Chapter 22
Vaccine Delivery with a Detoxified Bacterial Toxin
Diana Diaz-Are ´ valo, Yanping Chen, and Mingtao Zeng
Abstract
It is still a challenge to develop needle-free mucosal vaccines. Despite progress in the development of the
influenza vaccine, it must be reformulated annually because of antigenic changes in circulating influenza
viral strains. Due to seasonal drift and shift of circulating strains, the influenza vaccine does not always match
the circulating strains, and included adjuvants are not sufficient to induce a protective effect with long-lived
memory cells. The adjuvants play a major role in the immune responses to a vaccine. Interestingly, the
Bacillus anthracis detoxified anthrax edema toxin, which composes of protective antigen PA and
N-fragment of edema factor (EFn), has shown improved effects for humoral and cellular immune
responses. Here we describe the design of a universal influenza vaccine construct that consists of three
tandem M2e repeats of the influenza antigen plus HA2 and detoxified toxin EFn, which is associated with
the PA component, as well as the techniques used to corroborate protection. We present two major parts of
description to demonstrate the vaccine strategy, using detoxified anthrax toxin for intranasal delivery of
influenza antigen: (1) vaccine candidate design, production, and purification; (2) influenza virus microneutralization assay and cellular responses and lethal challenge with influenza viruses and B. anthracis
Sterne spores. In the methods detailed here, we used different versions of the M2e–HA2 proteins.
Key words Vaccine, Adjuvant, Bacillus anthracis, Edema toxin, Edema factor, Influenza
1 Introduction
Bacillus anthracis escapes from host immune responses using two
toxins: edema toxin (ET) and lethal toxin (LT). These toxins are
formed after lethal factor (LF) or edema factor (EF) bind to protective antigen (PA), and individually they are nontoxic. The PA
associated with EF or LF binds to ANTXR1 (tumor endothelial
marker 8) and ANTXR2 (capillary morphogenesis protein 2). This
protein heptamer complex forms a pore in the endosomal membrane facilitating the translocation of the toxins into the cytosol,
where they exert their toxic activities [1]. LF is a zinc-dependent
metalloprotease that cleaves six of the seven mitogen-activated
protein kinase (MAPK) kinases, and EF is a calcium- and
calmodulin-dependent adenylate cyclase that increases intracellular
cAMP concentrations [2]. It is known that the EF binding region
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_22, © Springer Science+Business Media, LLC, part of Springer Nature 2021
423
Vaccine Delivery with a Detoxified Bacterial Toxin
Diana Diaz-Are ´ valo, Yanping Chen, and Mingtao Zeng
Abstract
It is still a challenge to develop needle-free mucosal vaccines. Despite progress in the development of the
influenza vaccine, it must be reformulated annually because of antigenic changes in circulating influenza
viral strains. Due to seasonal drift and shift of circulating strains, the influenza vaccine does not always match
the circulating strains, and included adjuvants are not sufficient to induce a protective effect with long-lived
memory cells. The adjuvants play a major role in the immune responses to a vaccine. Interestingly, the
Bacillus anthracis detoxified anthrax edema toxin, which composes of protective antigen PA and
N-fragment of edema factor (EFn), has shown improved effects for humoral and cellular immune
responses. Here we describe the design of a universal influenza vaccine construct that consists of three
tandem M2e repeats of the influenza antigen plus HA2 and detoxified toxin EFn, which is associated with
the PA component, as well as the techniques used to corroborate protection. We present two major parts of
description to demonstrate the vaccine strategy, using detoxified anthrax toxin for intranasal delivery of
influenza antigen: (1) vaccine candidate design, production, and purification; (2) influenza virus microneutralization assay and cellular responses and lethal challenge with influenza viruses and B. anthracis
Sterne spores. In the methods detailed here, we used different versions of the M2e–HA2 proteins.
Key words Vaccine, Adjuvant, Bacillus anthracis, Edema toxin, Edema factor, Influenza
1 Introduction
Bacillus anthracis escapes from host immune responses using two
toxins: edema toxin (ET) and lethal toxin (LT). These toxins are
formed after lethal factor (LF) or edema factor (EF) bind to protective antigen (PA), and individually they are nontoxic. The PA
associated with EF or LF binds to ANTXR1 (tumor endothelial
marker 8) and ANTXR2 (capillary morphogenesis protein 2). This
protein heptamer complex forms a pore in the endosomal membrane facilitating the translocation of the toxins into the cytosol,
where they exert their toxic activities [1]. LF is a zinc-dependent
metalloprotease that cleaves six of the seven mitogen-activated
protein kinase (MAPK) kinases, and EF is a calcium- and
calmodulin-dependent adenylate cyclase that increases intracellular
cAMP concentrations [2]. It is known that the EF binding region
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_22, © Springer Science+Business Media, LLC, part of Springer Nature 2021
423