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Published on: October 11, 2022
Development of Rickettsia prowazekii DNA vaccine: cloning strategies
Christopher Coker1, Mahreen Majid, Suzana Radulovic
1Department of Microbiology and Immunology, School of Medicine, University of Maryland, 655 West Baltimore Street, Baltimore, Maryland 21201, USA.
Abstract:
Rickettsia prowazekii, the etiologic agent of louse-borne typhus, is listed as a category B agent under the select agent list of the United States Centers for Disease Control and Prevention. R. prowazekii was placed on the select agent list due to its potential to cause epidemic, high mortality in untreated and/or misdiagnosed cases, and ease of spread in vulnerable populations. Historically, R. prowazekii vaccines using crude antigen and/or inactivated rickettsia were partially protective but have been accompanied with undesirable toxic reactions and difficulties in standardization. The availability of the genome sequence of R. prowazekii allowed us to select genes that encode proteins with potential in immuno-protection against this human pathogen. We successfully PCR-amplified a group of genes involved in invasion (invA), cell division (fts), protein secretion (sec gene family), and virulence (ompA and ompB, virB gene family, cap and tlyA and tlyC). The generated PCR products were cloned into the Gateway cloning system and the cloned products will be introduced into Vical VR 1020-DV and VR 1012-DV DNA vaccine plasmids. Twenty-four target genes from R. prowazekii have been PCR amplified, of which fifteen have been introduced into the pENTR/SD/D-TOPO entry cloning vector.
Insights
Developing a new vaccine against Rickettsia prowazekii, the cause of typhus, involves identifying protective genes. This research PCR-amplifies and clones key R. prowazekii genes for potential DNA vaccine development.
Area of Science:
- Microbiology
- Vaccinology
- Molecular Biology
Background:
- Rickettsia prowazekii causes epidemic typhus, a significant public health concern.
- Current vaccines for R. prowazekii are limited by toxicity and standardization issues.
- R. prowazekii is a Category B select agent due to its epidemic potential and ease of spread.
Purpose of the Study:
- To identify and characterize genes of Rickettsia prowazekii with potential for immuno-protection.
- To develop a novel DNA vaccine strategy against R. prowazekii infections.
Main Methods:
- Genome sequencing of R. prowazekii to identify target genes.
- Polymerase Chain Reaction (PCR) amplification of selected virulence and invasion genes.
- Cloning of PCR products into the Gateway system for DNA vaccine plasmid integration.
Main Results:
- Successfully PCR-amplified 24 target genes from R. prowazekii.
- Fifteen amplified genes, including those involved in invasion, cell division, and virulence, were cloned into an entry vector.
- Selected genes encode proteins crucial for R. prowazekii pathogenesis.
Conclusions:
- Identification and cloning of R. prowazekii genes provide a foundation for developing subunit or DNA vaccines.
- This approach bypasses the limitations of traditional inactivated or crude antigen vaccines.
- Further research will focus on incorporating these genes into DNA vaccine plasmids for immunogenicity testing.
