Abstract
Bacillus anthracis has long been considered a potential biological warfare agent, and therefore, there is a need for a safe, low-cost and highly efficient anthrax vaccine with demonstrated long-term stability for mass vaccination in case of an emergency. Many efforts have been made towards developing an anthrax vaccine based on recombinant protective antigen (rPA) of B. anthracis, a key component of the anthrax toxin, produced using different expression systems. Plants represent a promising recombinant protein production platform due to their relatively low cost, rapid scalability and favorable safety profile. Previous studies have shown that full-length rPA produced in Nicotiana benthamiana (pp-PA83) is immunogenic and can provide full protection against lethal spore challenge; however, further improvement in the potency and stability of the vaccine candidate is necessary. PA of B. anthracis is not a glycoprotein in its native host; however, this protein contains potential N-linked glycosylation sites, which can be aberrantly glycosylated during expression in eukaryotic systems including plants. This glycosylation could affect the availability of certain key epitopes either due to masking or misfolding of the protein. Therefore, a non-glycosylated form of pp-PA83 was engineered and produced in N. benthamiana using an in vivo deglycosylation approach based on co-expression of peptide-N-glycosidase F (PNGase F) from Flavobacterium meningosepticum. For comparison, versions of pp-PA83 containing point mutations in six potential N-glycosylation sites were also engineered and expressed in N. benthamiana. The in vivo deglycosylated pp-PA83 (pp-dPA83) was shown to have in vitro activity, in contrast to glycosylated pp-PA83, and to induce significantly higher levels of toxin-neutralizing antibody responses in mice compared with glycosylated pp-PA83, in vitro deglycosylated pp-PA83 or the mutated versions of pp-PA83. These results suggest that pp-dPA83 may offer advantages in terms of dose sparing and enhanced immunogenicity as a promising candidate for a safe, effective and low-cost subunit vaccine against anthrax.
Highlights
Anthrax is an acute disease caused by the bacterium Bacillus anthracis, affecting both humans and animals
The most advanced vaccine candidates are based on recombinant protective antigen (PA) expressed in and purified from Escherichia coli [5], or PA prepared from an asporogenic, non-toxigenic, non-encapsulated strain of B. anthracis [6,7]. rPA-based vaccines have been shown to induce high-titers of anti-PA toxin-neutralizing antibody (TNA) responses in animals and protect rabbits and nonhuman primates against lethal B. anthracis challenge [12,13]; in some studies protection waned dramatically over 6 to 12 months [13], indicating a need for vaccine formulations that can induce stronger, more robust long-lasting immunity
Purified plantproduced glycosylated PA83 (pp-PA83) deglycosylated by treatment with commercial PNGase F in vitro was used as a control
Summary
Anthrax is an acute disease caused by the bacterium Bacillus anthracis, affecting both humans and animals. Anthrax Vaccine Adsorbed (BioThrax1), licensed in 1972, is the only U.S Food and Drug Administration (FDA)-licensed human anthrax vaccine in the U.S The vaccine contains the 83-kDa PA protein prepared from cell-free filtrates of microaerophilic cultures of an avirulent, non-encapsulated strain of B. anthracis, adsorbed to aluminum hydroxide gel as an adjuvant. The vaccine induces only limited protection and requires a lengthy course of administration to achieve protective immunity [3,4]. These drawbacks have led to increased efforts in recent years to develop new, second-generation anthrax vaccines, including recombinant live and recombinant subunit vaccines [5,6,7,8,9,10,11]. The most advanced vaccine candidates are based on recombinant PA (rPA) expressed in and purified from Escherichia coli [5], or PA prepared from an asporogenic, non-toxigenic, non-encapsulated strain of B. anthracis [6,7]. rPA-based vaccines have been shown to induce high-titers of anti-PA toxin-neutralizing antibody (TNA) responses in animals and protect rabbits and nonhuman primates against lethal B. anthracis challenge [12,13]; in some studies protection waned dramatically over 6 to 12 months [13], indicating a need for vaccine formulations that can induce stronger, more robust long-lasting immunity
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