Abstract
Anthrax vaccines primarily relying only on protective antigen (PA), the cell binding component in anthrax toxins provide incomplete protection when challenged with spores of virulent encapsulated Bacillus anthracis strains. Alternatively, formaldehyde inactivated spores (FIS) or recombinant spore components generate anti-spore immune responses that inhibit the early stages of infection and augment the PA protective efficacy. In the present study domain IV of PA was spliced with exosporium antigen BxpB via a flexible G4S linker to generate a single functional antigen r-PAbxpB that was further assessed for its protective efficacy against anthrax toxins and spore infection. Immunization of mice with r-PAbxpB elicited significantly high titer antibodies comprising IgG1:IgG2a isotypes in 1:1 ratio, balanced up-regulation of both Th1 (IL2, IL12, IFN-γ) and Th2 (IL4, IL5, IL10) cytokines and high frequencies of CD4+ and CD8+ T cell subsets. The anti-r-PAbxpB antibodies significantly enhanced spore phagocytosis, and killing within macrophages; inhibited their germination to vegetative cells and completely neutralized the anthrax toxins as evidenced by the 100% protection in passive transfer studies. Active immunization with r-PAbxpB provided 100 and 83.3% protection in mice I.P. challenged with 5 × LD100 LD of toxins and 5 × 104 cfu/ml Ames spores, respectively while the sham immunized group succumbed to infection in 48 h. Therefore, the ability of r-PAbxpB to generate protective immune responses against both spores and toxin and provide significant protection suggests it as an efficient vaccine candidate against B. anthracis infection.
Highlights
The Gram positive, spore forming bacilli Bacillus anthracis is the etiological agent of anthrax, a fatal zoonotic disease that primarily affects ungulates and humans and is categorized as Category A bio threat agent by Center for Disease Control and Prevention [1]
The gene was ligated into pRSET A vector by restriction ligation mediated cloning and transformed into E. coli BL21 (DE3) and the integrity of cloned gene in r-pRSETA-PAbxpB plasmid was authenticated by sequencing
The expression of the r-PAbxpB fusion protein (39.9 kDa) was confirmed in 12% SDS PAGE gel stained with Coomasie Blue (Figure 1)
Summary
The Gram positive, spore forming bacilli Bacillus anthracis is the etiological agent of anthrax, a fatal zoonotic disease that primarily affects ungulates and humans and is categorized as Category A bio threat agent by Center for Disease Control and Prevention [1]. The protective antigen (PA) forms a common host cell binding moiety for both Lethal factor (LF) and Edema factor (EF) components of the anthrax toxin and aids in their transport to the host cell cytosol The binding of this 83 kDa protein with the LF and EF happens through the domain IV (PAIV) while the other domains (I–III) facilitate proteolytic activation, heptamerization, and host cell binding [10]. The 17 kDa protein BxpB ( known as ExsFA) is located in the exosporium basal layer and is necessary for the residual attachment of BclA filaments to the exosporium thereby maintaining the exosporium assembly [16] This exosporium protein is not protective by itself, but augments the protective efficacy of PA based vaccines by enhancing spore uptake and killing by macrophages as evidenced by the increase in mean time to death against spore challenge [17]. The fusion protein was further characterized for its correlates of protection and ability to provide concurrent protection against both B. anthracis spores and toxins
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