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Generation of a novel chimeric PALFn antigen of Bacillus anthracis and its immunological characterization in mouse model

  • Applied genetics and molecular biotechnology
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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.

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Acknowledgments

The authors are thankful to the Director, Defence Research and Development Establishment, Gwalior, for providing financial support and all necessary facilities to carry out this work.

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Authors and Affiliations

  1. Microbiology Division, Defence Research and Development Establishment, Jhansi Road, Gwalior, Madhya Pradesh, 474002, India

    Nagendra Suryanarayana, Monika Verma, Kulanthaivel Thavachelvam, Bharti Mankere, Urmil Tuteja & Vanlal Hmuaka

  2. Pharmacology and Toxicology Division, Defence Research and Development Establishment, Jhansi Road, Gwalior, Madhya Pradesh, 474002, India

    Nandita Saxena

Authors
  1. Nagendra Suryanarayana
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  2. Monika Verma
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  3. Kulanthaivel Thavachelvam
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  4. Nandita Saxena
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  5. Bharti Mankere
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  6. Urmil Tuteja
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  7. Vanlal Hmuaka
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Correspondence to Vanlal Hmuaka.

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The Institutional Animal Ethics Committee (IAEC) of Defence Research and Development Establishment approved all the protocols for experiments conducted using mice (wide registration number 37/Go/C/1999/CPCSEA) and the Institutional Biosafety committee (IBSC) as per the institutional norms. The principles of good laboratory animal care were followed all through the experimental process. The mice were maintained in accordance with recommendations of the committee for the purpose of control and supervision of experiments on animals, Government of India, by providing food and water ad libitum.

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The authors declare that they have no conflict of interest.

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Suryanarayana, N., Verma, M., Thavachelvam, K. et al. Generation of a novel chimeric PALFn antigen of Bacillus anthracis and its immunological characterization in mouse model. Appl Microbiol Biotechnol 100, 8439–8451 (2016). https://doi.org/10.1007/s00253-016-7684-4

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