Development of Rickettsia prowazekii DNA vaccine: cloning strategies.

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.