to PA is localized at the N-terminal (1–254 residues), and its
toxicity is lost after removing the C-terminal [3, 4].
Influenza virus diseases are prevented with a seasonal vaccine;
however, due to the frequent occurrence of mutations (drift and
shift), some circulating virus strains may not match the seasonal
vaccine. The best way to effectively protect people, especially young
children and senior citizens, is to develop a universal vaccine with
conserved viral regions. The ion channel protein M2 is found in all
strains of the influenza A virus, and this structural protein is
incorporated into the viral lipid envelope. The 23-amino acid extracellular N-terminal domain (M2e) is highly conserved, making it an
attractive vaccine target for a broadly effective protein vaccine
[5]. Hemagglutinin 0 is a precursor glycoprotein anchor at the
viral membrane that its cleavage form by transmembrane anchor
close to carboxyl terminal, it cleavage two disulfide-linked subunits,
HA1 and HA2, eliminating an arginine residue, R329 [6]. HA2 is
more highly conserved between influenza viruses, and anti-HA2specific antibodies reduce the replication of influenza [7]. Also,
vaccines based on conserved HA2 regions can have prophylactic
efficacy [8, 9].
Taken together, anthrax and influenza induce cellular and
humoral immune responses, whereas PA induces T cell immunity
and low levels of anti-PA antibodies [10], indicating that the protection is associated with T cell responses. The same happens with
lethal factor, which induces specific IFNγ-producing CD4
+ T cells
in patients treated for cutaneous anthrax and generating longlasting immunity to anthrax [11]. However, the immunization of
BALB/c mice with PA and EF induces high antibody titers, which
demonstrated an adjuvant effect. Mice vaccinated with PA + EF had
higher titers than mice immunized with PA + LF [12]. The nasal
delivery of ET coadministered with OVA resulted in high titers of
OVA-specific IgA and IgG [13]. In addition, the nontoxic
N-terminal fragment of EF was evaluated as a vaccine candidate
against anthrax. This vaccine was replicated in an incompetent
adenoviral vector (Ad/EFn) encoding the N-terminal region
(1–254 amino acids) of the edema factor (EFn). This vaccine was
administered to mice three times with 10
8 plaque-forming units
(PFU)/dose and resulted in 37% and 57% of the protection after
challenge with B. anthracis Sterne strain spores at a dosage of
200 Â LD 50 and 100 Â LD 50 , respectively [14]. The N-terminal
fragments of EF and LF bind to PA to further translocate to the
cytosol. Taking advantage of this property, it is possible to use
EF + PA to carry any type of vaccine into the antigen-presenting
cells and increase the immune response, since EF has an adjuvant
property [13].
In this chapter, we describe two methods to analyze the functionality of detoxified anthrax edema toxin EFn + PA as a carrier
and adjuvant system. The first method describes the design,
424
Diana Diaz-Are ´ valo et al.
toxicity is lost after removing the C-terminal [3, 4].
Influenza virus diseases are prevented with a seasonal vaccine;
however, due to the frequent occurrence of mutations (drift and
shift), some circulating virus strains may not match the seasonal
vaccine. The best way to effectively protect people, especially young
children and senior citizens, is to develop a universal vaccine with
conserved viral regions. The ion channel protein M2 is found in all
strains of the influenza A virus, and this structural protein is
incorporated into the viral lipid envelope. The 23-amino acid extracellular N-terminal domain (M2e) is highly conserved, making it an
attractive vaccine target for a broadly effective protein vaccine
[5]. Hemagglutinin 0 is a precursor glycoprotein anchor at the
viral membrane that its cleavage form by transmembrane anchor
close to carboxyl terminal, it cleavage two disulfide-linked subunits,
HA1 and HA2, eliminating an arginine residue, R329 [6]. HA2 is
more highly conserved between influenza viruses, and anti-HA2specific antibodies reduce the replication of influenza [7]. Also,
vaccines based on conserved HA2 regions can have prophylactic
efficacy [8, 9].
Taken together, anthrax and influenza induce cellular and
humoral immune responses, whereas PA induces T cell immunity
and low levels of anti-PA antibodies [10], indicating that the protection is associated with T cell responses. The same happens with
lethal factor, which induces specific IFNγ-producing CD4
+ T cells
in patients treated for cutaneous anthrax and generating longlasting immunity to anthrax [11]. However, the immunization of
BALB/c mice with PA and EF induces high antibody titers, which
demonstrated an adjuvant effect. Mice vaccinated with PA + EF had
higher titers than mice immunized with PA + LF [12]. The nasal
delivery of ET coadministered with OVA resulted in high titers of
OVA-specific IgA and IgG [13]. In addition, the nontoxic
N-terminal fragment of EF was evaluated as a vaccine candidate
against anthrax. This vaccine was replicated in an incompetent
adenoviral vector (Ad/EFn) encoding the N-terminal region
(1–254 amino acids) of the edema factor (EFn). This vaccine was
administered to mice three times with 10
8 plaque-forming units
(PFU)/dose and resulted in 37% and 57% of the protection after
challenge with B. anthracis Sterne strain spores at a dosage of
200 Â LD 50 and 100 Â LD 50 , respectively [14]. The N-terminal
fragments of EF and LF bind to PA to further translocate to the
cytosol. Taking advantage of this property, it is possible to use
EF + PA to carry any type of vaccine into the antigen-presenting
cells and increase the immune response, since EF has an adjuvant
property [13].
In this chapter, we describe two methods to analyze the functionality of detoxified anthrax edema toxin EFn + PA as a carrier
and adjuvant system. The first method describes the design,
424
Diana Diaz-Are ´ valo et al.