Recombinant Bacillus anthracis anthrax protective antigen

Bacillus anthracis is a Gram-positive bacterium that can form spores in vitro, and the spores can survive in the soil for several years. Anthrax is divided into three types: cutaneous anthrax, inhalation anthrax, and gastrointestinal anthrax. It is a highly contagious disease that is transmitted between humans and animals, often occurring among herbivores, and is also a potential biological weapon. Vaccines and therapeutic antibodies are effective means of preventing anthrax before and after exposure. There are two types of vaccines currently in use: one is the attenuated live anthrax vaccine; the other is a vaccine with protective antigen (PA) as the main component, including the anthrax adsorbed vaccine in the United States and the anthrax precipitation vaccine in the United Kingdom. Due to problems with the route of administration or technical issues, the ingredients of these vaccines are uncertain or unstable. In recent years, a new generation of improved attenuated live vaccines, inactivated spore vaccines, plant vaccines, subunit vaccines, DNA vaccines, peptide-based/multi-epitope vaccines, chimeric antigen vaccines, etc. have been under development. The main means of post-exposure prevention is therapeutic antibodies. Currently, the FDA has approved three antitoxin antibodies against PA for inhalation anthrax, namely, raxibamab, obituximab, and polyclonal anthrax immunoglobulin IV. However, antitoxin antibodies cannot cross the blood-brain barrier, and their protective efficacy is significantly reduced with delayed administration after exposure and the development of meningitis. Given the limitations of anthrax immunotherapy (hyperimmune polyclonal serum, monoclonal antibodies), it is urgent to develop safer and more effective treatments.

Figure 1. Modified anthrax protective antigen (PA)-mediated endocytosis of LFN-C3. (Sources: Zahaf NI, et al.2017)

PA is a key component of anthrax toxin, encoded by the pag gene on the pXO1 plasmid of Bacillus anthracis. It is a calcium-binding monomer protein with a relative molecular mass of 83,000. PA can attach to host cells, bind to anthrax toxin receptors, and oligomerize on the cell membrane surface to form channels, bind to lethal factor (LF) or edema factor (EF), allowing it to enter the cytoplasm and cause infection. PA has been shown to effectively induce protective immunity. Vaccines are an effective means of pre-exposure prevention, and the long-term stability of vaccines is an ideal feature of anthrax vaccines. It has been reported that the deamidation of asparagine residues of recombinant protective antigens (rPA) may affect the stability and immunogenicity of rPA vaccine antigens during long-term storage. Therefore, PA mutants have also become a research hotspot in recent years. Compared with natural rPA, PA mutants are more stable. It has been confirmed that PA mutants can become strong candidate antigens for long-term stability. For people currently infected with anthrax, PA as an antigen will promote the formation of lethal toxin (LT) or edema toxin (ET), causing serious harm. A better choice is a PA mutant, which is non-functional and can induce higher protective antibodies while ensuring that PA is non-toxic.

PA can be divided into four domains. Domain 1 (amino acids 1-258) contains a furin cleavage site that leads to the release of the amino-terminal fragment (PA20) and heptamerization of the rest of the protein (PA63) through monomer interactions on the cell surface. PA63 is cleaved by furin, exposing the site for binding to LF or EF. Domain 2 (amino acids 259-487) facilitates the binding of PA63 monomers and this domain contacts the host cell receptor binding site. Domain 3 (amino acids 488-595) and domain 2 form a heptamer pore on the cell surface that allows LF or EF binding, resulting in the entry of the toxin complex into the cell via receptor-mediated endocytosis. Domain 4 (amino acids 596-735) contains the host cell receptor binding site, and once in the cytosol, LF and EF are able to carry out their respective damage-inducing processes. To explore the immunogenicity of each antigen, the researchers expressed full-length PA, domain 1, domains 2-4, and domain 4 proteins. The researchers used these four proteins to immunize four strains of mice (A/J, BALB/c, C57BL/6, and Swiss Webster) and found that the ability of the four proteins to produce antibodies and the ability to neutralize toxins in vitro varied among the mouse strains. The LT neutralizing antibody titer produced by full-length PA immunization was significantly increased. Interestingly, PA domain 1 immunization was highly immunogenic in C57BL/6 mice and induced LT neutralizing antibodies, confirming that domain 1 may contain neutralizing antibody reactive epitopes. Some scholars synthesized and expressed fusion proteins GST1, GST1b-2, GST1-2, GST1b-3, GST1-3, GST2-4, GST3-4, GST4 and GST1-4, which were fused with different overlapping polypeptide regions and N-terminal glutathione S-transferase (GST), and found that rPA truncated proteins GST1, GST1b-2 and GST1-2 were easily degraded in the absence of domain 3, indicating that domain 3 can stabilize domain 1 and domain 2. However, regardless of whether there is a protective effect, the qualitative immune response stimulated by immunization with the complete or partial domains of rPA is the same, while the protective effect of mice immunized with PA without expressing 4 is reduced, indicating that domain 4 may be the main antigen causing immune stimulation. Other researchers cloned and expressed PA domain 1, domain 1-2, domain 2-3, and domain 3-4. All four proteins induced high antibody titers, among which domain 3-4 showed the highest immune response to PA, confirming that domain 4 may be an effective antigen for future anthrax vaccines. Some studies have expressed individual domains of PA in the form of recombinant proteins in Escherichia coli, and prepared mouse polyclonal antisera for verification. The titer and isotype of specific antibodies for each domain were determined by ELISA. The results showed that the IgG1 isotype was dominant, and the antiserum against PA4 could effectively neutralize LT. No significant neutralization effect of LT was observed with other specific antibodies, indicating that antibodies against PA4 have the potential to be used as therapeutic antitoxin molecules and in combination with appropriate anti-anthrax antibiotics.

ATPA
Anthrax toxin protective antigen
Protective antigen component of anthrax toxin
Bacillus anthracis protective antigen