Animals ◽  
Selection Marker ◽  
Bacterial Artificial Chromosomes ◽  
Loss Of Function ◽  
Artificial Chromosomes ◽  
Target Effects

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

Homologous Recombination Using Bacterial Artificial Chromosomes

2015 ◽  
Vol 2015 (2) ◽  
pp. pdb.prot072397
Author(s):  
Cary Lai ◽  
Tobias Fischer ◽  
Elizabeth Munroe
Keyword(s):  
Homologous Recombination ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes

scholarly journals The Nucleoprotein Is Required for Efficient Coronavirus Genome Replication

2004 ◽  
Vol 78 (22) ◽  
pp. 12683-12688 ◽  
Author(s):  
Fernando Almazán ◽  
Carmen Galán ◽  
Luis Enjuanes
Keyword(s):  
Heterologous Protein ◽  
Cytopathic Effect ◽  
Human Cell Line ◽  
Genome Replication ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes ◽  
Restriction Sites ◽  
Unique Restriction ◽  
Transmissible Gastroenteritis ◽  
Transmissible Gastroenteritis Coronavirus

ABSTRACT The construction of a set of transmissible gastroenteritis coronavirus (TGEV)-derived replicons as bacterial artificial chromosomes is reported. These replicons were generated by sequential deletion of nonessential genes for virus replication, using a modified TGEV full-length cDNA clone containing unique restriction sites between each pair of consecutive genes. Efficient activity of TGEV replicons was associated with the presence of the nucleoprotein provided either in cis or in trans. TGEV replicons were functional in several cell lines, including the human cell line 293T, in which no or very low cytopathic effect was observed, and expressed high amounts of heterologous protein.

scholarly journals Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study

The Lancet ◽  
2010 ◽  
Vol 375 (9729) ◽  
pp. 1896-1905 ◽  
Author(s):  
Thomas W Geisbert ◽  
Amy CH Lee ◽  
Marjorie Robbins ◽  
Joan B Geisbert ◽  
Anna N Honko ◽  
...  
Keyword(s):  
Rna Interference ◽  
Ebola Virus ◽  
Proof Of Concept ◽  
Virus Challenge ◽  
Concept Study

scholarly journals Assessment of immunogenicity and protective efficacy of ZyCoV-D DNA vaccine candidates in Rhesus macaques against SARS-CoV-2 infection

2021 ◽  
Author(s):  
Pragya D Yadav ◽  
Sanjay Kumar ◽  
Kshitij Agarwal ◽  
Mukul Jain ◽  
Dilip R Patil ◽  
...  
Keyword(s):  
Vaccine Candidate ◽  
Protective Efficacy ◽  
Rhesus Macaques ◽  
Herd Immunity ◽  
Lavage Fluid ◽  
Injection System ◽  
Protective Measure ◽  
Vaccine Candidates ◽  
Virus Challenge ◽  
Syringe Needle

AbstractVaccines remain the key protective measure to achieve herd immunity to control the disease burden and stop COVID-19 pandemic. We have developed and assessed the immunogenicity and protective efficacy of two formulations (1mg and 2mg) of ZyCoV-D (a plasmid DNA based vaccine candidates) administered through Needle Free Injection System (NFIS) and syringe-needle (intradermal) in rhesus macaques with three dose vaccine regimens. The vaccine candidate 2mg dose administered using Needle Free Injection System (NFIS) elicited a significant immune response with development of SARS-CoV-2 S1 spike region specific IgG and neutralizing antibody (NAb) titers during the immunization phase and significant enhancement in the levels after the virus challenge. In 2 mg NFIS group the IgG and NAb titers were maintained and showed gradual rise during the immunization period (15 weeks) and till 2 weeks after the virus challenge. It also conferred better protection to macaques evident by the viral clearance from nasal swab, throat swab and bronchoalveolar lavage fluid specimens in comparison with macaques from other immunized groups. In contrast, the animals from placebo group developed high levels of viremia and lung disease following the virus challenge. Besides this, the vaccine candidate also induced increase lymphocyte proliferation and cytokines response (IL-6, IL-5).The administration of the vaccine candidate with NFIS generated a better immunogenicity response in comparison to syringe-needle (intradermal route). The study demonstrated immunogenicity and protective efficacy of the vaccine candidate, ZyCoV-D in rhesus macaques.

Sign in / Sign up

Export Citation Format

Share Document