title: Generation of a novel chimeric PALFn antigen of Bacillus anthracis and its immunological characterization in mouse model.
abstract: Bacillus anthracis chimeric molecule PALFn, comprising the immunodominant domains of protective antigen (PA) and lethal factor (LF), has been developed in the past and has been shown to confer enhanced protection against anthrax in mouse model when challenged with anthrax lethal toxin (LeTx). However, the immunological correlates for this chimeric antigen, both in terms of humoral as well as cell-mediated immune responses, have not been described in detail. To address this gap, we have determined the immunological responses both at humoral as well as cellular levels for the protection conferred by the novel chimeric antigen PALFn constructed in our laboratory in comparison to PA antigen. The biological functionality of the chimeric antigen was ascertained by the trypsin digestion assay. The trypsin cleavage activated the functionality of PALFn and rendered it to interact and bind with the LF molecule. Similarly, the LFn component in the chimera could independently interact and bind to the trypsin-activated wild-type PA. Further, it was observed that the PALFn-immunized mice sera could readily react to both PA and LF antigens while PA-immunized mice sera showed reaction to PA and PALFn alone and not to the individual LF antigen. The in vitro toxin neutralizing ability of PALFn antisera on macrophage cell line J774.1 was robust but with 1.3-fold lesser titer than PA-immunized antisera. PALFn-immunized mouse splenocytes showed a significant lymphocyte proliferation when stimulated with PALFn. There was a remarkable increase in the level of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin 10 (IL-10), interferon-γ (IFN- γ), and tumor necrosis factor α (TNFα) from PALFn- and PA-stimulated splenocytes. In addition, there was a significant increase in antigen-specific CD4+ and CD8+ T-cell counts from both PALFn- and PA-immunized mouse splenocytes. The results clearly demonstrate the ability of chimeric molecule PALFn in eliciting robust humoral and cell-mediated immune responses in mouse model that is parallel to the wild-type PA but has additional anti-LF antibody response. Considering the enhanced protection offered by the chimera PALFn, we can conclude that it can be a better alternative to the wild-type PA-based recombinant vaccine against anthrax.
authors.affiliation: Microbiology Division, Defence Research and Development Establishment, Jhansi Road, Gwalior, Madhya Pradesh, 474002, India.
authors.forename: Nagendra
authors.initials: N
authors.lastname: Suryanarayana
authors.affiliation: Microbiology Division, Defence Research and Development Establishment, Jhansi Road, Gwalior, Madhya Pradesh, 474002, India.
authors.forename: Monika
authors.initials: M
authors.lastname: Verma
authors.affiliation: Microbiology Division, Defence Research and Development Establishment, Jhansi Road, Gwalior, Madhya Pradesh, 474002, India.
authors.forename: Kulanthaivel
authors.initials: K
authors.lastname: Thavachelvam
authors.affiliation: Pharmacology and Toxicology Division, Defence Research and Development Establishment, Jhansi Road, Gwalior, Madhya Pradesh, 474002, India.
authors.forename: Nandita
authors.initials: N
authors.lastname: Saxena
authors.affiliation: Microbiology Division, Defence Research and Development Establishment, Jhansi Road, Gwalior, Madhya Pradesh, 474002, India.
authors.forename: Bharti
authors.initials: B
authors.lastname: Mankere
authors.affiliation: Microbiology Division, Defence Research and Development Establishment, Jhansi Road, Gwalior, Madhya Pradesh, 474002, India.
authors.forename: Urmil
authors.initials: U
authors.lastname: Tuteja
authors.affiliation: Microbiology Division, Defence Research and Development Establishment, Jhansi Road, Gwalior, Madhya Pradesh, 474002, India. vanlalh.drdo@gmail.com.
authors.forename: Vanlal
authors.initials: V
authors.lastname: Hmuaka
mesh_terms: D000818:Animals; D000881:Anthrax / Q000517:prevention & control; D022122:Anthrax Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D000907:Antibodies, Bacterial / Q000097:blood; D057134:Antibodies, Neutralizing / Q000097:blood; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D000992:Antitoxins / Q000097:blood; D001408:Bacillus anthracis / Q000276:immunology*; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology*; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival / Q000187:drug effects; D016207:Cytokines / Q000378:metabolism; D008214:Lymphocytes / Q000276:immunology; D008264:Macrophages / Q000187:drug effects / Q000502:physiology; D051379:Mice; D009500:Neutralization Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology*; D013154:Spleen / Q000276:immunology; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology
chemical_list: D022122:Anthrax Vaccines; D000907:Antibodies, Bacterial; D057134:Antibodies, Neutralizing; D000942:Antigens, Bacterial; D000992:Antitoxins; D001427:Bacterial Toxins; D016207:Cytokines; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; C030325:anthrax toxin
keywords: Bacillus anthracis; Chimeric PALFn antigen; Lethal factor; Protective antigen; Trypsin