Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants

Abstract

The gene encoding Bacillus anthracis protective antigen (PA) was modified by site-directed mutagenesis, subcloned into baculovirus and vaccinia virus plasmid transfer vectors (pAcYMl and pSC-1l, respectively), and inserted via homologous recombinations into baculovirus Autographa californica nuclear polyhedrosis virus or vaccinia virus (strains WR and Connaught). Expression of PA was detected in both systems by immunofluorescence assays with antisera from rabbits immunized with B. anthracis PA. Western blot (immunoblot) analysis showed that the expressed product of both systems was slightly larger (86 kilodaltons) than B. anthracis-produced PA (83.5 kilodaltons). Analysis of trypsin digests of virus-expressed and authentic PA suggested that the size difference was due to the presence of a signal sequence remaining with the virus-expressed protein. Immunization of mice with either recombinant baculovirus-infected Spodoptera frugiperda cells or with vaccinia virus recombinants elicited a high-titer, anti-PA antibody response.

Expression and secretion of the protective antigen ofBacillus anthracisinBacillus brevis

“…The yield of PA in B. anthracis is very low and even highly purified PA was contaminated with small amounts of EF and LF, which may cause occasional mild side effects [28]. Therefore, many researchers have tried to express the PA gene in other bacterial systems such as E. coli [14], Baculovirus [29], Vaccinia virus [29], and B. subtilis [15,16]. Recently, a new candidate vaccine was constructed in which the capsule of B. anthracis, a poly-c-D-glutamic acid, was conjugated to PA [30].…”

The development of new vaccines against Bacillus anthracis

“…In the medium term a subunit vaccine based on PA is the only candidate likely to receive licensing approval. As a consequence there have been numerous attempts to develop high level PA expression systems based on a variety of organisms; attenuated strains of B. anthracis (Belton and Strange 1954;Turnbull 1991;Ivins et al 1998), B. subtilis (Ivins and Welkos 1986;Baillie et al 1994Baillie et al , 1996Baillie et al , 1998, B. brevis (Oh et al 1998), Vaccinia andBaculovirus (Iancono-Connors et al 1990), Salmonella typhimurium (Coulson 1993) and E. coli (Vodkin and Leppla 1983;Singh et al 1989). While none of these systems is ideal, to date, the best reported yields have been achieved with Bacillus spp.…”

“…The PA gene, which is plasmid encoded, is expressed well in B. subtilis achieving levels of expression higher than those obtained with the current B. anthracis based system (Ivins and Welkos 1986). A drawback of using bacilli is their ability to produce degradative proteases and initial attempts to produce fullsize rPA and develop downstream puri®cation protocols were hampered by proteolytic degradation (Xu-Chu et al 1991;Baillie et al 1994Baillie et al , 1996Baillie et al , 1998.…”

“…Following delivery the organism either colonizes the mucosal surface or causes a limited infection during which the vaccine candidate is expressed and presented to the immune system: the Sterne animal vaccine represents such as vaccine. To date a number of live vector systems have been described for PA, attenuated B. anthracis (Barnard and Friedlander 1999;Cohen et al 2000), B. subtilis (Ivins and Welkos 1986), Salmonella (Coulson et al 1994), Lactobacillus (Zeger et al 1999) and vaccinia .…”

High‐level expression and single‐step purification of recombinant Bacillus anthracis protective antigen from Escherichia coli

“…More recently, PA has been expressed and purified from other expression hosts, such as Bacillus subtilis, baculovirus and Escherichia coli. rPA (recombinant PA) from all sources was demonstrated to induce similar immune responses in animals, such as mice, rabbits and rhesus macaque monkeys [10][11][12][13]. rPA expressed and purified from E. coli showed comparable biological activity to PA obtained from B. anthracis [14][15][16][17].…”